G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
breakpoint cluster region
G00000922 (Mus musculus)

Databases (7)

ENSG00000186716 (Ensembl human gene)
613 (Entrez Gene)
720 (G2Cdb plasticity & disease)
BCR (GeneCards)
151410 (OMIM)
Marker Symbol
HGNC:1014 (HGNC)
Protein Sequence
P11274 (UniProt)

Synonyms (4)

  • ALL
  • CML
  • D22S662
  • PHL

Literature (217)

Pubmed - human_disease

  • [Role of molecular screening for common fusion genes in the diagnosis and classification of leukemia].

    Zhu YL, Zhang Y, Zhu P, Yang Y, Du JW and Liu J

    Department of Haematology, Peking University First Hospital, Beijing 100034, China.

    Objective: To assess the value of common fusion genes analysis in the diagnosis and classification of leukemia by multiplex RT-PCR.

    Methods: The multiplex RT-PCR, including 8 parallel PCR reactions, could screen 86 mRNA breakpoints or splice variants at the same time, which was important for the diagnosis and prognosis of leukemia. Bone marrow samples from 161 cases of leukemia and 8 cases of myelodysplastic syndrome (MDS) were involved in the study. The distribution of common fusion genes in leukemia was analyzed by the method mentioned above in combination with clinical and morphological features.

    Results: Ten fusion genes were detected in 115 cases of leukemia, including AML1/ETO, PML/RAR alpha, PLZF/RAR alpha, dupMLL, MLL/AF6, MLL/AF10, CBFbeta/MYH11, BCR/ABL, Hox11, and EVI1 BCR/ABL was positive in all the 52 cases of chronic myeloid leukemia; PML/RAR alpha was found in 21 of 25 acute promyelocytic leukemia (APL), and PLZF/RAR alpha was detected in one case of APL. Sixteen cases of 17 AML1/ETO-positive acute leukemia (AL) belonged to FAB-M2 subtype, and one case was mixed leukemia. Three of 4 AL cases carrying CBFbeta/MYH11 were M4 subtype, and one was M5 subtype. MLL aberrations were found in 16 AL, in which all MLL/AF6 translocation existed in M5 subtype with classic monoblastic characters. Furthermore, BCR/ABL was detected in 5 acute lymphoblastic leukemia (ALL) cases. Fusion genes were also found in 2 MDS cases, of which AML1/ETO positive-MDS-RAEB progressed to AML rapidly.

    Conclusion: Screening of common fusion genes by multiplex RT-PCR is an important tool which could provide useful and reliable molecular genetic information for the diagnosis and treatment of leukemia.

    Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 2005;37;3;236-9

Pubmed - other

  • A new fused transcript in Philadelphia chromosome positive acute lymphocytic leukaemia.

    Fainstein E, Marcelle C, Rosner A, Canaani E, Gale RP, Dreazen O, Smith SD and Croce CM

    Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.

    The leukaemic cells of more than 90% of chronic myelogenous leukaemia (CML) patients and of 10% of acute lymphocytic leukaemia (ALL) patients carry the t(9:22) (q34:q11) translocation which generates the Philadelphia chromosome (Ph1). In CML the abl gene is translocated from chromosome 9 to the centre of the bcr gene on chromosome 22 and this results in production of chimaeric bcr-abl RNA translated into a protein of relative molecular mass (Mr) 210,000 (210K). Our data indicate that in ALL abl is translocated into the 5' region of the bcr gene. The consequence of this is the expression of a fused transcript in which the first exon of bcr is linked to the second abl exon. This transcript encodes a 190K protein kinase.


  • Structural organization of the bcr gene and its role in the Ph' translocation.

    Heisterkamp N, Stam K, Groffen J, de Klein A and Grosveld G

    The Philadelphia (Ph') chromosome, an abnormal chromosome 22 (ref. 1), is one of the best-known examples of a specific human chromosomal abnormality strongly associated with one form of human leukaemia, chronic myelocytic leukaemia (CML). The finding that a small region of chromosome 9 which includes the c-abl oncogene is translocated to chromosome 22 prompted studies to elucidate the molecular mechanisms involved in this disease. We have demonstrated previously that the chromosome 9 of one patient with CML contains a breakpoint 14 kilobases (kb) 5' of the most 5' v-abl-homologous exon. These data suggest a role for c-abl in CML, a theory supported by the presence of an abnormally sized abl messenger RNA and protein in the CML cell line K562. The region involved in the translocation on chromosome 22 has also been identified: all Ph'-positive patients examined to date have a breakpoint within a 5.8-kb region, for which we have proposed the name 'breakpoint cluster region' (bcr). To determine whether bcr contains protein-encoding regions, probes from bcr were tested for their ability to hybridize to complementary DNA sequences. A 0.6-kb HindIII/BamHI bcr restriction enzyme fragment proved suitable for isolating several cDNA clones from a human fibroblast cDNA library. Using bcr cDNA sequences, we obtained data strongly suggesting the presence of a chimaeric bcr/abl mRNA in the leukaemic cells of Ph'-positive CML patients. The recent isolation of cDNA clones containing bcr and abl sequences confirms this finding. Because the bcr part of the chimaeric mRNA could be required to induce the transforming activity of the human c-abl oncogene, we have now initiated studies to characterize the normal 'bcr gene' and to determine the effect of a translocation within its coding domain. We demonstrate that as a result of the Ph' translocation, a variable number of bcr exons are included in the chimaeric bcr/abl mRNA. The bcr gene sequences in this mRNA could be responsible for the transition of the abl cellular proto-oncogene into an oncogene.


  • Genetic susceptibility to distinct bladder cancer subphenotypes.

    Guey LT, García-Closas M, Murta-Nascimento C, Lloreta J, Palencia L, Kogevinas M, Rothman N, Vellalta G, Calle ML, Marenne G, Tardón A, Carrato A, García-Closas R, Serra C, Silverman DT, Chanock S, Real FX, Malats N and EPICURO/Spanish Bladder Cancer Study investigators

    Spanish National Cancer Research Centre, Madrid, Spain.

    Background: Clinical, pathologic, and molecular evidence indicate that bladder cancer is heterogeneous with pathologic/molecular features that define distinct subphenotypes with different prognoses. It is conceivable that specific patterns of genetic susceptibility are associated with particular subphenotypes.

    Objective: To examine evidence for the contribution of germline genetic variation to bladder cancer heterogeneity.

    The Spanish Bladder Cancer/EPICURO Study is a case-control study based in 18 hospitals located in five areas in Spain. Cases were patients with a newly diagnosed, histologically confirmed, urothelial cell carcinoma of the bladder from 1998 to 2001. Case diagnoses were reviewed and uniformly classified by pathologists following the World Health Organisation/International Society of Urological Pathology 1999 criteria. Controls were hospital-matched patients (n=1149).

    Measurements: A total of 1526 candidate variants in 423 candidate genes were analysed. Three distinct subphenotypes were defined according to stage and grade: low-grade nonmuscle invasive (n=586), high-grade nonmuscle invasive (n=219), and muscle invasive (n=246). The association between each variant and subphenotype was assessed by polytomous risk models adjusting for potential confounders. Heterogeneity in genetic susceptibility among subphenotypes was also tested.

    Two established bladder cancer susceptibility genotypes, NAT2 slow-acetylation and GSTM1-null, exhibited similar associations among the subphenotypes, as did VEGF-rs25648, which was previously identified in our study. Other variants conferred risks for specific tumour subphenotypes such as PMS2-rs6463524 and CD4-rs3213427 (respective heterogeneity p values of 0.006 and 0.004), which were associated with muscle-invasive tumours (per-allele odds ratios [95% confidence interval] of 0.56 [0.41-0.77] and 0.71 [0.57-0.88], respectively) but not with non-muscle-invasive tumours. Heterogeneity p values were not robust in multiple testing according to their false-discovery rate.

    Conclusions: These exploratory analyses suggest that genetic susceptibility loci might be related to the molecular/pathologic diversity of bladder cancer. Validation through large-scale replication studies and the study of additional genes and single nucleotide polymorphisms are required.

    Funded by: Intramural NIH HHS: ZIA CP010136-16

    European urology 2010;57;2;283-92

  • Mutation of ARHGAP9 in patients with coronary spastic angina.

    Takefuji M, Asano H, Mori K, Amano M, Kato K, Watanabe T, Morita Y, Katsumi A, Itoh T, Takenawa T, Hirashiki A, Izawa H, Nagata K, Hirayama H, Takatsu F, Naoe T, Yokota M and Kaibuchi K

    Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa-ku, Nagoya, Japan.

    Coronary artery spasm has an important function in the etiology of variant angina and other acute coronary syndromes. Abnormal activation of Rho-family GTPases has been observed in cardiovascular disorders, but the function of genetic variability in Rho-family GTPases remains to be evaluated in cardiovascular disorders. We examined the genetic variability of Rho-family GTPases and their regulators in coronary artery spasm. We performed a comprehensive candidate gene analysis of 67 single nucleotide polymorphisms with amino-acid substitution in Rho-family GTPases and their regulators in 103 unrelated Japanese patients with acetylcholine-induced coronary artery spasm and 102 control Japanese subjects without acetylcholine-induced coronary artery spasm. We noted an association of the single nucleotide polymorphism of ARHGAP9 (rs11544238, Ala370Ser) with coronary artery spasm (odds ratio =2.67). We found that ARHGAP9 inactivated Rac as RacGAP and that the mRNA level of ARHGAP9 was strongly detected in hematopoietic cells. ARHGAP9 negatively regulated cell migration. The Ala370Ser polymorphism counteracted ARHGAP9-reduced cell migration, spreading and adhesion. The Ala370Ser polymorphism in the ARHGAP9 gene is associated with coronary artery spasm. These data suggest that the polymorphism of ARHGAP9 has a critical function in the infiltration of hematopoietic cells into the endothelium and inflammation leading to endothelial dysfunction.

    Journal of human genetics 2010;55;1;42-9

  • Transglutaminase 2 regulates the GTPase-activating activity of Bcr.

    Yi SJ, Groffen J and Heisterkamp N

    Section of Molecular Carcinogenesis, Division of Hematology/Oncology, Childrens Hospital Los Angeles and the Saban Research Institute of Childrens Hospital, Los Angeles, California 90027, USA.

    Transglutaminase 2 (TG2) is a multifunctional protein that has been implicated in numerous pathologies including that of neurodegeneration and celiac disease, but the molecular interactions that mediate its diverse activities are largely unknown. Bcr and the closely related Abr negatively regulate the small G-protein Rac: loss of their combined function in vivo results in increased reactivity of innate immune cells. Bcr and Abr are GTPase-activating proteins that catalyze the hydrolysis of the GTP bound to Rac. However, how the Bcr and Abr GTPase-activating activity is regulated is not precisely understood. We here report a novel mechanism of regulation through direct protein-protein interaction with TG2. TG2 bound to the Rac-binding pocket in the GTPase-activating domains of Bcr and Abr, blocked Bcr activity and, through this mechanism, increased levels of active GTP-bound Rac and EGF-stimulated membrane ruffling. TG2 exists in at least two different conformations. Interestingly, experiments using TG2 mutants showed that Bcr exhibits preferential binding to the non-compacted conformation of TG2, in which its catalytic domain is exposed, but transamidation is not needed for the interaction. Thus, TG2 regulates levels of cellular GTP-bound Rac and actin cytoskeletal reorganization through a new mechanism involving direct inhibition of Bcr GTPase-activating activity.

    Funded by: NHLBI NIH HHS: HL060231, HL071945, P01 HL060231, R01 HL071945

    The Journal of biological chemistry 2009;284;51;35645-51

  • An accurate and rapid flow cytometric diagnosis of BCR-ABL positive acute lymphoblastic leukemia.

    Raponi S, De Propris MS, Wai H, Intoppa S, Elia L, Diverio D, Vitale A, Foà R and Guarini A

    Division of Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy.

    Tyrosine kinase inhibitors have profoundly modified the treatment and prognosis of chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia. A rapid and accurate detection of the BCR-ABL fusion protein is paramount today for an optimal management of Ph(+) acute lymphoblastic leukemia. We have utilized a recently described and commercialized immunoassay that identifies qualitatively the presence of the BCR-ABL protein in leukemic cell lysates. The BCR-ABL fusion protein is captured and detected by a cytometric bead assay and analyzed by flow cytometry. The assay was applied to 101 primary patient samples (94 acute leukemias and 7 chronic myeloid leukemia blast crisis) and the results of the immunoassay were concordant with those obtained by conventional molecular techniques. The method proved reliable, reproducible, of simple execution and it was successfully completed within four hours. This flow cytometric immunoassay has important implications for perfecting the management of Ph(+) acute lymphoblastic leukemia patients worldwide.

    Haematologica 2009;94;12;1767-70

  • PTEN identified as important risk factor of chronic obstructive pulmonary disease.

    Hosgood HD, Menashe I, He X, Chanock S and Lan Q

    Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.

    Common genetic variation may play an important role in altering chronic obstructive pulmonary disease (COPD) risk. In Xuanwei, China, the COPD rate is more than twice the Chinese national average, and COPD is strongly associated with in-home coal use. To identify genetic variation that may be associated with COPD in a population with substantial in-home coal smoke exposures, we evaluated 1261 single nucleotide polymorphisms (SNPs) in 380 candidate genes potentially relevant for cancer and other human diseases in a population-based case-control study in Xuanwei (53 cases; 107 controls). PTEN was the most significantly associated gene with COPD in a minP analysis using 20,000 permutations (P=0.00005). SNP-based analyses found that homozygote variant carriers of PTEN rs701848 (OR(TT)=0.12, 95% CI=0.03-0.47) had a significant decreased risk of COPD. PTEN, or phosphatase and tensin homolog, is an important regulator of cell cycle progression and cellular survival via the AKT signaling pathway. Our exploratory analysis suggests that genetic variation in PTEN may be an important risk factor of COPD in Xuanwei. However, due to the small sample size, additional studies are needed to evaluate these associations within Xuanwei and other populations with coal smoke exposures.

    Funded by: Intramural NIH HHS: Z99 CA999999

    Respiratory medicine 2009;103;12;1866-70

  • p210(Bcr-Abl) desensitizes Cdc42 GTPase signaling for SDF-1alpha-directed migration in chronic myeloid leukemia cells.

    Chang YC, Tien SC, Tien HF, Zhang H, Bokoch GM and Chang ZF

    Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.

    Chronic myeloid leukemia (CML) is a lethal hematological disorder caused by the p210(Bcr-Abl) oncogene. Previous studies have suggested that p210(Bcr-Abl) transformation contributes to homing and retention defects, typical of immature myeloid cells in CML, by attenuating chemotactic response to stromal-derived factor-1alpha (SDF-1alpha). As Rho family GTPases are key regulators of the cytoskeleton and have been previously found to interact with p210(Bcr-Abl), this study aimed to determine whether p210(Bcr-Abl) signaling affects SDF-1alpha chemotaxis through Rho GTPase signaling. We found that SDF-1alpha stimulated Cdc42 GTPase activation in myeloid progenitor 32D, but not in p210(Bcr-Abl)-transformed (32Dp210) cells. In fact, the basal level of active Cdc42 was elevated in 32Dp210 cells and mononuclear cells isolated from bone marrow of CML patients. Inhibition of p210(Bcr-Abl) kinase activity decreased basal Cdc42 activity and restored SDF-1alpha-induced Cdc42 and migration responses. Transduction of active Tat-Cdc42V12 abolished this reconstituted chemotactic response. As Cdc42 is particularly important in cytoskeletal remodeling and directional sensing, these results suggest that sustained activation of Cdc42 GTPase through p210(Bcr-Abl) tyrosine kinase signaling in CML cells contributes to defects in SDF-1alpha-chemotactic response due to desensitization of the actin polarization signal required for directional migration.

    Oncogene 2009;28;46;4105-15

  • Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment.

    Zheng X, Oancea C, Henschler R, Moore MA and Ruthardt M

    Department of Hematology, Laboratory for Tumor Stem Cell Biology, Goethe University, Frankfurt, Germany.

    Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome--Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185(BCR/ABL) from der22 and p96(ABL/BCR) from der9. The "major" breakpoint in CML encodes p210(BCR/ABL) and p40(ABL/BCR). Herein, we investigated the leukemogenic potential of the der9-associated p96(ABL/BCR) and p40(ABL/BCR) fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL.

    Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming--spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR.

    Both p96(ABL/BCR) and p40(ABL/BCR) increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96(ABL/BCR) and to a minor extent p40(ABL/BCR) forced the B-cell commitment of SL-cells and UCBC.

    Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.

    PloS one 2009;4;10;e7661

  • DNA vaccination against bcr-abl-positive cells in mice.

    Lucansky V, Sobotkova E, Tachezy R, Duskova M and Vonka V

    Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic.

    A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

    International journal of oncology 2009;35;4;941-51

  • Determining the rise in BCR-ABL RNA that optimally predicts a kinase domain mutation in patients with chronic myeloid leukemia on imatinib.

    Press RD, Willis SG, Laudadio J, Mauro MJ and Deininger MW

    Department of Pathology, Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97201, USA. pressr@ohsu.edu

    In imatinib-treated chronic myeloid leukemia (CML), secondary drug resistance is often caused by mutations in the BCR-ABL kinase domain (KD). As alternative therapies are available for imatinib resistance, early identification of mutations may prevent disease progression. Because most patients are routinely monitored by BCR-ABL quantitative polymerase chain reaction (PCR), it is important to define the optimal increase in BCR-ABL that should trigger mutation testing. Expert panels have provisionally recommended a 10-fold BCR-ABL increase as the trigger for mutation screening, acknowledging the lack of consensus. To address this question, we monitored 150 CML patients by quantitative PCR and DNA sequencing. Thirty-five different mutations were identified in 53 patients, and, during 22.5 months (median) of follow-up after sequencing, mutations were significantly predictive of shorter progression-free survival. An unbiased receiver operating characteristic analysis identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). The 2.6-fold threshold approximated the analytic precision limit of our PCR assay. In contrast, transcript rise cutoffs of 5-fold or greater had poor diagnostic sensitivity and no significant association with mutations. We conclude that the currently recommended 10-fold threshold to trigger mutation screening is insensitive and not universally applicable.

    Funded by: NCI NIH HHS: R21 CA095203; NHLBI NIH HHS: HL082978-01, R01 HL082978

    Blood 2009;114;13;2598-605

  • Impact of baseline BCR-ABL mutations on response to nilotinib in patients with chronic myeloid leukemia in chronic phase.

    Hughes T, Saglio G, Branford S, Soverini S, Kim DW, Müller MC, Martinelli G, Cortes J, Beppu L, Gottardi E, Kim D, Erben P, Shou Y, Haque A, Gallagher N, Radich J and Hochhaus A

    Institute of Medical and Veterinary Science, Hanson Center for Cancer Research, Department of Hematology, Adelaide, 5000, Australia. Timothy.hughes@imvs.sa.gov.au

    Purpose: Nilotinib is a second-generation tyrosine kinase inhibitor indicated for the treatment of patients with chronic myeloid leukemia (CML) in chronic phase (CP; CML-CP) and accelerated phase (AP; CML-AP) who are resistant to or intolerant of prior imatinib therapy. In this subanalysis of a phase II study of nilotinib in patients with imatinib-resistant or imatinib-intolerant CML-CP, the occurrence and impact of baseline and newly detectable BCR-ABL mutations were assessed.

    Baseline mutation data were assessed in 281 (88%) of 321 patients with CML-CP in the phase II nilotinib registration trial.

    Results: Among imatinib-resistant patients, the frequency of mutations at baseline was 55%. After 12 months of therapy, major cytogenetic response (MCyR) was achieved in 60%, complete cytogenetic response (CCyR) in 40%, and major molecular response (MMR) in 29% of patients without baseline mutations versus 49% (P = .145), 32% (P = .285), and 22% (P = .366), respectively, of patients with mutations. Responses in patients who harbored mutations with high in vitro sensitivity to nilotinib (50% inhibitory concentration [IC(50)] <or= 150 nM) or mutations with unknown nilotinib sensitivity were equivalent to those responses for patients without mutations (not significant). Patients with mutations that were less sensitive to nilotinib in vitro (IC(50) > 150 nM; Y253H, E255V/K, F359V/C) had less favorable responses, as 13%, 43%, and 9% of patients with each of these mutations, respectively, achieved MCyR; none achieved CCyR.

    Conclusion: For most patients with imatinib resistance and with mutations, nilotinib offers a substantial probability of response. However, mutational status at baseline may influence response. Less sensitive mutations that occurred at three residues defined in this study, as well as the T315I mutation, may be associated with less favorable responses to nilotinib.

    Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2009;27;25;4204-10

  • The gatekeeper mutation T315I confers resistance against small molecules by increasing or restoring the ABL-kinase activity accompanied by aberrant transphosphorylation of endogenous BCR, even in loss-of-function mutants of BCR/ABL.

    Mian AA, Schüll M, Zhao Z, Oancea C, Hundertmark A, Beissert T, Ottmann OG and Ruthardt M

    Department of Hematology, Goethe University, Frankfurt, Germany.

    In Philadelphia chromosome-positive (Ph+) leukemia BCR/ABL induces the leukemic phenotype. Targeted inhibition of BCR/ABL by kinase inhibitors leads to complete remission. However, patients with advanced Ph+ leukemia relapse and acquire resistance, mainly due to point mutations in BCR/ABL. The 'gatekeeper mutation' T315I is responsible for a general resistance to small molecules. It seems not only to decrease the affinity for kinase inhibitors, but to also confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor independence. Here, we show that T315I (i) requires autophosphorylation at tyrosine 177 in the BCR-portion to mediate resistance against the inhibition of oligomerization; (ii) restores the capacity to mediate factor-independent growth of loss-of-function mutants due to an increase in or activation of ABL-kinase; (iii) leads to phosphorylation of endogenous BCR, suggesting aberrant substrate activation by BCR/ABL harboring the T315I mutation. These data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL-T315I-positive blasts.

    Leukemia 2009;23;9;1614-21

  • BCR-ABL promotes the frequency of mutagenic single-strand annealing DNA repair.

    Fernandes MS, Reddy MM, Gonneville JR, DeRoo SC, Podar K, Griffin JD, Weinstock DM and Sattler M

    Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

    Intracellular oxidative stress in cells transformed by the BCR-ABL oncogene is associated with increased DNA double-strand breaks. Imprecise repair of these breaks can result in the accumulation of mutations, leading to therapy-related drug resistance and disease progression. Using several BCR-ABL model systems, we found that BCR-ABL specifically promotes the repair of double-strand breaks through single-strand annealing (SSA), a mutagenic pathway that involves sequence repeats. Moreover, our results suggest that mutagenic SSA repair can be regulated through the interplay between BCR-ABL and extrinsic growth factors. Increased SSA activity required Y177 in BCR-ABL, as well as a functional PI3K and Ras pathway downstream of this site. Furthermore, our data hint at a common pathway for DSB repair whereby BCR-ABL, Tel-ABL, Tel-PDGFR, FLT3-ITD, and Jak2V617F all increase mutagenic repair. This increase in SSA may not be sufficiently suppressed by tyrosine kinase inhibitors in the stromal microenvironment. Therefore, drugs that target growth factor receptor signaling represent potential therapeutic agents to combat tyrosine kinase-induced genomic instability.

    Funded by: NCI NIH HHS: CA134660-01, CA36167, CA66996, P01 CA066996, R01 CA036167, R01 CA134660, R37 CA036167; NIDDK NIH HHS: DK50654, P01 DK050654

    Blood 2009;114;9;1813-9

  • Depletion of Pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 by Bcr-Abl promotes chronic myelogenous leukemia cell proliferation through continuous phosphorylation of Akt isoforms.

    Hirano I, Nakamura S, Yokota D, Ono T, Shigeno K, Fujisawa S, Shinjo K and Ohnishi K

    Department of Internal Medicine III, Cancer Center, Hamamatsu University School of Medicine, Shizuoka, Japan.

    The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.

    The Journal of biological chemistry 2009;284;33;22155-65

  • Bcr-Abl-mediated redox regulation of the PI3K/AKT pathway.

    Naughton R, Quiney C, Turner SD and Cotter TG

    Tumour Biology Laboratory, Biochemistry Department, Bioscience Research Institute, University College, Cork, Ireland.

    Bcr-Abl causes chronic myelogenous leukemia, a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells. In this study, inducible expression of Bcr-Abl in TonB.210 cells is associated with increased production of intracellular reactive oxygen species (ROS), which is thought to play a role in survival signaling when generated at specific levels. Elevated ROS in Bcr-Abl-expressing cells were found to activate PI3k/Akt pathway members such as Akt and GSK3beta as well as downstream targets beta-catenin and Mcl-1. The activation of these proteins was inhibited by the flavoprotein inhibitor diphenyleneiodonium, which is commonly used to inhibit NADPH oxidase (Nox). This indicated that increased ROS might be related to increased activity of one member of the Nox family. Knock-down experiments using siRNA suggest that Nox-4 is the main source of increased ROS following Bcr-Abl expression. We showed that Bcr-Abl-induced ROS could also increase survival pathway signaling through redox inhibition of PP1alpha, a serine threonine phosphatase that negatively regulates the PI3k/Akt pathway. Overall our results demonstrate that Bcr-Abl expression increases Nox-4-generated ROS, which in turn increases survival signaling through PI3k/Akt pathway by inhibition of PP1alpha, thus contributing to the high level of resistance to apoptosis seen in these Bcr-Abl-expressing cells.

    Leukemia 2009;23;8;1432-40

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models.

    Boumendjel A, McLeer-Florin A, Champelovier P, Allegro D, Muhammad D, Souard F, Derouazi M, Peyrot V, Toussaint B and Boutonnat J

    Bâtiment Jean Roget, Faculté de Médecine, Grenoble, F-38700 France. ahcene.boumendjel@ujf-grenoble.fr

    Background: Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect.

    Methods: The in-vitro activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on in-vitro microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound in vivo. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours.

    Results: In the four human and the murine glioblastoma cell lines tested, 10 muM JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 x 105 M-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These in vitro studies were reinforced by our in vivo investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB.

    Conclusion: These in vitro and in vivo data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model.

    BMC cancer 2009;9;242

  • New mutations detected by denaturing high performance liquid chromatography during screening of exon 6 bcr-abl mutations in patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors.

    Mascarenhas CC, Cunha AF, Miranda EC, Zulli R, Silveira RA, Costa FF, Pagnano KB and De Souza CA

    Hematology and Hemotherapy Center, University of Campinas, SP, Brazil.

    Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinase (TK) inhibitors in patients with CML, conferring a poor prognosis. T315I (Treonine-->Isoleucine) is a mutation in the exon 6 of BCR-ABL gene that makes the protein resistant to kinase inhibitors currently used for treating CML. Denaturing High-performance liquid chromatography (D-HPLC) allows for high throughput mutation screening. In this study, we screened mutations in exon 6 of the BCR-ABL gene in patients presenting failure or sub optimal response according to Leukemia Net criteria and correlated the presence of mutations with clinical outcome. Genomic DNA was extracted from peripheral blood samples from 93 patients with CML (5 intolerant and 88 resistant). The PCR product was analysed by D-HPLC, and the patients samples with abnormal D-HLPC profiles were submitted to automated sequencing, using specific primers. Overall survival (OS) was calculated from the date of mutation analysis, for the whole group and for both groups (mutation versus no mutation). We screened mutations in exon 6 of the BCR-ABL gene in 93 CML TKI - resistant patients. Twenty-three out of 93 samples (25%) showed an abnormal elution profile. Automated sequencing confirmed the presence of a nucleotide change in 19 out of 23 cases: one polymorphism, T315T, seven known point mutations: T315I, F317L, V339L, M351T, E355G and F359V and three novel mutations: C305R, D325D and I360S. OS for the whole group was 80% in a median observation time of 30 months. OS for patients without the mutation was 87% and with the mutation was 56%, in a median observation time of 37 and 10 months, respectively (p < 0.0001, RR = 68). D-HPLC is a practical and sensitive method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimising therapy in CML. The screening of mutations in exon 6 is clinically relevant, once the presence of mutations confers a poor outcome. Early detection of emerging mutant clones may help in decision-making for alternative treatment.

    Leukemia & lymphoma 2009;50;7;1148-54

  • Sensitization of imatinib-resistant CML cells to TRAIL-induced apoptosis is mediated through down-regulation of Bcr-Abl as well as c-FLIP.

    Park SJ, Kim MJ, Kim HB, Kang CD and Kim SH

    Department of Biochemistry, Pusan National University School of Medicine, Yangsan 622-770, South Korea.

    Resistance to imatinib is commonly associated with reactivation of Bcr-Abl signalling. However, Bcr-Abl-independent signalling pathways may be activated and contributed to imatinib resistance in some CML (chronic myelogenous leukaemia) patients. We had isolated three imatinib-resistant K562/R1, R2 and R3 variants with gradual loss of Bcr-Abl from K562 cells to develop effective therapeutic strategies for imatinib-resistant CML. Interestingly, we found that these cells became highly sensitive to TRAIL (tumour necrosis factor-related apoptosis-inducing factor) in comparison with K562 cells showing high resistance to TRAIL. Treatment of K562/R3 cells with TRAIL resulted in activation of TRAIL receptor pathway by including caspase 8 activation, Bid cleavage, cytochrome c release and caspase 3 activation. These results were accompanied by down-regulation of c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-inhibitory protein} in imatinib-resistant K562 variants compared with K562 cells. Overexpression of c-FLIP in K562/R3 cells acquired TRAIL resistance and conversely, c-FLIP-silenced K562 cells became sensitive to TRAIL. Moreover, Bcr-Abl-silenced K562 cells showed down-regulation of c-FLIP and the subsequent overcome of TRAIL resistance. Taken together, our results demonstrated for the first time that the loss of Bcr-Abl in imatinib-resistant cells led to the down-regulation of c-FLIP and subsequent increase of TRAIL sensitivity, suggesting that TRAIL could be an effective strategy for the treatment of imatinib-resistant CML with loss of Bcr-Abl.

    The Biochemical journal 2009;420;1;73-81

  • Triptolide inhibits Bcr-Abl transcription and induces apoptosis in STI571-resistant chronic myelogenous leukemia cells harboring T315I mutation.

    Shi X, Jin Y, Cheng C, Zhang H, Zou W, Zheng Q, Lu Z, Chen Q, Lai Y and Pan J

    Department of Pathophysiology, Zhongshan School of Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.

    Purpose: Resistance to STI571 is an emerging problem for patients with chronic myelogenous leukemia (CML). Mutation in the kinase domain of Bcr-Abl is the predominant mechanism of the acquired resistance to STI571. In the present study, we investigated the effect of triptolide on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl.

    CML cell lines (KBM5 versus KBM5-T315I, BaF3-Bcr-Abl versus BaF3-Bcr-Abl-T315I) and primary cells from CML patients with clinical resistance to STI571 were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. Nude mouse xenograft model was also used to evaluate the antitumor activity.

    Results: Triptolide potently down-regulated the mRNA and protein levels of Bcr-Abl independently of the caspase or proteosome activation in CML cells. It induced mitochondrial-dependent apoptosis in Bcr-Abl-T315I CML cells and primary cells from CML patients with clinical resistance to STI571. Additionally, triptolide inhibited the growth of STI571-sensitive KBM5 and STI571-resistant KBM5-T315I CML cells in nude mouse xenografts. Triptolide also down-regulated the expression of survivin, Mcl-1, and Akt in CML cells, which suggests that it may have multiple targets.

    Conclusions: These findings suggest that triptolide is a promising agent to overcome STI571-resistant CML cells, and warrant a clinical trial of triptolide derivatives for CML with Bcr-Abl-T315I mutation.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;5;1686-97

  • Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage.

    Järås M, Johnels P, Agerstam H, Lassen C, Rissler M, Edén P, Cammenga J, Olofsson T, Bjerrum OW, Richter J, Fan X and Fioretos T

    Department of Clinical Genetics, Lund University, Sweden.

    Objective: The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells.

    We investigated and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood.

    Results: Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under in vitro conditions favoring B-cell differentiation, neither P190 nor P210 BCR/ABL1-expressing cells formed detectable levels of CD19-positive cells. Gene expression profiling revealed that P190 BCR/ABL1 and P210 BCR/ABL1 induced almost identical gene expression profiles.

    Conclusions: Our data suggest that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion.

    Experimental hematology 2009;37;3;367-75

  • Is p190 bcr-abl rearrangement necessary for acute transformation in some p210 CML of childhood?

    Scrideli CA, de Oliveira FM, Brassesco MS, de Paula Queiroz R, Bernardes JE, Valera ET and Tone LG

    Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14049-900 Ribeirão Preto (SP), Brazil.

    Chronic myeloid leukemia (CML) is a rare disease in childhood which is almost exclusively associated with bcr-abl p210 (M-bcr) rearrangements. It has been suggested that co-expression of p190 and p210 may be a pathway of CML progression in adult patients. We report two cases of pediatric patients with a diagnosis of CML who presented co-expression of the p210 and p190 transcripts during progression to the blastic phase. The present data suggest that p190 may be a secondary event in at least some cases of childhood CML, suggesting an association with progression to a blastic crisis in these patients.

    Leukemia research 2009;33;3;495-9

  • Molecular biology of bcr-abl1-positive chronic myeloid leukemia.

    Quintás-Cardama A and Cortes J

    Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. aquintas@mdanderson.org

    Chronic myeloid leukemia (CML) has been regarded as the paradigmatic example of a malignancy defined by a unique molecular event, the BCR-ABL1 oncogene. Decades of research zeroing in on the role of BCR-ABL1 kinase in the pathogenesis of CML have culminated in the development of highly efficacious therapeutics that, like imatinib mesylate, target the oncogenic kinase activity of BCR-ABL1. In recent years, most research efforts in CML have been devoted to developing novel tyrosine kinase inhibitors (TKIs) as well as to elucidating the mechanisms of resistance to imatinib and other TKIs. Nonetheless, primordial aspects of the pathogenesis of CML, such as the mechanisms responsible for the transition from chronic phase to blast crisis, the causes of genomic instability and faulty DNA repair, the phenomenon of stem cell quiescence, the role of tumor suppressors in TKI resistance and CML progression, or the cross-talk between BCR-ABL1 and other oncogenic signaling pathways, still remain poorly understood. Herein, we synthesize the most relevant and current knowledge on such areas of the pathogenesis of CML.

    Funded by: NCI NIH HHS: P30 CA016672

    Blood 2009;113;8;1619-30

  • BCR/ABL induces chromosomal instability after genotoxic stress and alters the cell death threshold.

    Dierov J, Sanchez PV, Burke BA, Padilla-Nash H, Putt ME, Ried T and Carroll M

    Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

    Earlier reports have suggested that the BCR/ABL oncogene, associated with chronic myeloid leukemia, induces a mutator phenotype; however, it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However, although BCR/ABL-expressing cell lines have an equivalent cell survival, they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples, normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells, but not normal progenitor cells, consistent with error-prone DNA repair. Taken together, these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair.

    Funded by: Intramural NIH HHS; NCI NIH HHS: K01 CA129151, R01 CA100885, R01CA100885

    Leukemia 2009;23;2;279-86

  • Correlation of BCR/ABL transcript variants with patients' characteristics in childhood chronic myeloid leukaemia.

    Adler R, Viehmann S, Kuhlisch E, Martiniak Y, Röttgers S, Harbott J and Suttorp M

    Division of Paediatric Haematology and Oncology, University Hospital Carl Gustav Carus, Technical University at Dresden, Dresden, Germany.

    The characteristic chromosomal translocation t(9;22)(q34;q11) in chronic myeloid leukaemia (CML) mainly results in the two different BCR/ABL fusion transcripts b2a2 or b3a2. Both transcript variants can occur simultaneously due to alternative splicing of the b3a2 transcript. Conflicting results have been reported on the influence of the transcripts on haematological findings at diagnosis and the course of the disease in adults while data concerning these topics on childhood CML are still missing. This paper reports on a correlation of BCR/ABL transcript variants with patients' characteristics in childhood CML.

    Transcript types were determined in 146 paediatric patients with CML enrolled in trial CML-paed-I. Fifty-five patients (38%) expressed b2a2, 53 patients (36%) b3a2 and 38 patients (26%) both transcripts, respectively. These findings were correlated with patients' characteristics (sex, age, WBC, Hb, platelet count, hepatosplenomegaly, etc.) assessed at diagnosis.

    Results: While the co-expression of both transcripts was evenly distributed among genders [b2a2 + b3a2: 22 females (28%), 16 males (24%)] a highly significant difference (P = 0.007) was found concerning the expression of the b2a2 transcript [34 male (51%) vs. 21 female (27%)] and vice versa of the b3a2 transcript [17 male (25%) vs. 36 female (45%)]. High platelet counts and the combination of high platelet counts in conjunction with pronounced leukocytosis were observed more often in patients expressing the b3a2 transcript.

    Conclusions: These findings demonstrate that in children like in adults specific BCR/ABL transcript types present at diagnosis are associated with distinct haematological alterations (e.g. a high platelet count with the transcript b3a2). However, the sex-dependent skewed distribution of the BCR/ABL transcript types observed so far in this paediatric cohort only deserves further investigation.

    European journal of haematology 2009;82;2;112-8

  • BCR-ABL fusion transcript types and levels and their interaction with secondary genetic changes in determining the phenotype of Philadelphia chromosome-positive leukemias.

    Jones D, Luthra R, Cortes J, Thomas D, O'Brien S, Bueso-Ramos C, Hai S, Ravandi F, de Lima M, Kantarjian H and Jorgensen JL

    Departments of Hematopathology, University of Texas M. D. Anderson Cancer Center, Houston, USA.

    It remains unresolved how different BCR-ABL transcripts differentially drive lymphoid and myeloid proliferation in Philadelphia chromosome-positive (Ph(+)) leukemias. We compared BCR-ABL transcript type and level with kinase domain (KD) mutation status, genotype, and phenotype in 1855 Ph(+) leukemias. Compared with e1a2/p190 BCR-ABL cases, de novo e13-e14a2/p210 Ph(+) lymphoid leukemia more frequently showed CML-type background, had higher blast-normalized BCR-ABL transcript levels, and more frequent persistent BCR-ABL transcript in the absence of detectable lymphoblasts. Secondary lymphoid blast transformation of CML was exclusively due to e13/e14a2/p210 BCR-ABL but was associated, at a much higher level than p210 myeloid transformation, with acquisition of new KD mutations and/or Ph genomic amplification. In contrast, myeloid blast transformation was more frequently accompanied by new acquisition of acute myeloid leukemia-type chromosomal aberrations, particularly involving the EVI1 and RUNX1 loci. Therefore, higher kinase activity by mutation, transcriptional up-regulation or gene amplification appears required for lymphoid transformation by p210 BCR-ABL.

    Funded by: NCI NIH HHS: P30 CA016672, P50 CA100632

    Blood 2008;112;13;5190-2

  • Transient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis.

    Shah NP, Kasap C, Weier C, Balbas M, Nicoll JM, Bleickardt E, Nicaise C and Sawyers CL

    Division of Hematology/Oncology, Department of Medicine, UCSF School of Medicine, San Francisco, CA 94143, USA. nshah@medicine.ucsf.edu

    The BCR-ABL inhibitor dasatinib achieves clinical remissions in chronic myeloid leukemia (CML) patients using a dosing schedule that achieves potent but transient BCR-ABL inhibition. In vitro, transient potent BCR-ABL inhibition with either dasatinib or imatinib is cytotoxic to CML cell lines, as is transient potent EGFR inhibition with erlotinib in a lung cancer cell line. Cytotoxicity correlates with the magnitude as well as the duration of kinase inhibition. Moreover, cytotoxicity with transient potent target inhibition is equivalent to prolonged target inhibition and in both cases is associated with BIM activation and rescued by BCL-2 overexpression. In CML patients receiving dasatinib once daily, response correlates with the magnitude of BCR-ABL kinase inhibition, thereby demonstrating the potential clinical utility of intermittent potent kinase inhibitor therapy.

    Funded by: Howard Hughes Medical Institute

    Cancer cell 2008;14;6;485-93

  • Intrinsic differences between the catalytic properties of the oncogenic NUP214-ABL1 and BCR-ABL1 fusion protein kinases.

    De Keersmaecker K, Versele M, Cools J, Superti-Furga G and Hantschel O

    Department of Molecular and Developmental Genetics, VIB, Leuven, Belgium.

    The NUP214-ABL1 fusion kinase has recently been identified in 6% of patients with T-cell acute lymphoblastic leukemia. In contrast to the more common oncogenic ABL1 fusion BCR-ABL1, NUP214-ABL1 localizes to the nuclear pore complexes and has attenuated transforming properties in hematopoietic cells and in mouse bone marrow transplant models. We have performed a thorough biochemical comparative analysis of NUP214-ABL1 and BCR-ABL1 and show that, despite their common tyrosine kinase domain, the two fusion proteins differ in many critical catalytic properties. NUP214-ABL1 has lower in vitro tyrosine kinase activity, which is in agreement with the absence of phosphorylation on its activation loop. NUP214-ABL1 was more sensitive to imatinib (Glivec) than BCR-ABL1 in vitro and in cells, indicating a different activation state and conformation of the two ABL1 fusion kinases. Using a peptide array, we identified differences in the spectrum and efficiency of substrate peptide phosphorylation and a differential involvement of Src kinases in downstream signaling. These results clearly indicate that different fusion partners of the same kinase can determine not only localization, but also critical functional properties of the enzyme such as inhibitor sensitivity and substrate preference, with subsequent differences in downstream signaling effectors and likely consequences in disease pathogenesis.

    Leukemia 2008;22;12;2208-16

  • An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259.

    Pene-Dumitrescu T, Peterson LF, Donato NJ and Smithgall TE

    Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

    Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of Hck and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of Hck by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (Hck-T338M). This substitution reduced Hck sensitivity to A-419259 by more than 30-fold without significantly affecting kinase activity in vitro. Expression of Hck-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of Hck-T338M kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type Hck retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34(+) CML cells with equal potency to imatinib. These data suggest that Hck has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.

    Funded by: NCI NIH HHS: CA101828, R01 CA101828, R01 CA101828-04; NIGMS NIH HHS: GM077629, R01 GM077629, R01 GM077629-02

    Oncogene 2008;27;56;7055-69

  • Detection of BCR-ABL gene mutations in Philadelphia chromosome positive leukemia patients resistant to STI-571 cancer therapy.

    Chien JH, Tang JL, Chen RL, Li CC and Lee CP

    Institute of Medical Science, Tzu Chi University, Taiwan, ROC.

    The ABL-BCR fusion protein is a constitutively activated tyrosine kinase thought to play a central role in chronic myeloid leukemia (CML) and Philadelphia (Ph) chromosome acute lymphoid leukemia (ALL). Targeting the tyrosine kinase activity of ABL-BCR has been shown to be a promising therapeutic strategy in treating this disorder. Among the tyrosine kinase inhibitors, STI571 is a very effective therapeutic agent when administered to CML patients in the stable chronic phase. However, it has been reported that many CML patients with blast cell crisis treated with STI571 relapsed and became resistant to STI571. In order to understand the possible molecular mechanisms underlying STI571 resistance caused by ABL gene mutations, we investigated 19 patients (18 CML patients and 1 Ph (+) ALL patient) who either relapsed after initial response or had no response to STI571 treatment. We used polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis, dHPLC, and direct DNA sequencing to analyze any possible mutations in exons 5 to 9 of the ABL gene. Our results showed that 5 out of 19 patients had various mutations between exons 5 and 7 of the ABL gene. The Ph (+) ALL patient had a Glu255Lys mutation in exon 5 and a Thr315Ile mutation in exon 7. The Glu255Lys substitution has a G to A change, and the Thr315Ile substitution has a C to T change in the ABL gene. The other unique mutations found in this study include: Tyr253His, Met351Thr, GAA tri-nucleotides insertion, and Leu213Pro.

    Leukemia research 2008;32;11;1724-34

  • Analysis and comparison of clinicohematological parameters and molecular and cytogenetic response of two Bcr/Abl fusion transcripts.

    Polampalli S, Choughule A, Negi N, Shinde S, Baisane C, Amre P, Subramanian PG, Gujral S, Prabhash K and Parikh P

    Department of Medical Oncology, Molecular Biology Laboratory, Tata Memorial Hospital, Mumbai, India. drsunitap@yahoo.co.in

    Different forms of p210 are produced by alternative splicing, namely b2a2 and b3a2. There have been many contrasting data establishing a relationship between the two Bcr/Abl transcripts and platelet counts and also response to treatment. However, the data published to date have been on a small group of patients. The aim of the present study was to determine whether there was any difference between clinical and hematological parameters at diagnosis between the two Bcr/Abl fusion transcripts in our population, and whether the two transcripts responded differently or similarly to imatinib treatment. RT-PCR was performed in 202 cases for detection of Bcr/Abl transcripts in newly diagnosed chronic myelogenous leukemia cases in one year. The two transcripts were compared and correlated with clinical, hematological and FISH data and with response to treatment. A total of 138 cases were of b3a2 and 64 were of b2a2 transcript. There was no correlation between the hematological parameters and the type of transcript. There was a significant association of blast crisis with b2a2, especially with myeloid blast crisis. When compared to FISH results, 10% of b3a2 were found to have a significant association with 5'Abl deletion as compared to 3% of b2a2. On analyzing the therapeutic response, we did not find any difference between the two transcripts. In conclusion, our findings confirm that the b3a2 type transcript is not significantly associated with thrombocytosis, that the short transcript, b2a2, occurs with acute phase, i.e., blast crisis, and that there is no difference in treatment response between the two transcripts. However, further studies are required to understand the molecular pathways involved in the Bcr/Abl mechanism.

    Genetics and molecular research : GMR 2008;7;4;1138-49

  • Pathway-based evaluation of 380 candidate genes and lung cancer susceptibility suggests the importance of the cell cycle pathway.

    Hosgood HD, Menashe I, Shen M, Yeager M, Yuenger J, Rajaraman P, He X, Chatterjee N, Caporaso NE, Zhu Y, Chanock SJ, Zheng T and Lan Q

    Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. hosgoodd@mail.nih.gov

    Common genetic variation may play an important role in altering lung cancer risk. We conducted a pathway-based candidate gene evaluation to identify genetic variations that may be associated with lung cancer in a population-based case-control study in Xuan Wei, China (122 cases and 111 controls). A total of 1260 single-nucleotide polymorphisms (SNPs) in 380 candidate genes for lung cancer were successfully genotyped and assigned to one of 10 pathways based on gene ontology. Logistic regression was used to assess the marginal effect of each SNP on lung cancer susceptibility. The minP test was used to identify statistically significant associations at the gene level. Important pathways were identified using a test of proportions and the rank truncated product methods. The cell cycle pathway was found as the most important pathway (P = 0.044) with four genes significantly associated with lung cancer (PLA2G6 minP = 0.001, CCNA2 minP = 0.006, GSK3 beta minP = 0.007 and EGF minP = 0.013), after adjusting for multiple comparisons. Interestingly, most cell cycle genes that were associated with lung cancer in this analysis were concentrated in the AKT signaling pathway, which is essential for regulation of cell cycle progression and cellular survival, and may be important in lung cancer etiology in Xuan Wei. These results should be viewed as exploratory until they are replicated in a larger study.

    Funded by: Intramural NIH HHS; NCI NIH HHS: TU2 CA105666

    Carcinogenesis 2008;29;10;1938-43

  • ABL single nucleotide polymorphisms may masquerade as BCR-ABL mutations associated with resistance to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

    Ernst T, Hoffmann J, Erben P, Hanfstein B, Leitner A, Hehlmann R, Hochhaus A and Müller MC

    III. Medizinische Klinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Wiesbadener Str. 7-11, 68305 Mannheim, Germany. thomas.ernst@med3.ma.uni-heidelberg.de

    The BCR-ABL K247R change is based on a rare single nucleotide polymorphism occurring likewise in healthy controls and non-hematologic cell types. Despite its juxtaposition to the P-loop, functional analysis showed no alteration compared to non-mutated BCR-ABL. We sought to investigate if other changes in the BCR-ABL kinase domain should be considered as single nucleotide polymorphisms rather than acquired mutations. A total of 911 chronic myeloid leukemia patients after failure or suboptimal response to imatinib were screened for BCR-ABL kinase domain mutations. Single nucleotide polymorphism analysis was based on the search for nucleotide changes in corresponding normal, non-translocated ABL alleles by ABL allele-specific PCR following mutation analysis. In addition to the K247R polymorphism we uncovered five new single nucleotide polymorphisms within the BCR-ABL kinase domain; two of them led to amino acid changes. Single nucleotide polymorphisms could theoretically contribute to primary but not to secondary resistance to tyrosine kinase inhibitors and must therefore be distinguished from acquired mutations. Novel point mutations should be confirmed by analyzing the normal ABL alleles to exclude polymorphisms.

    Haematologica 2008;93;9;1389-93

  • Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells.

    Kharas MG, Janes MR, Scarfone VM, Lilly MB, Knight ZA, Shokat KM and Fruman DA

    Department of Molecular Biology and Biochemistry, Center for Immunology, University of California, Irvine, Irvine, California, USA.

    Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.

    Funded by: NCI NIH HHS: T32 CA009054, T32 CA9054; NIAID NIH HHS: AI44009, AI50831, R01 AI044009, R01 AI050831

    The Journal of clinical investigation 2008;118;9;3038-50

  • Analysis of gene rearrangements using a fluorescence in situ hybridization method in Mexican patients with acute lymphoblastic leukemia: experience at a single institution.

    Pérez-Vera P, Salas C, Montero-Ruiz O, Frías S, Dehesa G, Jarquín B and Rivera-Luna R

    Department of Research in Human Genetics, Instituto Nacional de Pediatría, Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, Mexico City DF 04530, Mexico. pperezvera@yahoo.com

    We evaluated the prevalence of BCR/ABL, MLL, and ETV6/RUNX1 rearrangements as well as CDKN2A (alias p16) deletion in a group of Mexican children with acute lymphoblastic leukemia (ALL) to determine whether the changes coexist, and to compare the incidences found with other reports in the literature. To increase the detection of these abnormalities, we combined conventional cytogenetics and fluorescence in situ hybridization (FISH) analysis. Bone marrow samples were obtained from 59 consecutive children with ALL. FISH detected a total of 63 abnormalities with the selected probes, 34 of which were related to the conventional cytogenetic results. The most common abnormality was the p16 deletion (22.8%), followed by MLL and ETV6/RUNX1 rearrangements (8.7%), and the BCR/ABL fusion was the least frequent (2.7%). The coexistence of two recurrent abnormalities with specific prognostic significance in the same patient was not found. A lesser proportion of the p16 deletion in T-ALL patients was observed, probably related to the low prevalence of this subtype in our population. In addition, we confirmed the low frequency of the ETV6/RUNX1 fusion observed in Hispanics. Due to the different prevalence of these abnormalities in the Mexican population, similar studies should be conducted analyzing new rearrangements, to improve the adequate classification of the abnormalities and the stratification in prognostic groups.

    Cancer genetics and cytogenetics 2008;184;2;94-8

  • Bcr-Abl induces autocrine IGF-1 signaling.

    Lakshmikuttyamma A, Pastural E, Takahashi N, Sawada K, Sheridan DP, DeCoteau JF and Geyer CR

    Genomic Medicine and Pathobiology Research Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

    Bcr-Abl oncogene is responsible for the initial phase of chronic myelogenous leukemia (CML), which is effectively treated by the Bcr-Abl inhibitor imatinib. Over time patients become resistant to treatment and progress to blast crisis, an event that is driven by additional genetic and epigenetic aberrations. Recently, we showed that Riz1 expression decreases in blast crisis and that re-expression of Riz1 inhibits IGF-1 expression. IGF-1 signaling is required in many stages of hematopoiesis and inappropriate activation of autocrine IGF-1 signaling may facilitate transformation to blast crisis. We observed that in 8 out of 11 matched CML patient biopsies the IGF-1 expression is elevated in blast crisis. We examined mechanisms used by CML blast crisis cell lines to activate IGF-1 expression. We found that Bcr-Abl activates autocrine IGF-1 signaling using Hck and Stat5b. Inhibition of these signaling components using small molecule drugs or shRNA decreases proliferation and enhances apoptosis. Together, our study suggests that aberrant IGF-1 signaling is an important event in blast crisis transformation and it provides a mechanism to explain the activity of IGF-1R and Hck inhibitors in blocking CML blast crisis phenotypes.

    Oncogene 2008;27;27;3831-44

  • A BCR-JAK2 fusion gene as the result of a t(9;22)(p24;q11) in a patient with acute myeloid leukemia.

    Cirmena G, Aliano S, Fugazza G, Bruzzone R, Garuti A, Bocciardi R, Bacigalupo A, Ravazzolo R, Ballestrero A and Sessarego M

    Department of Hematology, University of Genoa, V.le Benedetto XV-6, 16132 Genoa, Italy.

    We report the occurrence of a BCR-JAK2 fusion gene in a case of acute myeloid leukemia (AML) resulting from a t(9;22)(p24;q11) translocation as the sole cytogenetic abnormality. The BCR-JAK2 fusion gene has the same breakpoint in BCR as is found in the BCR/ABL p210. The chimeric gene is the result of a reciprocal translocation between chromosomes 9 and 22, which implies a double break on chromosome 9; this has allowed generating an in-frame fusion transcript. Previously, BCR-JAK2 rearrangement was observed in a single case with atypical chronic myelogenous leukemia (CML), but in that case the breakpoint in the BCR was different.

    Cancer genetics and cytogenetics 2008;183;2;105-8

  • Characterization of BCR-ABL deletion mutants from patients with chronic myeloid leukemia.

    Sherbenou DW, Hantschel O, Turaga L, Kaupe I, Willis S, Bumm T, Press RD, Superti-Furga G, Druker BJ and Deininger MW

    Cell and Developmental Biology, Oregon Health & Science University, Portland, OR, USA.

    The BCR-ABL oncogenic tyrosine kinase causes chronic myeloid leukemia and is the target for imatinib therapy. During imatinib treatment, cells are selected in some patients with BCR-ABL kinase domain mutations that render decreased drug sensitivity. In addition, some patients express deletion mutants of BCR-ABL, apparently due to missplicing. Most commonly these deletion mutants lack a significant portion of the kinase domain that includes the P-loop. We describe a screen for such mutations in patients with CML and demonstrate that they are not oncogenic and are catalytically inactive. We hypothesized that coexpressing BCR-ABL deletion mutants has a dominant-negative effect on the native form through heterocomplex formation. However, upon coexpression of native and deletion mutant BCR-ABL in Ba/F3 cells, growth factor independence is maintained and signaling is activated normally. Despite this, these cells have increased imatinib sensitivity compared to cells expressing only native BCR-ABL. Thus, it will be important to investigate the prognostic impact of coexpression of deletion mutants in CML patients during imatinib treatment.

    Funded by: NHLBI NIH HHS: HL082978-01

    Leukemia 2008;22;6;1184-90

  • Effective killing of Gleevec-resistant CML cells with T315I mutation by a natural compound PEITC through redox-mediated mechanism.

    Zhang H, Trachootham D, Lu W, Carew J, Giles FJ, Keating MJ, Arlinghaus RB and Huang P

    Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

    Mutation of Bcr-Abl is an important mechanism by which chronic myelogenous leukemia (CML) cells become resistant to Gleevec. The T315I mutation is clinically significant since CML cells harboring this mutation are insensitive to Gleevec and other Bcr-Abl-targeted drugs. Identification of new agents capable of effectively killing CML cells with T315I mutation would have important therapeutic implications in Gleevec-resistant CML. Here, we showed that beta-phenylethyl isothiocyanate (PEITC), a natural compound found in vegetables, is effective in killing CML cells expressing T315I BCR-ABL. Treatment of leukemia cell lines harboring wild-type or mutant Bcr-Abl with 10 microM PEITC resulted in an elevated ROS stress and a redox-mediated degradation of the BCR-ABL protein, leading to massive death of the leukemia cells. Antioxidant NAC attenuated the PEITC-induced oxidative stress in CML cells and prevented the degradation of BCR-ABL, caspase-3 activation and cell death. We further showed that the ROS-induced degradation of BCR-ABL was mediated partially by caspase-3 and the proteasome pathway. The ability of PEITC to effectively kill T315I-positive CML cells was further confirmed using primary leukemia cells isolated from CML patients. Our results suggest that PEITC is a promising compound capable of killing Gleevec-resistant CML cells through a ROS-mediated mechanism and warrants further investigations.

    Funded by: NCI NIH HHS: CA085563, CA100428, CA109041, CA16672, P30 CA016672, P30 CA016672-24S39021, R01 CA085563, R01 CA100428, R01 CA109041

    Leukemia 2008;22;6;1191-9

  • Genetic and epigenetic silencing of microRNA-203 enhances ABL1 and BCR-ABL1 oncogene expression.

    Bueno MJ, Pérez de Castro I, Gómez de Cedrón M, Santos J, Calin GA, Cigudosa JC, Croce CM, Fernández-Piqueras J and Malumbres M

    Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), E-28029 Madrid, Spain.

    The mammalian genome contains several hundred microRNAs that regulate gene expression through modulation of target mRNAs. Here, we report a fragile chromosomal region lost in specific hematopoietic malignancies. This 7 Mb region encodes about 12% of all genomic microRNAs, including miR-203. This microRNA is additionally hypermethylated in several hematopoietic tumors, including chronic myelogenous leukemias and some acute lymphoblastic leukemias. A putative miR-203 target, ABL1, is specifically activated in these hematopoietic malignancies in some cases as a BCR-ABL1 fusion protein (Philadelphia chromosome). Re-expression of miR-203 reduces ABL1 and BCR-ABL1 fusion protein levels and inhibits tumor cell proliferation in an ABL1-dependent manner. Thus, miR-203 functions as a tumor suppressor, and re-expression of this microRNA might have therapeutic benefits in specific hematopoietic malignancies.

    Funded by: Worldwide Cancer Research: 08-0188

    Cancer cell 2008;13;6;496-506

  • [Polymorphisms in the breakpoint cluster region of bcr gene].

    Tian H and Zhou SY

    Department of Hematology, Fuzhou General Hospital, Nanjing Military Area, Fuzhou 350025, Fujian Province, China.

    This study was aimed to explore the single nucleotide polymorphism (SNPs) of breakpoint cluster region of bcr gene in Chinese people and the relationship between SNPs and chronic myelogenous leukemia (CML). A 3.12 kb region spanning from exon 13 to exon 15 in the bcr region were screened by DNA pooling and denaturing high performance liquid chromatography (dHPLC), and the results were verified by sequencing. The results indicated that 6 novel SNP sites and 2 bases different from reference sequence were confirmed in the region studied, and the frequency of 6 novel SNP sites in studied population was obtained, one SNP of which was found in exon 13 and caused a nonsynonymous mutation. The gene frequencies of novel SNPs had no significant difference between CML and control people. It is concluded that sequence polymorphisms in the major breakpoint cluster region of bcr gene are found, most of which are SNPs, No relationship can be confirmed between SNPs and CML disease.

    Zhongguo shi yan xue ye xue za zhi 2008;16;3;659-62

  • Requirement of c-Myb for p210(BCR/ABL)-dependent transformation of hematopoietic progenitors and leukemogenesis.

    Lidonnici MR, Corradini F, Waldron T, Bender TP and Calabretta B

    Department of Cancer Biology, and Kimmel Cancer Center, Thomas Jefferson University Medical College, Philadelphia, PA 19107, USA.

    The c-Myb gene encodes a transcription factor required for proliferation and survival of normal myeloid progenitors and leukemic blast cells. Targeting of c-Myb by antisense oligodeoxynucleotides has suggested that myeloid leukemia blasts (including chronic myelogenous leukemia [CML]-blast crisis cells) rely on c-Myb expression more than normal progenitors, but a genetic approach to assess the requirement of c-Myb by p210(BCR/ABL)-transformed hematopoietic progenitors has not been taken. We show here that loss of a c-Myb allele had modest effects (20%-28% decrease) on colony formation of nontransduced progenitors, while the effect on p210(BCR/ABL)-expressing Lin(-) Sca-1(+) and Lin(-) Sca-1(+)Kit(+) cells was more pronounced (50%-80% decrease). Using a model of CML-blast crisis, mice (n = 14) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/w) marrow cells developed leukemia rapidly and had a median survival of 26 days, while only 67% of mice (n = 12) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/d) marrow cells died of leukemia with a median survival of 96 days. p210(BCR/ABL)-transduced c-Myb(w/w) and c-Myb(w/d) marrow progenitors expressed similar levels of the c-Myb-regulated genes c-Myc and cyclin B1, while those of Bcl-2 were reduced. However, ectopic Bcl-2 expression did not enhance colony formation of p210(BCR/ABL)-transduced c-Myb(w/d) Lin(-)Sca-1(+)Kit(+) cells. Together, these studies support the requirement of c-Myb for p210(BCR/ABL)-dependent leukemogenesis.

    Funded by: NCI NIH HHS: CA95111, R01 CA085842, R01 CA095111, T32 CA009683, T32-CA09683-14; NIAID NIH HHS: R01 AI059294

    Blood 2008;111;9;4771-9

  • BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region.

    Wozniak K, Piaskowski S, Gresner SM, Golanska E, Bieniek E, Bigoszewska K, Sikorska B, Szybka M, Kulczycka-Wojdala D, Zakrzewska M, Zawlik I, Papierz W, Stawski R, Jaskolski DJ, Och W, Sieruta M, Liberski PP and Rieske P

    Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Czechoslowacka 8/10, 92-216 Lodz, Poland.

    Neurofibromin 2 (NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), alpha-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH (P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.

    Cancer genetics and cytogenetics 2008;183;1;14-20

  • Many sequence variants affecting diversity of adult human height.

    Gudbjartsson DF, Walters GB, Thorleifsson G, Stefansson H, Halldorsson BV, Zusmanovich P, Sulem P, Thorlacius S, Gylfason A, Steinberg S, Helgadottir A, Ingason A, Steinthorsdottir V, Olafsdottir EJ, Olafsdottir GH, Jonsson T, Borch-Johnsen K, Hansen T, Andersen G, Jorgensen T, Pedersen O, Aben KK, Witjes JA, Swinkels DW, den Heijer M, Franke B, Verbeek AL, Becker DM, Yanek LR, Becker LC, Tryggvadottir L, Rafnar T, Gulcher J, Kiemeney LA, Kong A, Thorsteinsdottir U and Stefansson K

    deCODE Genetics, 101 Reykjavik, Iceland. daniel.gudbjartsson@decode.is

    Adult human height is one of the classical complex human traits. We searched for sequence variants that affect height by scanning the genomes of 25,174 Icelanders, 2,876 Dutch, 1,770 European Americans and 1,148 African Americans. We then combined these results with previously published results from the Diabetes Genetics Initiative on 3,024 Scandinavians and tested a selected subset of SNPs in 5,517 Danes. We identified 27 regions of the genome with one or more sequence variants showing significant association with height. The estimated effects per allele of these variants ranged between 0.3 and 0.6 cm and, taken together, they explain around 3.7% of the population variation in height. The genes neighboring the identified loci cluster in biological processes related to skeletal development and mitosis. Association to three previously reported loci are replicated in our analyses, and the strongest association was with SNPs in the ZBTB38 gene.

    Nature genetics 2008;40;5;609-15

  • Suppression of interferon (IFN)-inducible genes and IFN-mediated functional responses in BCR-ABL-expressing cells.

    Katsoulidis E, Sassano A, Majchrzak-Kita B, Carayol N, Yoon P, Jordan A, Druker BJ, Fish EN and Platanias LC

    Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Northwestern University Medical School and Lakeside Veterans Affairs Medical Center, Chicago, Illinois 60611, USA.

    The interferons (IFNs) are cytokines that play key roles in host defense against viral infections and immune surveillance against cancer. We report that BCR-ABL transformation of hematopoietic cells results in suppression of IFN-dependent responses, including transcription of IFN-inducible genes and generation of IFN-mediated antiviral effects. BCR-ABL transformation suppresses expression of several IFN-regulated genes containing IFN-sensitive response element (ISRE) or GAS elements in their promoters, including Isg15, Irf1, Irf9, and Ifit2 (interferon-induced protein with tetratricopeptide repeats 2). Suppression of transcription of ISRE-containing genes is also seen in cells expressing various BCR-ABL kinase domain mutants, including T315I, H396P, Y253F, and E255K, but not kinase-defective BCR-ABL. Such effects are associated with impaired IFN-dependent phosphorylation of Stat1 on Tyr(701) and Stat3 on Tyr(705) and defective binding of Stat complexes to ISRE or GAS elements. Beyond suppression of Stat activities, BCR-ABL inhibits IFN-inducible phosphorylation/activation of the p38 MAPK, suggesting a dual mechanism by which this abnormal fusion protein blocks IFN transcriptional responses. The inhibitory activities of BCR-ABL ultimately result in impaired IFNalpha-mediated protection against encephalomyocarditis virus infection and reversal of IFN-dependent growth suppression. Altogether, our data provide evidence for a novel mechanism by which BCR-ABL impairs host defenses and promotes malignant transformation, involving dual suppression of IFN-activated signaling pathways.

    Funded by: NCI NIH HHS: CA100579, CA121192, CA77816

    The Journal of biological chemistry 2008;283;16;10793-803

  • Deletions adjacent to BCR and ABL1 breakpoints occur in a substantial minority of chronic myeloid leukemia patients with masked Philadelphia rearrangements.

    De Melo VA, Milojkovic D, Marin D, Apperley JF, Nacheva EP and Reid AG

    Department of Haematology, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

    Deletions at the t(9;22) breakpoint regions, found in 15% of chronic myeloid leukemia patients (CML) with an overt Philadelphia (Ph) translocation, are associated with an adverse disease prognosis in patients receiving interferon-alpha therapy. The incidence of deletions has been shown to vary for different cytogenetic subgroups of CML, with a significantly higher incidence of deletion in patients with a variant Ph translocation. To date, however, the frequency of such deletions in the subgroup of CML patients in whom the BCR/ABL1 fusion arises via submicroscopic chromosomal insertion (masked Ph) has not been investigated. We report the evaluation of 14 patients with masked Ph-positive CML for the presence of deletions extending 3' from BCR and 5' from ABL1 using two triple-color BCR/ABL probes. Deletions were identified in 3 patients (21%), encompassing sequences 5' to ABL1 in two of these and sequences 3' to BCR in the remaining patient, thus demonstrating that the phenomenon is a significant feature of the masked Ph CML subgroup. Furthermore, our findings are consistent with the notion that loss of genomic material is a potential side effect of any DNA breakage event at the 9q34.1 and 22q11.2 chromosomal regions, regardless of the subsequent mechanism of chromosomal rearrangement.

    Cancer genetics and cytogenetics 2008;182;2;111-5

  • Mechanisms of genesis of variant translocation in chronic myeloid leukemia are not correlated with ABL1 or BCR deletion status or response to imatinib therapy.

    Richebourg S, Eclache V, Perot C, Portnoi MF, Van den Akker J, Terré C, Maareck O, Soenen V, Viguié F, Laï JL, Andrieux J, Corm S, Roche-Lestienne C and Fi-LMC Group

    Laboratory of Medical Genetics, Hospital Jeanne de Flandre, University Hospital Regional Center-CHRU, Avenue Eugène Avinée, 59037 Lille cedex, France.

    Many published studies have indicated that various mechanisms could be involved in the genesis of variant chronic myelogeneous leukemia (CML) translocations. These are mainly one-step or two-step mechanisms, associated or not with deletions adjacent to the translocation junction on der(9) or der(22) chromosomes (or both). Based on the mechanism of genesis, it has been suggested that the complexity may affect the occurrence of ABL1 and BCR deletions (either or both), or may be associated with the CML disease course, and thus could determine the response to imatinib therapy. Through a retrospective molecular cytogenetic study of 41 CML patients with variant Philadelphia chromosome (Ph), we explored the genesis of these variant rearrangements and analyzed the correlation with deletion status and imatinib efficiency. Our results confirmed that the one-step mechanism is the most frequent, evidenced in 30 of 41 patients (73%); 3 patients demonstrated other more complex multistep events and 8 patients (19.5%) harbored ABL1 or BCR deletions that are not significantly associated with the complexity of translocation genesis. We also found no association between one-step, two-step, or multistep mechanisms and the response to imatinib therapy.

    Cancer genetics and cytogenetics 2008;182;2;95-102

  • Kinase domain mutants of Bcr enhance Bcr-Abl oncogenic effects.

    Perazzona B, Lin H, Sun T, Wang Y and Arlinghaus R

    Department of Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

    Bcr-Abl acquires its transforming ability through its upregulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. In chronic myelogenous leukemia (CML) cells, Bcr-Abl phosphorylates Bcr on tyrosine residues reducing its kinase activity. Overexpression of BCR in BCR-ABL+ cells produces a phosphoserine form of Bcr, which inhibits the oncogenic effects of BCR-ABL. To investigate the inhibitory effects of Bcr on Bcr-Abl, we expressed BCR/GFP in TonB210 cells, which contain a tetracycline-inducible BCR-ABL. In nude mice injected with cell clones of TonB210/BCR/GFP, tumor formation was delayed, and tumors were 50% smaller compared with the TonB210/GFP. In addition, TonB210/ BCR/GFP cells had little colony-forming ability in soft agar compared with TonB210/GFP cells. In contrast, a point mutant of BCR (Y360F), which disrupts its kinase activity, not only blocked Bcr's inhibitory effects but also enhanced the oncogenic effects of Bcr-Abl in a solid tumor model and in soft agar colony assays. Similar effects were observed with a second BCR kinase domain mutant, S354A. These results indicate that the inhibitory function of Bcr directed toward Bcr-Abl requires its kinase function.

    Funded by: NCI NIH HHS: CA049639-150024, CA49639, P01 CA049639, P01 CA049639-150024

    Oncogene 2008;27;15;2208-14

  • A multiplex PCR for improved detection of typical and atypical BCR-ABL fusion transcripts.

    Burmeister T and Reinhardt R

    Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, Medizinische Klinik III, Hindenburgdamm 30, 12200 Berlin, Germany. thomas.burmeister@charite.de

    RT-PCR is the method of choice for detecting BCR-ABL in CML and ALL. The three predominant mRNA transcripts found are e1a2 (in ALL), e13a2, and e14a2 (in CML and ALL). However, a number of "atypical"BCR-ABL transcripts (e1a3, e13a3, e14a3, e19a2, e6a2, e8a2, etc.) resulting from chromosomal breakpoints outside ABL intron 1 or BCR intron 1, 13 or 14, respectively, have been reported. These atypical transcripts may escape detection when using methods that are optimized to detect just the typical ones. We present here a novel, fast, and reliable multiplex PCR for improved detection of typical and atypical BCR-ABL transcripts.

    Leukemia research 2008;32;4;579-85

  • Expression of BCR-ABL1 oncogene relative to ABL1 gene changes overtime in chronic myeloid leukemia.

    Gupta M, Milani L, Hermansson M, Simonsson B, Markevärn B, Syvänen AC and Barbany G

    Molecular Medicine, Department of Medical Sciences, Uppsala University, Sweden.

    Using a quantitative single nucleotide polymorphism (SNP) assay we have investigated the changes in the expression of the BCR-ABL1 oncogene relative to the wild-type ABL1 and BCR alleles in cells from chronic myeloid leukemia (CML) patients not responding to therapy. The results show a progressive increase in the BCR-ABL1 oncogene expression at the expense of decreased expression of the ABL1 allele, not involved in the fusion. No relative changes in the expression of the two BCR alleles were found. These results demonstrate that allele-specific changes in gene expression, with selective, progressive silencing of the wild-type ABL1 allele in favor of the oncogenic BCR-ABL1 allele occur in CML patients with therapy-resistant disease.

    Biochemical and biophysical research communications 2008;366;3;848-51

  • Activity of the Bcr GTPase-activating domain is regulated through direct protein/protein interaction with the Rho guanine nucleotide dissociation inhibitor.

    Kweon SM, Cho YJ, Minoo P, Groffen J and Heisterkamp N

    Section of Molecular Carcinogenesis, Division of Hematology/Oncology, The Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, California 90027, USA.

    The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Little is known about how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert Rac-GTP to Rac-GDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to Rac-GTP and catalyzes conversion of the bound GTP to GDP on Rac. The presence of activated Rac diminished the Bcr/RhoGDIalpha interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of Rac-GTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, indicating that binding of RhoGDIalpha and Rac-GTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates BcrGAP activity.

    Funded by: NHLBI NIH HHS: HL060231, HL71945

    The Journal of biological chemistry 2008;283;6;3023-30

  • A possible association between missense polymorphism of the breakpoint cluster region gene and lithium prophylaxis in bipolar disorder.

    Masui T, Hashimoto R, Kusumi I, Suzuki K, Tanaka T, Nakagawa S, Suzuki T, Iwata N, Ozaki N, Kato T, Takeda M, Kunugi H and Koyama T

    Department of Psychiatry, Hokkaido University Graduate School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo, 060-8638, Hokkaido, Japan.

    Lithium is one of the most commonly used drugs for the treatment of bipolar disorder. To prescribe lithium appropriately to patients, predictors of response to this drug were explored, and several genetic markers are considered to be good candidates. We previously reported a significant association between genetic variations in the breakpoint cluster region (BCR) gene and bipolar disorder. In this study, we examined a possible relationship between response to maintenance treatment of lithium and Asn796Ser single-nucleotide polymorphism in the BCR gene. Genotyping was performed in 161 bipolar patients who had been taking lithium for at least 1 year, and they were classified into responders for lithium mono-therapy and non-responders. We found that the allele frequency of Ser796 was significantly higher in non-responders than in responders. Further investigation is warranted to confirm our findings.

    Progress in neuro-psychopharmacology & biological psychiatry 2008;32;1;204-8

  • Ph+/VE-cadherin+ identifies a stem cell like population of acute lymphoblastic leukemia sustained by bone marrow niche cells.

    Wang L, O'Leary H, Fortney J and Gibson LF

    Department of Pediatrics, and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, WV 26506, USA.

    Although leukemic stem cells (LSCs) show a symbiotic relationship with bone marrow microenvironmental niches, the mechanism by which the marrow microenvironment contributes to self-renewal and proliferation of LSCs remains elusive. In the present study, we identified a unique subpopulation of Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) cells coexpressing markers of endothelial cells (including VE-cadherin, PECAM-1, and Flk-1) and committed B-lineage progenitors. After long-term coculture with bone marrow stromal cells, tumor cells formed hematopoietic colonies and cords, expressed early stem- cell markers, and showed endothelial sprouting. Gene expression profiles of LSCs were altered in the presence of stromal cell contact. Stromal cell contact promoted leukemic cell VE-cadherin expression, stabilized beta-catenin, and up-regulated Bcr-abl fusion gene expression. Our study indicates that these specific tumor cells are uniquely positioned to respond to microenvironment-derived self-renewing and proliferative cues. Ph(+)/VE-cadherin(+) tumor subpopulation circumvents the requirement of exogenous Wnt signaling for self-renewal through stromal cell support of leukemic cell VE-cadherin expression and up-regulated Bcr-abl tyrosine kinase activity. These data suggest that strategies targeting signals in the marrow microenvironment that amplify the Bcr-abl/VE-cadherin/beta-catenin axis may have utility in sensitizing drug-resistant leukemic stem cells.

    Funded by: NCI NIH HHS: R01 CA134573; NCRR NIH HHS: P20 RR016440, RR 16440; NHLBI NIH HHS: R01 HL 056888, R01 HL056888

    Blood 2007;110;9;3334-44

  • Immunohistochemical study of N-epsilon-carboxymethyl lysine (CML) in human brain: relation to vascular dementia.

    Southern L, Williams J and Esiri MM

    Department of Clinical Neurology, University of Oxford, West Wing, John Radcliffe Hospital, Oxford UK. louise.southern@gwmail.jr2.ox.ac.uk

    Background: Advanced glycation end-products (AGEs) and their receptor (RAGE) occur in dementia of the Alzheimer's type and diabetic microvascular disease. Accumulation of AGEs relates to risk factors for vascular dementia with ageing, including hypertension and diabetes. Cognitive dysfunction in vascular dementia may relate to microvascular disease resembling that in diabetes. We tested if, among people with cerebrovascular disease, (1) those with dementia have higher levels of neuronal and vascular AGEs and (2) if cognitive dysfunction depends on neuronal and/or vascular AGE levels.

    Methods: Brain Sections from 25 cases of the OPTIMA (Oxford Project to Investigate Memory and Ageing) cohort, with varying degrees of cerebrovascular pathology and cognitive dysfunction (but only minimal Alzheimer type pathology) were immunostained for Nepsilon-(carboxymethyl)-lysine (CML), the most abundant AGE. The level of staining in vessels and neurons in the cortex, white matter and basal ganglia was compared to neuropsychological and other clinical measures.

    Results: The probability of cortical neurons staining positive for CML was higher in cases with worse cognition (p = 0.01) or a history of hypertension (p = 0.028). Additionally, vascular CML staining related to cognitive impairment (p = 0.02) and a history of diabetes (p = 0.007). Neuronal CML staining in the basal ganglia related to a history of hypertension (p = 0.002).

    Conclusion: CML staining in cortical neurons and cerebral vessels is related to the severity of cognitive impairment in people with cerebrovascular disease and only minimal Alzheimer pathology. These findings support the possibility that cerebral accumulation of AGEs may contribute to dementia in people with cerebrovascular disease.

    BMC neurology 2007;7;35

  • The immunogenicity of Bcr-Abl expressing dendritic cells is dependent on the Bcr-Abl kinase activity and dominated by Bcr-Abl regulated antigens.

    Scheich F, Duyster J, Peschel C and Bernhard H

    Department of Hematology/Oncology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.

    In Ph(+) chronic myeloid leukemia (CML), the constitutively active Bcr-Abl kinase leads to the up-regulation and activation of multiple genes, which may subsequently result in the expression of leukemia-associated antigens. In this study, we investigated the immunogenicity of Bcr-Abl-regulated antigens by stimulating CD8(+) T lymphocytes with autologous dendritic cells transfected with RNA coding for Bcr-Abl wild-type or a kinase-deficient mutant. Significant HLA class I-restricted T-cell responses were detected against antigens regulated by the Bcr-Abl kinase, but not toward the Bcr-Abl protein itself. The T-cell repertoire of a patient with CML in major molecular remission due to imatinib mesylate was also dominated by T cells directed against Bcr-Abl-regulated antigens. These results encourage the development of immunotherapeutic approaches against Bcr-Abl-regulated antigens for the treatment of CML patients with residual disease following therapy with Bcr-Abl kinase inhibitors.

    Blood 2007;110;7;2556-60

  • Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells.

    Giehl M, Fabarius A, Frank O, Erben P, Zheng C, Hafner M, Hochhaus A, Hehlmann R and Seifarth W

    III. Medizinische Universitätsklinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany.

    Centrosomes play fundamental roles in mitotic spindle organization, chromosome segregation and maintenance of genetic stability. Recently, we have shown that centrosome aberrations occur early in chronic myeloid leukemia (CML) and are induced by imatinib in normal fibroblasts in vitro. To investigate the influence of BCR-ABL on centrosomes, we performed long-term in vitro experiments employing the conditionally p210BCR-ABL-expressing (tetracycline-inducible promoter) human monocytic cell line U937p210BCR-ABL/c6 as a model of CML chronic phase. Centrosome hypertrophy was detectable after 4 weeks of transgene expression onset, increasing up to a rate of 25.7% aberrant cells within 13 weeks of propagation. This concurred with clonal expansion of aneuploid cells displaying a hyperdiploid phenotype with 57 chromosomes. Partial reversibility of centrosome aberrations (26-8%) was achieved under prolonged propagation (14 weeks) after abortion of induction and bcr-abl silencing using small interfering RNA. Therapeutic doses of imatinib did not revert the aberrant phenotype, but counteracted the observed reverting effect of bcr-abl gene expression switch off. Suggesting a mechanistic model that features distinct abl-related tyrosine kinase activity levels as essential determinants of centrosomal integrity, this is the first report mechanistically linking p210BCR-ABL oncoprotein activity to centrosomal hypertrophy.

    Leukemia 2007;21;9;1971-6

  • Identification of a novel e8/a4 BCR/ABL fusion transcript in a case of a transformed Sézary syndrome.

    Callet-Bauchu E, Salles G, Gazzo S, Dalle S, Berger F and Hayette S

    This report deals with a case of Sézary syndrome, a rare peripheral T-cell lymphoproliferative disorder, in which cytogenetic analysis performed during the disease transformation revealed the presence of a t(9;22) (q34;q11.2) translocation. Molecular analyses identified a new transcript, an e8a4 BCR-ABL fusion mRNA which could be responsible for the disease transformation.

    Haematologica 2007;92;9;1277-8

  • Sequential ABL kinase inhibitor therapy selects for compound drug-resistant BCR-ABL mutations with altered oncogenic potency.

    Shah NP, Skaggs BJ, Branford S, Hughes TP, Nicoll JM, Paquette RL and Sawyers CL

    Division of Hematology/Oncology, Department of Medicine, UCSF School of Medicine, San Francisco, California, USA.

    Molecularly targeted kinase inhibitor cancer therapies are currently administered sequentially rather than simultaneously. We addressed the potential long-term impact of this strategy in patients with chronic myelogenous leukemia (CML), which is driven by the fusion oncogene BCR-ABL. Analysis of BCR-ABL genotypes in CML patients who relapsed after sequential treatment with the ABL inhibitors imatinib and dasatinib revealed evolving resistant BCR-ABL kinase domain mutations in all cases. Twelve patients relapsed with the pan-resistant T315I mutation, whereas 6 patients developed novel BCR-ABL mutations predicted to retain sensitivity to imatinib based on in vitro studies. Three of these patients were retreated with imatinib (or the chemically related compound nilotinib) and responded; however, selection for compound mutants (2 or 3 BCR-ABL mutations in the same molecule) can substantially limit the potential effectiveness of retreating patients with inhibitors that have previously failed. Furthermore, drug-resistant mutations, when compounded, can increase oncogenic potency relative to the component mutants in transformation assays. The Aurora kinase inhibitor VX-680, currently under clinical evaluation based on its activity against the T315I mutation, is also effective against the other commonly detected dasatinib-resistant mutation in our analysis, V299L. Our findings demonstrate the potential hazards of sequential kinase inhibitor therapy and suggest a role for a combination of ABL kinase inhibitors, perhaps including VX-680, to prevent the outgrowth of cells harboring drug-resistant BCR-ABL mutations.

    The Journal of clinical investigation 2007;117;9;2562-9

  • Frequency of BCR-ABL fusion oncogene in Pakistani childhood acute lymphoid leukemia (ALL) patients reflects ethnic differences in molecular genetics of ALL.

    Iqbal Z, Iqbal M and Akhter T

    Journal of pediatric hematology/oncology 2007;29;8;585

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • BCR-tyrosine 177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in chronic myelogenous leukemia.

    Chu S, Li L, Singh H and Bhatia R

    Department of Hematopoietic Stem Cell and Leukemia Research, Division of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010, USA.

    Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the BCR/ABL gene. BCR/ABL signaling has been studied in cell lines and murine models, but the transforming effects of BCR/ABL are highly dependent on cellular context, and mechanisms responsible for the transformation of primitive human hematopoietic cells remain poorly understood. Current targeted therapies fail to eliminate malignant CML progenitors, and improved understanding of crucial molecular mechanisms of progenitor transformation may facilitate the development of improved therapeutic approaches. We investigated the role of BCR/ABL tyrosine 177 (BCR/ABL-Y177) in CML progenitor transformation by comparing the effects of expression of Y177-mutated BCR/ABL, wild-type BCR/ABL, or green fluorescent protein alone on normal CD34(+) cells. We show that BCR/ABL-Y177 plays a critical role in CML progenitor expansion, proliferation, and survival. BCR/ABL expression results in enhanced Ras and Akt activity but reduced mitogen-activated protein kinase activity in human hematopoietic cells, which is reversed by BCR/ABL-Y177 mutation. Blocking BCR/ABL-Y177-mediated signaling enhances targeting of CML progenitors by imatinib mesylate. Our studies indicate that BCR/ABL-Y177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in CML.

    Funded by: NCI NIH HHS: R01 CA 95684; NCRR NIH HHS: 5M01 RR 00043; NHLBI NIH HHS: R01 HL 77847

    Cancer research 2007;67;14;7045-53

  • Ubp43 regulates BCR-ABL leukemogenesis via the type 1 interferon receptor signaling.

    Yan M, Luo JK, Ritchie KJ, Sakai I, Takeuchi K, Ren R and Zhang DE

    Division of Oncovirology, Department of Molecular, The Scripps Research Institute, La Jolla, CA 92037, USA.

    Interferon (IFN) signaling induces the expression of interferon-responsive genes and leads to the activation of pathways that are involved in the innate immune response. Ubp43 is an ISG15-specific isopeptidase, the expression of which is activated by IFN. Ubp43 knock-out mice are hypersensitive to IFN-alpha/beta and have enhanced resistance to lethal viral and bacterial infections. Here we show that in addition to protection against foreign pathogens, Ubp43 deficiency increases the resistance to oncogenic transformation by BCR-ABL. BCR-ABL viral transduction/transplantation of wild-type bone marrow cells results in the rapid development of a chronic myeloid leukemia (CML)-like myeloproliferative disease; in contrast, a significantly increased latency of disease development is observed following BCR-ABL viral transduction/transplantation of Ubp43-deficient bone marrow cells. This resistance to leukemic development is dependent on type 1 IFN (IFN-alpha/beta) signaling in Ubp43-deficient cells. Increased levels of type 1 IFN are also detected in the serum of CML mice. These results suggest that inhibition of Ubp43-negative effect on IFN signaling can potentiate the response to increased endogenous IFN levels in innate immune responses against cancer development, indicating that pharmacological inhibition of Ubp43 may be of benefit in cancers and others diseases in which interferon is currently prescribed.

    Funded by: NCI NIH HHS: CA102625, R01 CA102625

    Blood 2007;110;1;305-12

  • Role of BCR/ABL gene-expression levels in determining the phenotype and imatinib sensitivity of transformed human hematopoietic cells.

    Modi H, McDonald T, Chu S, Yee JK, Forman SJ and Bhatia R

    Department of Hematopoietic Stem Cell and Leukemia Research, Division of Hematology and Hematopoietic Cell Transplantation (HCT), City of Hope National Medical Center, Duarte, CA 91010, USA.

    Increased levels of Bcr-Abl expression in chronic myelogenous leukemia (CML) cells are associated with disease progression and imatinib (IM) resistance. However, it is not clear if these associations are a direct result of elevated Bcr-Abl expression. We used a human transduction model of CML to directly investigate the role of varying Bcr-Abl expression levels in determining the phenotype and IM sensitivity of hematopoietic cells. CD34(+) cells were transduced with vectors coexpressing Bcr-Abl and GFP, and cells expressing low and high levels of GFP and Bcr-Abl (BA(lo) and BA(hi)) were selected. BA(hi) cells demonstrated enhanced activation of downstream proliferative and antiapoptotic signaling and enhanced proliferation and survival compared to BA(lo) cells. Freshly isolated BA(hi) CD34(+) cells and cell lines demonstrated increased IM-mediated growth inhibition likely reflecting Bcr-Abl dependence for growth and survival. CD34(+) cells expressing BCR/ABL kinase-mutant genes demonstrated resistance to IM-mediated inhibition of proliferation and viability, which was not enhanced by increased expression of BCR/ABL kinase-mutant genes. We conclude that Bcr-Abl overexpression results in increased proliferation and antiapoptotic signaling in CD34(+) cells, but may not play a direct role in IM resistance in progenitor cells expressing either wild-type or mutant BCR/ABL genes.

    Funded by: NCI NIH HHS: R01 CA095684, R01 CA95684; NHLBI NIH HHS: R01 HL077847, R01 HL77847

    Blood 2007;109;12;5411-21

  • Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study.

    Ray A, Cowan-Jacob SW, Manley PW, Mestan J and Griffin JD

    Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.

    Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 microM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate-resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

    Funded by: NCI NIH HHS: CA 66996

    Blood 2007;109;11;5011-5

  • Upregulation of the TGFbeta signalling pathway by Bcr-Abl: implications for haemopoietic cell growth and chronic myeloid leukaemia.

    Møller GM, Frost V, Melo JV and Chantry A

    School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

    Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by uncontrolled growth of progenitor cells expressing the tyrosine kinase fusion gene product, Bcr-Abl. At present, little is known regarding how TGFbeta, and downstream Smad transcription factors, influence CML cell proliferation in the context of Bcr-Abl expression. Here we show that ectopic Bcr-Abl expression dramatically increases TGFbeta/Smad-dependent transcriptional activity in Cosl cells, and that this may be due to enhancement of Smad promoter activity. Bcr-Abl expressing TF-1 myeloid cells are more potently growth arrested by TGFbeta compared to the parental TF-1 cell line. Additionally, growth of Bcr-Abl-expressing CD34+ cells from chronic phase CML patients is inhibited by TGFbeta and, interestingly, treatment of a non-proliferating CD34+ CML cell sub-population with the TGFbeta kinase inhibitor SB431542 enhanced cell death mediated by the Bcr-Abl inhibitor imatinib. Our data suggest that the expression of Bcr-Abl leads to hyper-responsiveness of myeloid cells to TGFbeta, and we hypothesise that this novel cross-regulatory mechanism might play an important role in maintaining the transformed progenitor cell population in CML.

    FEBS letters 2007;581;7;1329-34

  • aCGH local copy number aberrations associated with overall copy number genomic instability in colorectal cancer: coordinate involvement of the regions including BCR and ABL.

    Bartos JD, Gaile DP, McQuaid DE, Conroy JM, Darbary H, Nowak NJ, Block A, Petrelli NJ, Mittelman A, Stoler DL and Anderson GR

    Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States.

    In order to identify small regions of the genome whose specific copy number alteration is associated with high genomic instability in the form of overall genome-wide copy number aberrations, we have analyzed array-based comparative genomic hybridization (aCGH) data from 33 sporadic colorectal carcinomas. Copy number changes of a small number of specific regions were significantly correlated with elevated overall amplifications and deletions scattered throughout the entire genome. One significant region at 9q34 includes the c-ABL gene. Another region spanning 22q11-q13 includes the breakpoint cluster region (BCR) of the Philadelphia chromosome. Coordinate 22q11-q13 alterations were observed in 9 of 11 tumors with the 9q34 alteration. Additional regions on 1q and 14q were associated with overall genome-wide copy number changes, while copy number aberrations on chromosome 7p, 7q, and 13q21.1-q31.3 were found associated with this instability only in tumors from patients with a smoking history. Our analysis demonstrates there are a small number of regions of the genome where gain or loss is commonly associated with a tumor's overall level of copy number aberrations. Our finding BCR and ABL located within two of the instability-associated regions, and the involvement of these two regions occurring coordinately, suggests a system akin to the BCR-ABL translocation of CML may be involved in genomic instability in about one-third of human colorectal carcinomas.

    Funded by: NCI NIH HHS: P30 CA016056, P30-CA16056, R01 CA074127, R01 CA074127-07, R01-CA74127

    Mutation research 2007;615;1-2;1-11

  • Bcr-Abl signaling through the PI-3/S6 kinase pathway inhibits nuclear translocation of the transcription factor Bach2, which represses the antiapoptotic factor heme oxygenase-1.

    Yoshida C, Yoshida F, Sears DE, Hart SM, Ikebe D, Muto A, Basu S, Igarashi K and Melo JV

    Department of Haematology, Imperial College London, Hammersmith Hospital, UK.

    The malignant phenotype of chronic myeloid leukemia (CML) is due to the abnormal tyrosine kinase activity of the Bcr-Abl oncoprotein. We have previously reported that expression of the Bach2 transcription factor, which induces apoptosis in response to oxidative stress, is greatly reduced in CML cells. Because these cells are resistant to apoptosis, we tested whether Bach2 could also be regulated through posttranslational mechanisms that promote inhibition of the apoptotic response to mutagenic stimuli in CML. We found that Bach2 is phosphorylated on S521 via the phosphatidylinositol-3/S6 kinase pathway, and substitution of this site to alanine leads to nuclear accumulation of the protein, indicating that this phosphorylation is important for its subcellular localization. Ectopic expression of the S521 mutant imparts greater impairment to CML cell growth than the wild-type factor. Furthermore, we showed that Bach2 transcriptionally represses heme oxygenase-1, an antiapoptotic factor up-regulated in CML. Because CML cells are known to produce high levels of intracellular reactive oxygen species, overexpression of heme oxygenase-1 resulting from inhibition of Bach2 activity may contribute to their genomic instability and leukemic phenotype.

    Blood 2007;109;3;1211-9

  • Detection in primary chronic myeloid leukaemia cells of p210BCR-ABL1 in complexes with adaptor proteins CBL, CRKL, and GRB2.

    Patel H, Marley SB and Gordon MY

    Department of Haematology, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK.

    Chronic myeloid leukemia (CML) arises as a consequence of the expression of a chimeric fusion protein, p210BCR-ABL1, which is localized to the cytoplasm and has constitutive protein tyrosine kinase activity. Extensive publications report that p210BCR-ABL1 complexed with multiple cytoplasmic proteins can modulate several cell signaling pathways. However, while altered signaling states can be demonstrated in primary CML material, most of the reported analytical work on complexed proteins has been done in cell lines expressing p210BCR-ABL1. This has been necessary because primary hemopoietic cell lysates contain a degradative activity which rapidly and permanently destroys p210BCR-ABL1, precluding accurate p210BCR-ABL1 quantification by Western blotting or investigation of coimmunoprecipitating proteins in primary cells. This degradative activity has proven intractable to inhibition by conventional protease inhibitors. We show here that the degradative activity in primary cells is associated with cell lysosomes and is most likely to be an acid-dependent hydrolase. By lysing primary hemopoietic cells at high pH, we have demonstrated substantial inhibition of the p210BCR-ABL1-degradative activity and now report, to the best of our knowledge, the first published demonstration by coimmunoprecipitation of the association between p210BCR-ABL1 and cytoplasmic effector proteins in primary CML material.

    Genes, chromosomes & cancer 2006;45;12;1121-9

  • The CML-related oncoprotein BCR/ABL induces expression of histidine decarboxylase (HDC) and the synthesis of histamine in leukemic cells.

    Aichberger KJ, Mayerhofer M, Vales A, Krauth MT, Gleixner KV, Bilban M, Esterbauer H, Sonneck K, Florian S, Derdak S, Pickl WF, Agis H, Falus A, Sillaber C and Valent P

    Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, AKH-Wien, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

    Basophil numbers are typically elevated in chronic myeloid leukemia (CML) and increase during disease progression. Histamine is an essential mediator and marker of basophils and is highly up-regulated in CML. We examined the biochemical basis of histamine synthesis in CML cells. The CML-specific oncoprotein BCR/ABL was found to promote expression of histidine decarboxylase (HDC) and synthesis of histamine in Ba/F3 cells. Moreover, the BCR/ABL tyrosine kinase inhibitors imatinib (STI571) and nilotinib (AMN107) decreased histamine levels and HDC mRNA expression in BCR/ABL-transformed Ba/F3 cells, in the CML-derived basophil cell line KU812, and in primary CML cells. Synthesis of histamine was found to be restricted to the basophil compartment of the CML clone and to depend on signaling through the PI3-kinase pathway. CML cells also expressed histamine receptors (HRs), including HR-1, HR-2, HR-4, and histamine-binding CYP450 isoenzymes which also serve as targets of HR antagonists. The HR-1 antagonists loratadine and terfenadine, which bind to CYP450, were found to counteract proliferation of CML cells, whereas no growth inhibition was observed with the HR-1 antagonist fexofenadine which is not targeted or metabolized by CYP450. Moreover, DPPE, an inhibitor of histamine-binding CYP450 isoenzymes, produced growth inhibition in CML cells. Together, these data show that BCR/ABL promotes histamine production in CML cells and that certain HR-targeting drugs exert antileukemic effects on CML cells.

    Blood 2006;108;10;3538-47

  • BCR and its mutants, the reciprocal t(9;22)-associated ABL/BCR fusion proteins, differentially regulate the cytoskeleton and cell motility.

    Zheng X, Güller S, Beissert T, Puccetti E and Ruthardt M

    Laboratory for Tumor Stem Cell Biology, Department of Hematology, J.W. Goethe University, Frankfurt, Germany. xiaomin.zheng@em.uni-frankfurt.de <xiaomin.zheng@em.uni-frankfurt.de&gt;

    Background: The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome--Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR.

    Methods: We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1alpha gradient.

    Results: Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells.

    Conclusion: Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.

    BMC cancer 2006;6;262

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Comprehensive analysis of BCR-ABL transcript types in Korean CML patients using a newly developed multiplex RT-PCR.

    Goh HG, Hwang JY, Kim SH, Lee YH, Kim YL and Kim DW

    Division of Hematology, St. Mary's Hospital, The Catholic University of Korea, Youngdeungpo-gu, Seoul, Korea.

    Diagnosis of chronic myeloid leukemia (CML) is based on the detection of BCR-ABL gene or Philadelphia chromosome (Ph chromosome), and fusion proteins with different sizes are encoded depending on the breakpoint in the BCR gene. In general, 3 breakpoint cluster regions in the BCR gene have been described: major (M-bcr), minor (m-bcr), and micro (mu-bcr). This study was designed to determine the frequency of BCR-ABL transcripts using one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). Bone marrow (BM) or peripheral blood (PB) samples at diagnosis from 548 patients were obtained with a referring diagnosis of Ph-positive (Ph+) CML, and multistep RT-PCR and newly developed one-step multiplex RT-PCR were applied on each sample. Compared with the previous multistep RT-PCR, one-step multiplex RT-PCR with the primers is the more rapid and accurate method to identify the BCR-ABL breakpoints. Most patients (538/548, 98.18%) were found to have b3a2 or b2a2, and total frequency of occurrence of c3a2, e1a2, b2a3, b1a1, and e1a3 or coexpression of b2a2 and b3a2 was less than 2.00%. No differences were observed between women and men. As the multiplex RT-PCR technique distinguishes BCR-ABL transcripts in all samples with high sensitivity and specificity, it easily could be applied at early stages of diagnosis. The incidence of one or the other rearrangement in CML patients varies in different reported series, and the frequency in each type of BCR-ABL transcript in Korean CML patients seems to be different from those of Western countries.

    Translational research : the journal of laboratory and clinical medicine 2006;148;5;249-56

  • Molecular profiling of chronic myeloid leukemia in eastern India.

    Mondal BC, Bandyopadhyay A, Majumdar S, Mukhopadhyay A, Chandra S, Chaudhuri U, Chakrabarti P, Bhattacharyya S and Dasgupta UB

    Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, Kolkata, India.

    Molecular breakpoint of the BCR-ABL fusion gene has been characterized for 122 chronic myeloid leukemia patients. Out of 122 cases, 33 b2a2, 69 b3a2, 2 e1a2, and 2 e19a2 cases have been detected. Six coexpressed both b2a2 and b3a2 transcripts. All the coexpressing samples had an A>G polymorphism at the putative splice branchpoint in intron 13. The T>C polymorphism in exon 13, reported to be linked to coexpression, was not present in all the coexpressing patients. No correlation of transcript type with platelet count was detected. Those expressing b2a2 transcript were diagnosed at relatively younger age and with higher white blood cell count, in agreement with other reports. However, the correlation was not statistically significant.

    American journal of hematology 2006;81;11;845-9

  • Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity.

    Meyn MA, Wilson MB, Abdi FA, Fahey N, Schiavone AP, Wu J, Hochrein JM, Engen JR and Smithgall TE

    Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.

    Funded by: NCI NIH HHS: CA101828; NCRR NIH HHS: RR016480; NIGMS NIH HHS: GM70590

    The Journal of biological chemistry 2006;281;41;30907-16

  • Absence of the JAK2 mutation V617F in CD34+ hematopoietic stem and progenitor cells from patients with BCR-ABL-positive CML in chronic phase and blast crisis.

    Kronenwett R, Gräf T, Neumann F, Pechtel S, Steidl U, Diaz-Blanco E and Haas R

    In this study, we examined highly enriched CD34+ cells from patients with BCR-ABL-positve CML in chronic phase or blast crisis for the JAK2 V617F activation mutation by sequencing. The cells examined did not bear the mutation irrespective of the disease stage. This finding suggests that the previously described increase of expression and kinase activity of JAK2 in CML cells does not result from the JAK2 V617F activation mutation and that transformation into blast crisis is not associated with the occurrence of this mutation.

    Leukemia research 2006;30;10;1323-4

  • Adaphostin and bortezomib induce oxidative injury and apoptosis in imatinib mesylate-resistant hematopoietic cells expressing mutant forms of Bcr/Abl.

    Dasmahapatra G, Nguyen TK, Dent P and Grant S

    Department of Medicine, Virginia Commonwealth University/Massey Cancer Center, Richmond, VA 23298, USA.

    Effects of the tyrphostin adaphostin and bortezomib were examined in Bcr/Abl+ leukemia cell resistant to imatinib mesylate secondary to Bcr/Abl point mutations. Adaphostin was equally effective in inducing mitochondrial damage, caspase activation, JNK activation, and Raf-1, phospho-Stat3 and -Stat5 inactivation in mutant and wild-type cells, but differentially down-regulated phospho-Bcr/Abl. Adaphostin and bortezomib synergistically induced apoptosis in wild-type and mutant cells, including T315I mutants. Notably, adaphostin+/-bortezomib potently induced ROS and lethality in mutant cells, effects attenuated by the antioxidant NAC. These findings indicate that adaphostin+/-bortezomib circumvent imatinib resistance due to Bcr/Abl point mutations most likely through ROS generation.

    Funded by: NCI NIH HHS: CA 100866, CA 63753, CA 93738

    Leukemia research 2006;30;10;1263-72

  • Detection and quantification of the abelson tyrosine kinase domains of the BCR-ABL gene translocation in chronic myeloid leukaemia using genomic quantitative real-time polymerase chain reaction.

    Gullo CA, Chuah CT, Hwang WY and Teoh GK

    Multiple Myeloma Research Laboratory (MMRL), SingHealth Research Facilities, Singapore. gcrcg@cgh.com.sg

    Introduction: Since undetectable BCR-ABL mRNA transcription does not always indicate eradication of the Ph+ CML clone and since transcriptionally silent Ph+ CML cells exist, quantitation by genomic PCR of bcr-abl genes can be clinically useful. Furthermore, hotspot mutations in the Abelson tyrosine kinase (ABLK) domain of the bcr-abl gene translocation in Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) cells confer resistance on the specific kinase blocking agent, STI571.

    Genomic DNA from K562, CESS and patient CML cells were amplified using rapid cycle quantitative real-time polymerase chain reaction for the gene regions spanning the mutation hotspots. In assays for ABLK exons 4 or 6, exonic or intronic PCR primers were used.

    Results: We show that separation of cycle threshold (CT) values for log-fold amplicon quantification was 2.9 cycles for ABLK exon 4, and 3.8 cycles for exon 6 with rapid amplification times. K562 CML cells were found to have a approximately 2 log-fold ABLK gene amplification. In contrast, patient CML cells had CT differences of 2.2 for both exon, suggesting that there was no significant ABLK gene amplification. DNA sequencing confirmed that neither K562 nor patient CML cells contained ABLK hotspot mutations. Messenger RNA transcription analysis permitted the assessment of BCR-ABL transcription, which was qualitatively correlated to genomic amplification.

    Conclusions: This novel Q-PCR assay was found to have high fidelity and legitimacy, and potentially useful for monitoring minimal residual disease, transcriptionally silent Ph+ CML cells, and bcr-abl gene amplification.

    Annals of the Academy of Medicine, Singapore 2006;35;10;680-7

  • Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate.

    Jabbour E, Kantarjian H, Jones D, Talpaz M, Bekele N, O'Brien S, Zhou X, Luthra R, Garcia-Manero G, Giles F, Rios MB, Verstovsek S and Cortes J

    Department of Leukemia, The University of Texas, UT MD Anderson Cancer Center, Houston, TX 77030, USA.

    Mutations of the BCR-ABL kinase domain are a common mechanism of resistance to imatinib in chronic myeloid leukemia. We screened for mutations 171 patients failing imatinib therapy. Sixty-six mutations in 23 amino acids were identified in 62 (36%) patients not responding to imatinib. Phosphate-binding loop (P-loop) mutations were the most frequent (n=24; 36%). By multivariate analysis, factors associated with development of mutations were older age (P=0.026) prior interferon therapy (P=0.026), and accelerated phase or blast phase at time of imatinib failure (P=0.001). After a median follow-up of 38 months (range, 4-68 months) from the start of imatinib therapy, seven patients with non-P-loop and two with P-loop mutation died. By multivariate analysis, development of clonal evolution and higher percentage of peripheral blood basophils were associated with worse survival from the time of imatinib failure. Mutation status had no impact on survival. When survival was measured from the time therapy started, non-P-loop mutations together with duration of response and transformation at the time of failure to imatinib were associated with shorter survival. In conclusion, P-loop mutations were not associated with poor outcome, suggesting that the prognosis of patients who fail imatinib is multifactorial.

    Leukemia 2006;20;10;1767-73

  • Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells.

    Krause DS, Lazarides K, von Andrian UH and Van Etten RA

    Molecular Oncology Research Institute, Tufts-New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111, USA.

    In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.

    Funded by: NHLBI NIH HHS: HL56949, P01 HL056949

    Nature medicine 2006;12;10;1175-80

  • A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation.

    McCallum L, Price S, Planque N, Perbal B, Pierce A, Whetton AD and Irvine AE

    Department of Haematology, Centre for Cancer Research and Cell Biology, Queen's University Belfast, University Floor, Tower Block, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, United Kingdom.

    Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

    Blood 2006;108;5;1716-23

  • A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands.

    Giallourakis C, Cao Z, Green T, Wachtel H, Xie X, Lopez-Illasaca M, Daly M, Rioux J and Xavier R

    Massachusetts General Hospital, Center for Computational and Integrative Biology, Harvard University Medical School, Boston, Massachusetts 02114, USA.

    PDZ domain-containing proteins and their interaction partners are mutated in numerous human diseases and function in complexes regulating epithelial polarity, ion channels, cochlear hair cell development, vesicular sorting, and neuronal synaptic communication. Among several properties of a collection of documented PDZ domain-ligand interactions, we discovered embedded in a large-scale expression data set the existence of a significant level of co-regulation between PDZ domain-encoding genes and these ligands. From this observation, we show how integration of expression data, a comparative genomics catalog of 899 mammalian genes with conserved PDZ-binding motifs, phylogenetic analysis, and literature mining can be utilized to infer PDZ complexes. Using molecular studies we map novel interaction partners for the PDZ proteins DLG1 and CARD11. These results provide insight into the diverse roles of PDZ-ligand complexes in cellular signaling and provide a computational framework for the genome-wide evaluation of PDZ complexes.

    Funded by: NIDDK NIH HHS: P30 DK040561, P30 DK040561-11

    Genome research 2006;16;8;1056-72

  • Distinct biological impact of dephosphorylation vs. downregulation of p210Bcr-Abl: implications for imatinib mesylate response and resistance.

    Kurzrock R, Talpaz M, Li L and Estrov Z

    Division of Cancer Medicine, The University of Texas, M.D. Anderson Cancver Center, Houston, Texas 77230-1402, USA. rkurzroc@mdanderson.org

    Imatinib mesylate suppresses phosphorylation of its kinase target, Bcr-Abl. We hypothesized that loss of p210Bcr-Abl (the kinase target) may lead to imatinib mesylate resistance. We studied K562 cells [chronic myelogenous leukemia (CML) blast crisis line] and MO7E/MBA-1 cells (with MBA-1 cells representing MO7E cells stably transfected with BCR-ABL). Imatinib mesylate resistance developed when p210Bcr-Abl expression was abolished. Furthermore, K562 cells were significantly more growth suppressed after imatinib mesylate exposure than after downregulation of Bcr-Abl expression. Signaling pathways which were functional in the absence of Bcr-Abl expression (NF-kappaB and mitogen-activated protein kinase activation or the growth factor pathway) were disrupted when p210Bcr-Abl was present but dephosphorylated, suggesting that an intact, but enzymatically inactive Bcr-Abl, may interfere with critical growth/signaling pathways. Downregulation of p210Bcr-Abl may be a mechanism by which imatinib mesylate resistance emerges. Samples from three of 15 patients with imatinib mesylate-resistant CML blast crisis had undetectable levels of p210Bcr-Abl. We conclude that retention of a dephosphorylated p210Bcr-Abl has a biologic impact distinct from that of downregulation/loss of p210Bcr-Abl and, in a subset of patients, loss of the target of the kinase inhibitor may lead to imatinib mesylate resistance.

    Leukemia & lymphoma 2006;47;8;1651-64

  • Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression.

    Nakanishi T, Shiozawa K, Hassel BA and Ross DD

    Program in Experimental Therapeutics, University of Maryland Marlene and Stewart Greenebaum Cancer Center (UMGCC), Baltimore, 21201, USA.

    Imatinib, a potent tyrosine kinase inhibitor, is effluxed from cells by the breast cancer resistance protein (BCRP/ABCG2), yet published studies to date fail to demonstrate resistance to imatinib cytotoxicity in BCRP-overexpressing cells in vitro. We investigated cellular resistance to imatinib in BCR-ABL-expressing cells transduced and selected to overexpress BCRP (K562/BCRP-MX10). These cells exhibited a 2- to 3-fold increase in resistance to imatinib (P < .05) and a 7- to 12-fold increase in resistance to mitoxantrone, a known BCRP substrate. Resistance to imatinib was completely abolished by the specific BCRP inhibitor fumitremorgin C. Studies of the mechanism of the diminished resistance to imatinib compared with mitoxantrone revealed that imatinib decreased the expression of BCRP in K562/BCRP-MX10 cells without affecting mRNA levels. BCRP levels in cells that do not express BCR-ABL were not affected by imatinib. Loss of BCRP expression was accompanied by imatinib-induced reduction of phosphorylated Akt in the BCRP-expressing K562 cells. The phosphoinositol-3 kinase (PI3K) inhibitor LY294002 also decreased BCRP levels in K562/BCRP-MX10 cells. These studies show that BCRP causes measurable imatinib resistance, but this effect is attenuated by imatinib-mediated inhibition of BCR-ABL, which in turn downregulates overall BCRP levels posttranscriptionally via the PI3K-Akt pathway.

    Blood 2006;108;2;678-84

  • Generation of the BCR/ABL fusion gene in a Philadelphia chromosome-negative chronic myeloid leukaemia: insertion of 5.6 Mb of 9q34 into the BCR region of chromosome 22.

    Valle L, Fernández V, Pérez-Pons C, Sánchez FG, Benítez J and Urioste M

    Familial Cancer Unit, Human Cancer Genetics Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain. lvalle@cnio.es

    This report describes a chronic myelogenous leukaemia patient with an apparently normal bone marrow karyotype but BCR/ABL fusion-gene-positive. Commercial FISH probes showed an atypical pattern and the BCR/ABL fusion transcript was detected by RT-PCR, but not the reciprocal ABL/BCR. Consecutive FISH assays clarified the mechanism of the masked Ph. The ABL gene and the following 5.6-5.7 Mb of 9q are inserted into the BCR region of chromosome 22. There is no transference of 22q material to chromosome 9 or to any other chromosomes. Clinical features and evolution of the patient are similar to those cases with classic Ph chromosome.

    Hematological oncology 2006;24;2;86-8

  • Bcr-Abl reduces endoplasmic reticulum releasable calcium levels by a Bcl-2-independent mechanism and inhibits calcium-dependent apoptotic signaling.

    Piwocka K, Vejda S, Cotter TG, O'Sullivan GC and McKenna SL

    Department of Biochemistry, BioSciences Institute, University College Cork, Cork, Ireland.

    The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia (CML). Several studies have suggested that the expression levels of Bcr-Abl are elevated at disease progression to blast crisis and that this plays a significant role in the achievement of drug resistance. We have established cell lines expressing low and high levels of Bcr-Abl to study the molecular mechanisms involved in disease progression and drug resistance. It is now known that the endoplasmic reticulum (ER) can play a major role in the regulation of apoptosis. We therefore investigated whether Bcr-Abl expression modulates ER homeostasis and interferes with ER-mediated apoptotic pathways to promote survival. Bcr-Abl-expressing cells exhibit a decreased amount of free releasable calcium in the ER as well as a weaker capacitative calcium entry response, relative to parental cells. This effect is independent of Bcl-2, which is a known modulator of ER calcium homeostasis. The reduction in ER releasable calcium results in inhibition of the ER/mitochondria-coupling process and mitochondrial calcium uptake. This study demonstrates a novel downstream consequence of Bcr-Abl signaling. The ability to negate calcium-dependent apoptotic signaling is likely to be a major prosurvival mechanism in Bcr-Abl-expressing cells.

    Blood 2006;107;10;4003-10

  • Inducible activation of CEBPB, a gene negatively regulated by BCR/ABL, inhibits proliferation and promotes differentiation of BCR/ABL-expressing cells.

    Guerzoni C, Bardini M, Mariani SA, Ferrari-Amorotti G, Neviani P, Panno ML, Zhang Y, Martinez R, Perrotti D and Calabretta B

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson Medical College, 233 South and 10th Street, Philadelphia, PA 19107, USA.

    Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebpbeta mRNA. Constitutive expression or conditional activation of wild-type CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because expression of CEBPB (and CEBPA) is low in the blast crisis (BC) stage of chronic myelogenous leukemia (CML) and is inversely correlated with BCR/ABL tyrosine kinase levels, these findings point to the therapeutic potential of restoring C/EBP activity in CML-BC and, perhaps, other types of acute leukemia.

    Funded by: NCI NIH HHS: CA 95111, CA 95512, R01 CA095512; PHS HHS: P01 78890

    Blood 2006;107;10;4080-9

  • Serial measurement of BCR-ABL transcripts in the peripheral blood after allogeneic stem cell transplantation for chronic myeloid leukemia: an attempt to define patients who may not require further therapy.

    Kaeda J, O'Shea D, Szydlo RM, Olavarria E, Dazzi F, Marin D, Saunders S, Khorashad JS, Cross NC, Goldman JM and Apperley JF

    Department of Haematology, Imperial College at Hammersmith Hospital, London, UK.

    We identified 243 patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) who had BCR-ABL transcripts monitored by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) after allogeneic stem cell transplantation for a median of 84.3 months. Individual patients were regarded as having achieved molecular relapse (MR) if the BCR-ABL/ABL ratio exceeded 0.02% on 3 occasions or reached 0.05% on 2 occasions. Patients were allocated to 1 of 4 categories: (1) 36 patients were "persistently negative" or had a single low-level positive result; (2) 51 patients, "fluctuating positive, low level," had more than 1 positive result but never more than 2 consecutive positive results; (3) 27 patients, "persistently positive, low level," had persisting low levels of BCR-ABL transcripts but never more than 3 consecutive positive results; and (4) 129 patients relapsed. In 107 of these, relapse was based initially only on molecular criteria; in 72 (67.3%) patients the leukemia progressed to cytogenetic or hematologic relapse either prior to or during treatment with donor lymphocyte infusions. We conclude that the pattern of BCR-ABL transcript levels after allograft is variable; only a minority of patients with fluctuating or persistent low levels of BCR-ABL transcripts satisfied our definitions of MR, whereas the majority of patients who did so were likely to progress further.

    Blood 2006;107;10;4171-6

  • Identification of transcriptional targets associated with the expression of p210 Bcr-Abl.

    Hickey FB and Cotter TG

    Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland.

    Objectives: Chronic myeloid leukaemia is caused by the expression of the p210 Bcr-Abl fusion protein which results from the Philadelphia translocation, t(9;22). This oncogene has been the focus of extensive research. However, the molecular mechanisms responsible for the haematological malignancy are not fully understood. The main objective of the current study was to identify novel transcriptional targets of Bcr-Abl.

    Methods: In order to achieve this, microarrays were employed in order to conduct a genome-wide expression analysis comparing 32D cells with a transfected clone expressing high levels of p210 Bcr-Abl. Quantitative RT-PCR was employed in order to confirm the observed increase/decrease in expression for a number of the deregulated genes.

    This comparison identified 138 genes of known function showing altered expression in response to Bcr-Abl-mediated signalling. Among the genes found to be upregulated in response to p210 Bcr-Abl were aldolase 1A and phosphofructokinase, both of which encode key enzymes in the glycolytic pathway. As a consequence of this, we demonstrate that the rate of glycolysis is significantly increased in Bcr-Abl expressing cells in a PI3K-dependent manner. Our results also indicate altered expression of genes involved in cell proliferation, cell adhesion and cell signalling.

    European journal of haematology 2006;76;5;369-83

  • Quantitative real-time RT-PCR monitoring of BCR-ABL in chronic myelogenous leukemia shows lack of agreement in blood and bone marrow samples.

    Stock W, Yu D, Karrison T, Sher D, Stone RM, Larson RA and Bloomfield CD

    University of Chicago Cancer Research Center, Chicago, IL 60637, USA. wstock@medicine.bsd.uchicago.edu

    Molecular monitoring of the BCR-ABL transcript in chronic myelogenous leukemia (CML) using quantitative RT-PCR provides clinicians with important diagnostic and prognostic information. To determine whether molecular detection and monitoring of CML is comparable using peripheral blood (PB) and bone marrow (BM) aspirate samples, we performed a prospective study using quantitative real-time RT-PCR (QRT-PCR) of paired PB and BM samples from 41 patients with CML entered onto a single Cancer and Leukemia Group B (CALGB) treatment study. QRT-PCR analysis of PB and BM samples was performed prior to initiation of, and during, treatment with homoharringtonine and cytarabine on a CALGB study for previously untreated CML. Statistical analyses demonstrated good agreement of PB and BM pre-treatment samples. However, using the Bland-Altman statistical method that measures true agreement between PB and BM values, we found that there was only modest agreement of BCR-ABL measurements in PB and BM for samples obtained during treatment. PB values obtained during treatment tended to be lower than the corresponding BM values [average difference = -0.37 (p<0.001) in 36 paired samples] and the 95% limits of agreement ranged from -1.23 to 0.48. Nevertheless, our study demonstrates that BM and PB QRT-PCR values followed a similar trend during treatment (Spearman correlation coefficient, 0.83; 95% CI, 0.70, 0.96). Our data suggest that, quantitatively, PB and BM measurements of BCR-ABL are frequently disparate. Since BM values tended to be higher than PB values, BM sampling provides the most accurate assessment of minimal residual disease (MRD). Based on these results, we caution against interchanging BM with PB sampling for MRD monitoring during treatment of CML since this may lead to misinterpretation of treatment results.

    Funded by: NCI NIH HHS: CA16058, CA31946, CA77658, U10-CA101140

    International journal of oncology 2006;28;5;1099-103

  • A Ph-negative chronic myeloid leukemia with a complex BCR/ABL rearrangement and a t(6;9)(p21;q34.1).

    Todorić-Zivanović B, Marisavljević D, Surace C, Cemerikić V, Marković O, Krtolica K, Tatomirović Z, Cikota B, Magić Z and Rocchi M

    Military Medical Academy, Bezanijska Kosa, Belgrade, Serbia and Montenegro. btodoric@hotmail.com

    Chronic myeloid leukemia (CML) is a clonal malignant disorder of a pluripotent hematopoetic stem cell characterized by the presence of the Philadelphia (Ph) chromosome in more than 90% of patients. Cryptic or "masked" BCR/ABL gene rearrangements may be found in cases with a normal karyotype and in cases with the complex karyotype, in which typical t(9;22) is not visible at the microscopic level. Those rearrangements can now be detected by fluorescence in situ hybridization. Here, we report on a novel and complex Ph chromosome-negative CML case with a t(6;9)(p21;q34.1) in which the BCR/ABL fusion gene is located at 6p21.

    Cancer genetics and cytogenetics 2006;166;2;180-5

  • Bcr interferes with beta-catenin-Tcf1 interaction.

    Ress A and Moelling K

    Institute of Medical Virology, University of Zurich, Gloriastrasse 30, CH-8006 Zurich, Switzerland.

    The beta-catenin/Tcf complex is a downstream effector of the Wnt signalling pathway. It is a transcription complex, which activates gene expression and contributes to proliferation and tumor progression. Tcf1 in complex with beta-catenin is able to activate beta-catenin-dependent gene expression. We demonstrate that expressed Bcr is able to bind the transcription factor Tcf1 to disrupt the Tcf1/beta-catenin complex. Phosphorylation of Bcr by the tyrosine kinase pp60(src) can lead to dissociation of the transcriptionally inactive Bcr/Tcf1 complex. Thus two independent mechanisms may regulate Tcf/beta-catenin-mediated transcription via Bcr: binding to beta-catenin as we have previously shown and to Tcf1 as shown here.

    FEBS letters 2006;580;5;1227-30

  • The Philadelphia chromosome as a secondary abnormality in inv(3)(q21q26) acute myeloid leukemia at diagnosis: confirmation of p190 BCR-ABL mRNA by real-time quantitative polymerase chain reaction.

    Han JY and Theil KS

    Department of Clinical Pathology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. jyhan@daunet.donga.ac.uk

    The Philadelphia chromosome (Ph) as a secondary cytogenetic abnormality is a rare event. It is observed mostly as an additional, late-appearing cytogenetic change during the evolution of acute leukemia and its presentation as a secondary change at the onset of disease is much rarer. We describe here a patient with acute myelogenous leukemia (AML) who had Ph as a secondary chromosome abnormality at diagnosis. Cytogenetic analysis showed an abnormal karyotype, 45,XY,inv(3)(q21q26),-7[4]/45,idem, t(9;22)(q34;q11.2). The p190 variety of BCR-ABL rearrangements was confirmed by a real-time reverse-transcriptase polymerase chain reaction using fluorescent probes. To our knowledge, the minor BCR-ABL fusion gene involving a secondary Ph superimposed on inv(3) and monosomy 7 has not been reported in AML at diagnosis. Along with the identification of more cases, it will be possible to understand the exact role of this secondary Ph in a multistep leukemogenesis.

    Cancer genetics and cytogenetics 2006;165;1;70-4

  • Deletion of the derivative chromosome 9 in chronic myeloid leukemia.

    Campbell LJ

    Cytogenetics Department, St Vincent's Hospital, Victoria, Australia.

    With the development of fluorescence in situ hybridization (FISH), it was possible to detect the BCR-ABL fusion signal in both metaphase spreads and interphase cells of patients with chronic myeloid leukemia (CML). However, the use of FISH to detect residual disease in patients with CML post therapy was limited by the false positive rate using the early single fusion probes. Therefore, dual fusion probes that created a fusion signal on the derivative chromosome 9 in addition to the fusion sifnal on the Philadelphia chromosome or derivative chromosome 22 were developed. Using these second-generation probes, it was discovered that a significant proportion of CML cases has a sub-microscopic deletion at the site of the ABL-BCR fusion. This chapter outlines a testing strategy to identify deleltions of the derivative chromosome 9 and to use combinations of probes to identify residual disease in these cases.

    Methods in molecular medicine 2006;125;107-14

  • Detection of BCR-ABL mutations and resistance to imatinib mesylate.

    Branford S and Hughes T

    Division of Molecular Pathology, Institute of Medical and Veterinary Science, South Australia.

    The major mechanism of imatinib resistance for patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with mutations in the Bcr-Abl fusion tyrosine kinase that reduce the capacity of imatinib to inhibit kinase activity. The early detection of such mutations may allow timely treatment intervention to prevent or overcome resistance. Direct sequencing of the BCR-ABL kinase domain is relatively rapid and allows detection of emerging mutations at a sensitivity of approx 20%. Mutations have been detected over a range of 242 amino acids, which spans the entire kinase domain. For optimal sensitivity, the kinase domain of the abnormal gene should be isolated by reverse-transcription (RT) polymerase chain reaction (PCR) amplification using primers that hybridize to the BCR and ABL genes. The quality of the RNA is assessed by real-time quantitative PCR prior to analysis, and BCR-ABL levels are determined. Only RNA of adequate quality is used to ensure accurate and reproducible mutation analysis. Depending on the level of BCR-ABL transcripts, a one- or two-step PCR is required to amplify the kinase domain. Direct sequencing with dye terminator chemistry is performed using PCR-purified products. The sequence is compared to an ABL kinase domain reference sequence using sequencing analysis software, which aligns the sequences and highlights single or multiple mutations.

    Methods in molecular medicine 2006;125;93-106

  • Natural history of Southeast Asian chronic myeloid leukemia patients with different BCR-ABL gene variants.

    Auewarakul CU, Huang S, Yimyam M and Boonmoh S

    Department of Medicine, Division of Hematology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. sciaw@mahidol.ac.th

    Little evidence exists regarding the prognostic impact of the major BCR-ABL gene variants (e13a2 and e14a2) in chronic myeloid leukemia (CML) patients diagnosed and treated in the developing Asian countries. In this study, 139 Thai CML patients were followed for a median period of 3 years (range 18-43 months). Clinical presentations of both BCR-ABL gene variant groups (73% e14a2+ and 27% e13a2+) were similar, although e14a2+ patients tended to be older (42 vs. 37 years) and had higher white blood cell counts than e13a2+ patients. The majority of patients in both groups presented with Sokal stage 2-3 (score >0.8) and were categorized as Hasford's intermediate- to high-risk groups (score >780). All patients received oral chemotherapy and 13% underwent allogeneic stem cell transplantation. None received oral tyrosine kinase inhibitors. In the conventional chemotherapy group, the overall survival (OS) rate was slightly better in e14a2+ than in e13a2+ patients (p = n.s.). The median survival in e14a2+ and e13a2+ patients who did not receive stem cell transplantation was 49 and 33 months, respectively (p = n.s.). The type of blastic crisis in e14a2+ and e13a2+ patients was similar, being predominantly myeloid. In conclusion, CML patients in Thailand, despite being much younger, had a comparable OS with those in the Western countries, with no different OS between e14a2+ and e13a2+ patients. Future studies should focus on the impact of novel oral BCR-ABL tyrosine kinase inhibitors on the outcome of Thai CML patients with different BCR-ABL gene variants.

    Acta haematologica 2006;116;2;114-9

  • Detection of single nucleotide insertion of BCR/ABL region in imatinib-resistant human myelogenous leukemia SR-1 cells.

    Park TH, Kwon HC, Kim HJ, Han JY, Jeong JS, Han HY, Seo C, Kwak JY and Park JI

    Department of Internal Medicine, Dong-A University, College of Medicine, Busan 602-715, Korea.

    Imatinib mesylate is a selective Bcr/Abl kinase inhibitor and an effective anticancer agent for Bcr/Abl-positive chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Mutations within the BCR/ABL kinase domain are the most commonly identified mechanism associated with relapse. To overcome the imatinib resistance in CML, many investigators have tried to clarify molecular mechanism for imatinib resistance in cells of patients who failed to respond to imatinib. Our aim was to invesitigate underlying mechanism for imatinib resistance in SR-1 cells, which were derived from a CML patient in blast crisis. We detected the new mutation of BCR/ABL, resulting in premature termination and loss of BCR/ABL fusion protein expression, which might be possible mechanism for the resistance to imatinib in SR-1 cells.

    Experimental & molecular medicine 2005;37;5;507-11

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • BCR kinase phosphorylates 14-3-3 Tau on residue 233.

    Clokie SJ, Cheung KY, Mackie S, Marquez R, Peden AH and Aitken A

    School of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, UK.

    The breakpoint cluster region protein, BCR, has protein kinase activity that can auto- and trans-phosphorylate serine, threonine and tyrosine residues. BCR has been implicated in chronic myelogenous leukaemia as well as important signalling pathways, and as such its interaction with 14-3-3 is of major interest. 14-3-3tau and zeta isoforms have been shown previously to be phosphorylated in vitro and in vivo by BCR kinase on serine and threonine residue(s) but site(s) were not determined. Phosphorylation of 14-3-3 isoforms at distinct sites is an important mode of regulation that negatively affects interaction with Raf kinase and Bax, and potentially influences the dimerization of 14-3-3. In this study we have further characterized the BCR-14-3-3 interaction and have identified the site phosphorylated by BCR. We show here that BCR interacts with at least five isoforms of 14-3-3 in vivo and phosphorylates 14-3-3tau on Ser233 and to a lesser extent 14-3-3zeta on Thr233. We have previously shown that these two isoforms are also phosphorylated at this site by casein kinase 1, which, in contrast to BCR, preferentially phosphorylates 14-3-3zeta.

    The FEBS journal 2005;272;15;3767-76

  • e6a2 BCR/ABL1 fusion with cryptic der(9)t(9;22) deletions in a patient with chronic myeloid leukemia.

    Roti G, La Starza R, Gorello P, Gottardi E, Crescenzi B, Martelli MF and Mecucci C

    This is the first report of e6a2 and e1a2 BCR/ABL1 positive chronic myeloid leukemia (CML) with cryptic deletions of the 5'ABL1 and 3'BCR in separate clones which differ in genomic regions of the deleted der(9). Both deletions were detected throughout monitoring. Imatinib mesylate stabilized this CML with rare genetic aberrations for a relatively long time.

    Haematologica 2005;90;8;1139-41

  • Bcr-Abl regulates osteopontin transcription via Ras, PI-3K, aPKC, Raf-1, and MEK.

    Hickey FB, England K and Cotter TG

    Department of Biochemistry, Biosciences Institute, University College Cork, Ireland.

    Chronic myeloid leukemia (CML) is caused by the constitutively active Bcr-Abl tyrosine kinase. This fusion protein is generated by the Philadelphia translocation t(9;22). CML is a progressive condition that invariably advances from a drug-sensitive to a drug-resistant, aggressive, acute leukemia. The mechanisms responsible for this progression are largely unknown; however, in many cases, progression is accompanied by an increase in Bcr-Abl expression. Osteopontin (OPN) expression has been shown to be involved in the progression and increased aggression and invasiveness of many solid tumors. Here, we demonstrate that OPN expression is induced in a model of leukemia, and we describe the identification of specific signaling pathways required for the induction of OPN expression by p210 Bcr-Abl. We have determined that high levels of Bcr-Abl activate a signaling cascade involving the sequential activation of Ras, phosphatidylinositol-3 kinase, atypical protein kinase C, Raf-1, and mitogen-activated protein kinase kinase, leading to the ultimate expression of OPN. Our results suggest that these molecules represent a single pathway and also that there is no redundancy in this pathway, as inhibition of any individual component results in a block in the induction of OPN. The data presented here define for the first time the ability of Bcr-Abl to stimulate the expression of OPN and also identify the signaling pathway involved. This may not only prove important in understanding the mechanisms of progression of CML but also highlights a pathway that may prove significant in many other cases of oncogenesis, where OPN expression is implicated.

    Journal of leukocyte biology 2005;78;1;289-300

  • The breakpoint cluster region gene on chromosome 22q11 is associated with bipolar disorder.

    Hashimoto R, Okada T, Kato T, Kosuga A, Tatsumi M, Kamijima K and Kunugi H

    Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. rhashimo@ncnp.go.jp

    Background: Although the pathogenesis of bipolar disorder remains unclear, heritable factors have been shown to be involved. The breakpoint cluster region (BCR) gene is located on chromosome 22q11, one of the most significant susceptibility loci in bipolar disorder linkage studies. The BCR gene encodes a Rho GTPase activating protein, which is known to play important roles in neurite growth and axonal guidance.

    Methods: We examined patients with bipolar disorder (n = 171), major depressive disorder (n = 329) and controls (n = 351) in Japanese ethnicity for genetic association using eleven single nucleotide polymorphisms (SNPs), including a missense one (A2387G; N796S), in the genomic region of BCR.

    Results: Significant allelic associations with bipolar disorder were observed for three SNPs, and associations with bipolar II disorder were observed in ten SNPs including N796S SNP (bipolar disorder, p = .0054; bipolar II disorder p = .0014). There was a significant association with major depression in six SNPs. S796 allele carriers were in excess in bipolar II patients (p = .0046, odds ratio = 3.1, 95% CI 1.53-8.76). Furthermore, we found a stronger evidence for association with bipolar II disorder in a multi-marker haplotype analysis (p = .0002).

    Conclusions: Our results suggest that genetic variations in the BCR gene could confer susceptibility to bipolar disorder and major depressive disorder.

    Biological psychiatry 2005;57;10;1097-102

  • Chronic myeloid leukaemia: an investigation into the role of Bcr-Abl-induced abnormalities in glucose transport regulation.

    Barnes K, McIntosh E, Whetton AD, Daley GQ, Bentley J and Baldwin SA

    School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK. k.barnes@leeds.ac.uk

    In chronic myeloid leukaemia (CML) expression of the chimeric tyrosine kinase, Bcr-Abl, promotes the inappropriate survival of haemopoietic stem cells by a nonautocrine mechanism in the absence of IL-3. Stimulation of glucose uptake appears to play an important role in the suppression of apoptosis by this cytokine in normal haemopoietic cells. To investigate whether the cell survival mechanisms mediated by the oncoprotein and cytokine showed any similarities, we employed a haemopoietic cell line, TonB210, engineered for inducible expression of Bcr-Abl. Tyrosine kinase expression in cytokine-deprived cells was found to mimic the effect of IL-3 in maintaining a higher V(max) for hexose uptake. In both IL-3- treated cells and those expressing Bcr-Abl, high rates of hexose uptake were associated with the retention at the cell surface of approximately 80% of the total cellular content of the GLUT1 glucose transporter. In contrast, treatment of Bcr-Abl-expressing cells for 6 h with the Bcr-Abl kinase inhibitor Glivec (10 muM), in the absence of IL-3, led to internalization of approximately 90% of the cell-surface transporters and drastically decreased (4.4+/-0.9 (mean+/-s.e.m., 4)-fold) the V(max) for hexose uptake, without significant effect on the K(m) for this process or on the total cellular transporter content. These effects were not the result of any significant loss in cell viability, and preceded the onset of apoptosis caused by inhibition of Bcr-Abl. Both IL-3 treatment and expression of Bcr-Abl led to enhanced phosphorylation of Akt (protein kinase B). The stimulation of transport by IL-3 and Bcr-Abl in TonB210 cells was inhibitable by phosphatidylinositol 3-kinase inhibitors, indicating the involvement of this kinase in the signal transduction pathway. These findings suggest that inhibition of glucose transport plays an important role in the therapeutic action of Glivec, and that the signal transduction pathways involved in transport stimulation by Bcr-Abl may offer novel therapeutic targets for CML.

    Oncogene 2005;24;20;3257-67

  • Interpretation of submicroscopic deletions of the BCR or ABL gene should not depend on extra signal-FISH: problems in interpretation of submicroscopic deletion of the BCR or ABL gene with extra signal-FISH.

    Kim YR, Cho HI, Yoon SS, Park S, Kim BK, Lee YK, Chun H, Kim HC and Lee DS

    Department of Laboratory Medicine, Cheju National University College of Medicine, Jeju, Korea.

    Several groups have demonstrated that a submicroscopic gene deletion in Ph+ chronic myelogenous leukemia (CML) is associated with a poor prognosis and reduced response to treatment. To assess the variation between detection methods in the interpretation of a submicroscopic gene deletion, we performed an extra signal (ES)-FISH BCR/ABL and double-FISH (D-FISH) BCR/ABL on frozen bone marrow cells from 79 patients with CML (63 in the chronic phase, 6 in the accelerated phase, and 10 in blast crisis) and 30 patients with a BCR/ABL-negative myeloproliferative disorder as determined by RT-PCR. The normal cutoff values were 0.22% for ES-FISH and 0.25% for D-FISH. The cutoff values for false-positive signals from a juxtaposition of the BCR and ABL gene were 11% in ES-FISH and 13% in D-FISH. Of the 14 patients who showed an ABL gene deletion by ES-FISH, 5 had an ABL deletion only, 5 had both a BCR and an ABL deletion, but 4 proved to have a classic BCR/ABL rearrangement without a submicroscopic deletion, as determined by D-FISH. Discrepant results between ES- and D-FISH were observed in 12 of the 79 patients (15.8%), and the main causes of a discrepancy were a false-positive ABL deletion (4 of 12, 33%), a variant Philadelphia chromosome (3 of 12, 25%), an inversion of derivative chromosome 9 at the very breakpoint of the ABL gene (9q32) (1 of 12, 8.3%), a cryptic variant Ph chromosome (1 of 12, 8.3%), and a marker chromosome (1 of 12, 8.3%). Although there was no significant difference in the sensitivity for the detection of the fusion signal between ES- and D-FISH, ES-FISH showed a high percentage of cells with false-positive fusion signals (1 orange, 1 green, 1 yellow), which makes it difficult to interpret the submicroscopic ABL deletion. In conclusion, an interpretation of the submicroscopic deletions of the BCR or ABL gene should not depend on ES-FISH.

    Genes, chromosomes & cancer 2005;43;1;37-44

  • Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides.

    Aichberger KJ, Mayerhofer M, Krauth MT, Skvara H, Florian S, Sonneck K, Akgul C, Derdak S, Pickl WF, Wacheck V, Selzer E, Monia BP, Moriggl R, Valent P and Sillaber C

    Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, AKH-Wien, Waehringer Guertel 18-20, A-1097 Vienna, Austria.

    Antiapoptotic members of the bcl-2 family have recently been implicated in the pathogenesis of chronic myeloid leukemia (CML), a hematopoietic neoplasm associated with the BCR/ABL oncogene. We have examined expression of MCL-1 in primary CML cells and BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed myeloid cell leukemia-1 (mcl-1) mRNA and the MCL-1 protein in a constitutive manner. The BCR/ABL inhibitor imatinib (=STI571) decreased the expression of MCL-1 in these cells. Correspondingly, BCR/ABL enhanced mcl-1 promoter activity, mcl-1 mRNA expression, and the MCL-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of MCL-1 in Ba/F3 cells was counteracted by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Identical results were obtained for constitutive expression of MCL-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of MCL-1 as a survival-related target in CML cells, mcl-1 siRNA and mcl-1 antisense oligonucleotides (ASOs) were applied. The resulting down-regulation of MCL-1 was found to be associated with a substantial decrease in viability of K562 cells. Moreover, the mcl-1 ASO was found to synergize with imatinib in producing growth inhibition in these cells. Together, our data identify MCL-1 as a BCR/ABL-dependent survival factor and interesting target in CML.

    Blood 2005;105;8;3303-11

  • Solution structure of AF-6 PDZ domain and its interaction with the C-terminal peptides from Neurexin and Bcr.

    Zhou H, Xu Y, Yang Y, Huang A, Wu J and Shi Y

    Hefei National Laboratory for Physical Sciences at Microscale, School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China.

    AF-6 is a key molecule essential for structure organization of cell-cell junction of polarized epithelia. It belongs to a novel cell-cell adhesion system. The AF-6 PDZ domain mediates interactions by binding to a specific amino acid sequence in target proteins. Here we report the solution structure of the AF-6 PDZ domain determined by NMR. Previously, the AF-6 PDZ domain was considered to be a class II PDZ domain. However we found that a unique hydrophilic amino acid, Gln70, at position alphaB1 makes the alphaB/betaB groove of the AF-6 PDZ domain significantly different from that of the canonical class II PDZ domain. The AF-6 PDZ domain does not have the second hydrophobic binding pocket, and the N-terminal end of alphaB is closer to betaB. Using BIACORE and NMR chemical shift perturbation experiments, we have studied the binding characteristics of the PDZ domain to the C-terminal peptide of Neurexin, KKNKDKEYYV, and that of Bcr, KRQSILFSTEV. The C-terminal peptide of Neurexin is a class II ligand, whereas that of Bcr is a class I ligand. The dissociation constants of these ligands were 4.08 x 10(-7) and 2.23 x 10(-6) m, respectively. Each of the four C-terminal positions in Neurexin and Bcr may contribute to the interaction. The three-dimensional models of the AF-6 PDZ-Neurexin C-terminal peptide complex and the AF-6 PDZ-Bcr C-terminal peptide complex were built up by molecular dynamics simulations. Unlike the canonical class II PDZ domain, Ala74 at alphaB5 rather than the residue at alphaB1 makes direct hydrophobic contact with the side chain of Tyr at the -2 position of the ligand.

    The Journal of biological chemistry 2005;280;14;13841-7

  • Derivative chromosome 9 deletions are a significant feature of childhood Philadelphia chromosome positive acute lymphoblastic leukaemia.

    Robinson HM, Martineau M, Harris RL, Barber KE, Jalali GR, Moorman AV, Strefford JC, Broadfield ZJ, Cheung KL and Harrison CJ

    Leukaemia Research Fund Cytogenetics Group, Cancer Sciences Division, University of Southampton, Southampton, UK.

    Deletions from the derivative chromosome 9, der(9), of the translocation, t(9;22)(q34;q11), at the site of the ABL/BCR fusion gene, have been demonstrated by fluorescence in situ hybridisation (FISH), in both Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL). In CML they occur in 10-15% of cases and appear to indicate a worse prognosis, whereas in ALL, the situation is unclear. This study presents the findings of dual fusion FISH used to detect such deletions in a series of 27 BCR/ ABL-positive childhood ALL patients. Metaphase FISH was essential for the accurate interpretation of interphase FISH signal patterns. Three cases (11%) had a single fusion signal, resulting from deletions of the der(9). Three other patients with variant translocations and one with an insertion, also had a single fusion, but with no evidence of deletions. Gain of a fusion in approximately one-third of patients indicated a second Ph, which appears to be a diagnostic marker of Ph-positive ALL. This study shows that the incidence of deletions from the der(9) in childhood ALL is at least as high as that reported for CML.

    Leukemia 2005;19;4;564-71

  • [Acute lymphoblastic leukemias with aberrations of BCR-ABL genes].

    Parovichnikova EN, Savchenko VG, Verniuk MA, Vinogradova OA, Misiurin AV, Vorob'ev IA, Domracheva EV, Tikhonova LIu, Rukavitsyn OA, Rossiev VA, Kliasova GA, Turkina AG, Liubimova LS, Mendeleeva LP and Isaev VG

    Aim: To develop an original therapeutic strategy in Ph-positive acute lymphoblastic leukemia (ALL).

    In November 2001 Hematological Research Center (HRC) initiated the study of chimeric BCR-ABL gene. During the first stage of the study (November 2001-July 2004), 18 primary ALL patients were recruited in HRC, from July 2004 to January 2005--16 patients in HRC, N.N. Burdenko Central Military Hospital, regional Samara hospital. The diagnosis of Ph-positive ALL was established in detection of translocation t(9;22) by standard cytogenetic test or fluorescent hibridization in situ with double signal (D-FISH), or by polymerase chain reaction with reverse transcription (RT-PCR). In detection of aberration of BCR-ABL gene the patients received stem hemopoietic cells, from June 2004 imatinib was added to chemotherapy in the period of induction and consolidation.

    Results: Incidence rate of BCR-ABL-positive ALL by standard cytogenetic test and D-FISH makes up 20%, by RT-PCR--25%. Differences in chimeric transcripts detectability by different methods may be explained by different sensitivity of the methods. Complete hematological remissions were achieved in the majority of the patients (6 of 8) irrespective of imatinib administration. Achievement of molecular remission in BCR-ABL-positive ALL occurs also in standard chemotherapy but molecular remissions begin 2-4 months later than clinicohematological ones.

    Conclusion: In using imatinib combination with chemotherapy, molecular remission can be achieved simultaneously with hematological one. Long-term results will be analysed later.

    Terapevticheskii arkhiv 2005;77;7;11-6

  • Bcr-Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release.

    Deming PB, Schafer ZT, Tashker JS, Potts MB, Deshmukh M and Kornbluth S

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, NC 27710, USA.

    Bcr-Abl, activated in chronic myelogenous leukemias, is a potent cell death inhibitor. Previous reports have shown that Bcr-Abl prevents apoptosis through inhibition of mitochondrial cytochrome c release. We report here that Bcr-Abl also inhibits caspase activation after the release of cytochrome c. Bcr-Abl inhibited caspase activation by cytochrome c added to cell-free lysates and prevented apoptosis when cytochrome c was microinjected into intact cells. Bcr-Abl acted posttranslationally to prevent the cytochrome c-induced binding of Apaf-1 to procaspase 9. Although Bcr-Abl prevented interaction of endogenous Apaf-1 with the recombinant prodomain of caspase 9, it did not affect the association of endogenous caspase 9 with the isolated Apaf-1 caspase recruitment domain (CARD) or Apaf-1 lacking WD-40 repeats. These data suggest that Apaf-1 recruitment of caspase 9 is faulty in the presence of Bcr-Abl and that cytochrome c/dATP-induced exposure of the Apaf-1 CARD is likely defective. These data provide a novel locus of Bcr-Abl antiapoptotic action and suggest a distinct mechanism of apoptosomal inhibition.

    Funded by: NCI NIH HHS: R01 CA102702; NINDS NIH HHS: R01 NS042197, R01 NS42197

    Molecular and cellular biology 2004;24;23;10289-99

  • Bcr (breakpoint cluster region) protein binds to PDZ-domains of scaffold protein PDZK1 and vesicle coat protein Mint3.

    Malmberg EK, Andersson CX, Gentzsch M, Chen JH, Mengos A, Cui L, Hansson GC and Riordan JR

    Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, 413 90 Gothenburg, Sweden.

    The breakpoint cluster region protein (Bcr) is a large soluble oligomeric multidomain protein best known because of its involvement in chronic myelogenous leukemia (CML). A chromosomal translocation between its gene and that of the c-abl kinase ('Philadelphia chromosome') plays a major causative role in that malignancy. Thus most attention has been paid to the role of the protein in hemopoietic cells. However, Bcr is also expressed in other cell types including epithelia. Bcr is generally considered to be a cytoplasmic protein but in addition to its kinase and GTPase exchange and activating domains it contains potentially membrane-interacting pleckstrin homology and C2 domains as well as a PDZ-binding C terminus mediating an interaction with a PDZ-domain protein at intercellular junctions of epithelial cells. We have examined the ability of Bcr to interact with other epithelial PDZ proteins and found specific binding to both the apical PDZK1 protein and the Golgi-localized Mint3. The former is important in the organization of several apical functions and the latter in vesicular trafficking in the secretory pathway. Hence these findings extend the interactions and likely signaling impact of Bcr in epithelia from the cytosol to at least these two membrane compartments.

    Journal of cell science 2004;117;Pt 23;5535-41

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Second case of CML with aberrant BCR-ABL fusion transcript (e8/a2) with insertion of an inverted ABL intron 1b sequence.

    Sugimoto T, Ijima K, Hisatomi H, Murayama T, Mizuno I, Hato A, Imoto S, Nishimura R and Koizumi T

    Clinical Research Division, Hyogo Medical Center for Adults, Akashi, Japan.

    We found a case of Ph-positive chronic myelogenous leukemia (CML) patient with an atypical BCR-ABL transcript that was undetectable by a routine reverse transcription polymerase chain reaction (RT-PCR) for major BCR-ABL. Additional RT-PCR and sequence analyses have demonstrated that the aberrant transcript consists of a fusion of BCR exon 8 (e8) and ABL exon 2 (a2) with an insertion of a 55-bp inverted sequence of intron 1b between them. The nucleotide sequences of the aberrant transcript were identical to those of a previously reported CML patient. These are the only two CML cases in the literature with identical aberrant BCR-ABL transcripts.

    American journal of hematology 2004;77;2;164-6

  • Fluorescent in situ hybridization analysis of Philadelphia chromosome-negative chronic myeloid leukemia with the bcr/abl fusion gene.

    Monma F, Nishii K, Yamamori S, Hosokai N, Nakazaki T, Lorenzo F, Usui E, Sakakura M, Miyashita H, Fujieda A, Ohishi K, Katayama N and Shiku H

    Second Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie, Japan.

    This report describes a patient with Philadelphia chromosome-negative (Ph-) but bcr/abl fusion gene-positive chronic myeloid leukemia (CML) and a molecular analysis of the mechanisms behind the Ph status. Spectral karyotyping-fluorescent in situ hybridization (SKY-FISH) analysis showed no abnormal translocation; however, a bcr/abl fusion gene was detected by reverse transcriptase-polymerase chain reaction analysis. FISH analysis showed that signals from the 9q and 22q subtelomere probes were detected on the der(9) and der(22) chromosomes, respectively. On the other hand, FISH analysis of the abl and bcr genes with dual fusion probes, which can detect the bcr/abl fusion gene on both the der(9) and der(22) chromosomes, showed the signal for bcr/abl fusion on the der(22) chromosome but not on the der(9) chromosome. These results indicate that insertion of the abl gene into the bcr region on the der(22) chromosome or retranslocation between the der(9) chromosome and the der(22) chromosome may have caused the Ph CML in this case.

    International journal of hematology 2004;80;2;155-8

  • [Polymorphism analysis of 5' promotor region of BCR gene].

    Tian H, Zheng WY, Fu YG, Lin JH, Lu FJ and Zhou SY

    Department of Hematology, Fuzhou General Hospital, Fuzhou, Fujian, 350025, P.R.China.

    BCR-ABL fusion gene is regarded as the molecular hallmark of chronic myelogenous leukemia (CML), and its expression is controlled by the BCR gene promoter. This study was designed to investigate the polymorphism of the promoter region of BCR gene, and its possible correlation with the disease.

    Methods: A 1.13 kb fragment of BCR gene 5' promotor region was amplified and sequenced from 30 CML patients and 19 controls. Transcription factor binding sites and repeat sequences in this region were analyzed using softwares and online tools.

    Results: Four novel single nucleotide polymorphisms (SNPs) and 3 bases different from the reference sequence were detected in the region studied. Among these 2 novel SNPs and 1 different base were located in or near several bases of binding sites. The gene frequencies of the novel SNPs had no significant difference between CML and control people.

    Conclusion: Sequence polymorphisms were found in the 5' promotor region of BCR gene, most of them being SNPs. No relativity can be validated between the SNPs and the disease. But it appears that some SNPs might have the probability of bringing influence to the transcription and expression of the gene.

    Ai zheng = Aizheng = Chinese journal of cancer 2004;23;7;812-5

  • A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase.

    Million RP, Harakawa N, Roumiantsev S, Varticovski L and Van Etten RA

    Molecular Oncology Research Institute, Tufts-New England Medical Center, 750 Washington St., Box 5609, Boston, MA 02111, USA.

    A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.

    Funded by: NCI NIH HHS: CA09595, CA90576, R01 CA090576, T32 CA009595

    Molecular and cellular biology 2004;24;11;4685-95

  • Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry.

    Brill LM, Salomon AR, Ficarro SB, Mukherji M, Stettler-Gill M and Peters EC

    Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA. lbrill@gnf.org

    Protein tyrosine phosphorylation cascades are difficult to analyze and are critical for cell signaling in higher eukaryotes. Methodology for profiling tyrosine phosphorylation, considered herein as the assignment of multiple protein tyrosine phosphorylation sites in single analyses, was reported recently (Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443-448). The technology platform included the use of immunoprecipitation, immobilized metal affinity chromatography (IMAC), liquid chromatography, and tandem mass spectrometry. In the present report, we show that when using complex mixtures of peptides from human cells, methylation improved the selectivity of IMAC for phosphopeptides and eliminated the acidic bias that occurred with unmethylated peptides. The IMAC procedure was significantly improved by desalting methylated peptides, followed by gradient elution of the peptides to a larger IMAC column. These improvements resulted in assignment of approximately 3-fold more tyrosine phosphorylation sites, from human cell lysates, than the previous methodology. Nearly 70 tyrosine-phosphorylated peptides from proteins in human T cells were assigned in single analyses. These proteins had unknown functions or were associated with a plethora of fundamental cellular processes. This robust technology platform should be broadly applicable to profiling the dynamics of tyrosine phosphorylation.

    Analytical chemistry 2004;76;10;2763-72

  • Interferon consensus sequence binding protein (ICSBP; IRF-8) antagonizes BCR/ABL and down-regulates bcl-2.

    Burchert A, Cai D, Hofbauer LC, Samuelsson MK, Slater EP, Duyster J, Ritter M, Hochhaus A, Müller R, Eilers M, Schmidt M and Neubauer A

    Klinikum der Philipps Universität Marburg, Klinik für Hämatologie, Onkologie und Immunologie, Marburg, Germany.

    BCR/ABL is the causative genetic aberration in chronic myelogenous leukemia (CML). Mice lacking expression of the interferon (IFN) consensus sequence binding protein (ICSBP), an IFN gamma-inducible transcription factor of the interferon regulatory factor (IRF) family, develop a disease similar to human CML. Mounting evidence suggests a role for ICSBP in the pathogenesis of CML. However, the underlying mechanisms are largely unknown. By stable and conditional expression of ICSBP in wild-type and BCR/ABL-transformed 32D cells (32D/wt and 32D/BA), we found that ICSBP inhibited BCR/ABL-mediated leukemogenesis in vivo. Moreover, ICSBP also overrode BCR/ABL-mediated morphology changes, chemotherapy, and imatinib resistance, as well as BCR/ABL-induced repression of differentiation. Some of these ICSBP effects may be explained in part by an ICSBP-mediated repression of bcl-2, a major antiapoptotic target of BCR/ABL, on transcriptional and protein level. Using reporter gene assays and electrophoretic mobility shift assays we identified that the bcl-2 promoter activity was inhibited by ICSBP by way of a fragment containing 2 characteristic ICSBP-responsive elements. An inverse correlation between ICSBP and bcl-2 expression was confirmed in vivo. Collectively, our findings suggest that ICSBP antagonizes BCR/ABL by down-regulation of bcl-2 and implicates a central role for ICSBP in the pathogenesis of CML, as well as a therapeutic target to overcome drug resistance in bcl-2-dependent tumors.

    Blood 2004;103;9;3480-9

  • Identification of genes differentially regulated by the P210 BCR/ABL1 fusion oncogene using cDNA microarrays.

    Håkansson P, Segal D, Lassen C, Gullberg U, Morse HC, Fioretos T and Meltzer PS

    Department of Clinical Genetics, University Hospital, Lund, Sweden.

    Objective: The t(9;22) translocation is associated with more than 95% of cases of chronic myeloid leukemia. The resulting fusion of the BCR and ABL1 loci produces the constitutively active BCR/ABL1 tyrosine kinase. A wide range of signal transduction molecules are activated by BCR/ABL1, including MYC, PI-3 kinase, and different STAT molecules. In contrast, relatively few genes are known to be regulated by BCR/ABL1 at the level of transcription.

    In an effort to better understand the transcriptional program activated by BCR/ABL1, we used cDNA microarrays to evaluate the relative expression of approximately 6450 human genes in U937 myelomonocytic cells expressing P210 BCR/ABL1 via a tetracycline-inducible promoter.

    Results: We confirmed the previously reported up-regulation of the PIM1 and JUN oncogenes by BCR/ABL1. In addition, we identified 59 more genes up-regulated by BCR/ABL1. Interestingly, roughly one third of these were genes previously reported to be interferon (IFN)-responsive, including the OAS1, IFIT1, IFI16, ISGF3G, and STAT1 genes. An additional seven BCR/ABL1-regulated genes were found to be IFN-responsive in U937 cells. The expression profile also included genes encoding transcription factors, kinases, and signal transduction molecules, as well as genes regulating cell growth, differentiation, apoptosis, and cell adhesion, features previously suggested to be affected by BCR/ABL1.

    Conclusion: These observations shed novel insight into the mechanism of BCR/ABL1 action and provide a range of targets for further investigation.

    Experimental hematology 2004;32;5;476-82

  • p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells.

    Salesse S, Dylla SJ and Verfaillie CM

    Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA.

    Chronic myelogenous leukemia (CML) is a malignancy of the human hematopoietic stem cell (HSC) caused by the p210BCR/ABL oncoprotein. Although alternative splicing of pre-mRNA is a critical determinant of a cell's protein repertoire, it has not been associated with CML pathogenesis. We identified a BCR/ABL-dependent increase in expression of multiple genes involved in pre-mRNA splicing (eg SRPK1, RNA Helicase II/Gu, and hnRNPA2/B1) by subtractive hybridization of cDNA from p210BCR/ABL-eGFP vs eGFP-transduced umbilical cord blood CD34+ cells. beta1-integrin signaling is important to HSC maintenance and proliferation/differentiation, and is abnormal in CML. As an example of how changes in pre-mRNA processing might contribute to CML pathogenesis, we observed alternative splicing of a gene for a beta1-integrin-responsive nonreceptor tyrosine kinase (PYK2), resulting in increased expression of full-length Pyk2 in BCR/ABL-containing cells. Treatment of p210BCR/ABL-positive cells with the Abl-specific tyrosine kinase inhibitor STI571 reverted PYK2 splicing to a configuration more consistent with normal cells, and correlated with decreased expression of BCR/ABL-induced proteins involved in pre-mRNA processing. Whether altered PYK2 splicing contributes to CML pathogenesis remains undetermined; however, we propose that generic changes in pre-mRNA splicing as a result of p210BCR/ABL kinase activity may contribute to CML pathogenesis.

    Funded by: NCI NIH HHS: CA09138; NHLBI NIH HHS: R01 HL-49930; NIDDK NIH HHS: R01 DK-53673

    Leukemia 2004;18;4;727-33

  • The BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in pre-B acute lymphoblastic leukemia cells.

    Klein F, Feldhahn N, Harder L, Wang H, Wartenberg M, Hofmann WK, Wernet P, Siebert R and Müschen M

    Laboratory for Molecular Stem Cell Biology, Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-Universität Düsseldorf, 40225, Germany.

    The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre-B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre-B ALL and normal bone marrow pre-B cells by serial analysis of gene expression, many genes involved in pre-B cell receptor signaling are silenced in the leukemia cells. Although normal pre-B cells are selected for the expression of a functional pre-B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre-B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre-B cell receptor signaling and the expression of a truncated isoform of the pre-B cell receptor-associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre-) B cell receptor.

    The Journal of experimental medicine 2004;199;5;673-85

  • Detection of bcr-abl gene expression at a low level in blood cells of some patients with essential thrombocythemia.

    Hsu HC, Tan LY, Au LC, Lee YM, Lieu CH, Tsai WH, You JY, Liu MD and Ho CK

    Division of Hematology, Department of Medicine, Taipei Veterans General Hospital, Taiwan. hchsu@vghtpe.gov.tw

    The major bcr-abl fusion gene is seen as a major marker of chronic myeloid leukemia (CML). However, whether the bcr-abl transcript can be detected in patients with essential thrombocythemia (ET) is still a matter of controversy. We detected the messenger RNA expression of the bcr-abl gene using reverse transcription-polymerase chain reaction in peripheral-blood leukocytes (PBLs) from 63 patients with myeloproliferative disorders (including CML, ET, and polycythemia vera [PV]) and 51 normal, healthy volunteers. The bcr-abl transcript was detected in 4 of the 30 ET patients (13.3%), 17 of the 17 CML patients (100%), none of the 16 PV patients (0%), and 1 of the 51 normal subjects (1.9%). Compared with the normal controls, ET patients have a greater tendency to express the bcr-abl transcript in PBLs (P=.06, Fisher's exact test). Further semiquantitative analysis showed that the intensity of bcr-abl transcript expression in 4 ET patients and a normal individual was 10(3) to 10(4) times less than that in the CML patients. We conclude that the bcr-abl transcript can be detected in the PBLs of Philadelphia chromosome (Ph)-negative ET patients but that the level of expression is markedly less than that in CML patients. The clinical significance of this finding merits further investigation.

    The Journal of laboratory and clinical medicine 2004;143;2;125-9

  • Endoplasmic reticulum stress initiates apoptotic death induced by STI571 inhibition of p210 bcr-abl tyrosine kinase.

    Pattacini L, Mancini M, Mazzacurati L, Brusa G, Benvenuti M, Martinelli G, Baccarani M and Santucci MA

    Istituto di Ematologia e Oncologia Medica, Lorenzo e Ariosto Seràgnoli, Università di Bologna-Medical School, S. Orsola Hospital, Via Massarenti 9, Bologna 40138, Italy.

    The endoplasmic reticulum (ER) is the site where proteins destined to either secretion or different subcellular compartments assemble and the major storage of intracellular Ca(2+). The ER stress resulting from a variety of toxic insults leads to apoptosis. Here, we showed that the apoptotic death triggered by STI571, an inhibitor of the p210 bcr-abl tyrosine kinase, in murine myeloid progenitors transducing the p210 bcr-abl tyrosine kinase of Chronic Myeloid Leukemia (CML) proceeds from ER stress. The Bcl-2 dowmodulation and inactivation induced by the binding to its antagonist: Bad, the release of caspase 12 from the ER membranes in its active form and of Ca(2+) from the ER pool addressed towards ER a sensor of STI571-induced pro-apoptotic signal.

    Leukemia research 2004;28;2;191-202

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Induction of apoptosis by apicidin, a histone deacetylase inhibitor, via the activation of mitochondria-dependent caspase cascades in human Bcr-Abl-positive leukemia cells.

    Cheong JW, Chong SY, Kim JY, Eom JI, Jeung HK, Maeng HY, Lee ST and Min YH

    Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Korea.

    Purpose: Apicidin, a histone deacetylase inhibitor, is a novel cyclic tetrapeptide that exhibits potent antiproliferative activity against various cancer cell lines. The aim of this study was to examine the potential of apicidin to induce apoptosis in human Bcr-Abl-positive leukemia cells and to assess the mechanism of apicidin-induced apoptosis.

    Cells were exposed to various concentrations of apicidin for 2-72 h, after which the levels of apoptosis, histone acetylation, mitochondrial damage, caspase activation, and Bcr-Abl expression were assessed.

    Results: Apicidin induced apoptosis in K562 cells in a concentration- and time-dependent manner. Similarly, apicidin notably induced the apoptosis in the primary leukemic blasts obtained from chronic myelogenous leukemia patients in blast crisis. The acetylated histone H4 levels increased in a concentration-dependent manner in the K562 cells. However, the timing of cell death caused by apicidin did not exactly correlate with the histone deacetylase inhibitory effect. The disruption of the mitochondrial membrane potential, cytochrome c release into the cytosol, and the mitochondrial Bax translocation were notably demonstrated after the apicidin treatment. Apicidin induced the proteolytic cleavage of procaspase-9, -3, -8, and poly(ADP-ribose) polymerase. Pretreatment of the K562 cells with the caspase-3 inhibitor, DEVD-CHO, completely inhibited the apicidin-induced apoptosis, suggesting that apicidin-induced apoptosis was caspase-dependent. The Fas/Fas ligand death receptor pathway was not involved in the apicidin-mediated apoptosis in K562 cells. Pretreatment of the cells with the caspase-9 inhibitor LEHD-fmk abrogated the apicidin- induced cleavage of procaspase-3, -8, and poly(ADP-ribose) polymerase. The p210 Bcr-Abl protein levels were notably decreased after the apicidin treatment, with near complete loss after 48 h. Reverse transcription-PCR assay demonstrated that the Bcr-Abl mRNA level was also remarkably decreased in a time-dependent manner.

    Conclusions: These results indicate that apicidin effectively induces the apoptosis of Bcr-Abl-positive leukemia cells through the activation of the mitochondrial pathway-dependent caspase cascades. The down-regulation of Bcr-Abl mRNA might also be one of the mechanisms implicated in the apicidin-mediated apoptosis in the K562 cells. This study provides the rationale to additionally investigate apicidin as a potential therapeutic agent for the drug-resistant Bcr-Abl-positive leukemia cells.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2003;9;13;5018-27

  • Leukemia-associated translocation products able to activate RAS modify PML and render cells sensitive to arsenic-induced apoptosis.

    Puccetti E, Beissert T, Güller S, Li JE, Hoelzer D, Ottmann OG and Ruthardt M

    Med. Klinik III/Abtl. Hämatologie, Johann Wolfgang Goethe-Universität, D-60590 Frankfurt, Germany.

    Since the 19th century, arsenic (As2O3) has been used in the treatment of chronic myelogenous leukemia (CML) characterized by the t(9;22) translocation. As2O3 induces complete remissions in patients with acute promyelocytic leukemia. The response to As2O3 is genetically determined by the t(15;17)-or the t(9;22)-specific fusion proteins PML/RARalpha or BCR/ABL. The PML portion of PML/RARalpha is crucial for the sensitivity to As2O3. PML is nearly entirely contained in PML/RARalpha. PML is upregulated by oncogenic RAS in primary fibroblasts. The aberrant kinase activity of BCR/ABL leads to constitutive activation of RAS. Therefore, we hypothesized that BCR/ABL could increase sensitivity to As2O2-induced apoptosis by modifying PML expression. To disclose the mechanism of As2O3-induced apoptosis in PML/RARalpha- and BCR/ABL-expressing cells, we focused on the role of PML for As2O3-induced cell death. Here we report that (i) sensitivity to As2O3-induced apoptosis of U937 cells can be increased either by overexpression of PML, or by conditional expression of activated RAS; (ii) also the expression of the t(8;21)-related AML-1/ETO increased sensitivity to As2O3-induced apoptosis; (iii) both BCR/ABL and AML-1/ETO activated RAS and modified the PML expression pattern; (iv) the expression of either BCR/ABL or AML-1/ETO rendered U937 cells sensitive to interferon alpha-induced apoptosis. In summary, these data suggest a crucial role of factors able to upregulate PML for As2O2-induced cell death.

    Oncogene 2003;22;44;6900-8

  • Expression of BCR/ABL and BCL-2 in myeloid progenitors leads to myeloid leukemias.

    Jaiswal S, Traver D, Miyamoto T, Akashi K, Lagasse E and Weissman IL

    Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

    Chronic myelogenous leukemia is a myeloproliferative disorder (MPD) that, over time, progresses to acute leukemia. Both processes are closely associated with the t(9;22) chromosomal translocation that creates the BCR/ABL fusion gene in hematopoietic stem cells (HSCs) and their progeny. Chronic myelogenous leukemia is therefore classified as an HSC disorder in which a clone of multipotent HSCs is likely to be malignantly transformed, although direct evidence for malignant t(9;22)+ HSCs is lacking. To test whether HSC malignancy is required, we generated hMRP8p210BCR/ABL transgenic mice in which expression of BCR/ABL is absent in HSCs and targeted exclusively to myeloid progenitors and their myelomonocytic progeny. Four of 13 BCR/ABL transgenic founders developed a chronic MPD, but only one progressed to blast crisis. To address whether additional oncogenic events are required for progression to acute disease, we crossed hMRP8p210BCR/ABL mice to apoptosis-resistant hMRP8BCL-2 mice. Of 18 double-transgenic animals, 9 developed acute myeloid leukemias that were transplantable to wild-type recipients. Taken together, these data indicate that a MPD can arise in mice without expression of BCR/ABL in HSCs and that additional mutations inhibiting programmed cell death may be critical in the transition of this disease to blast-crisis leukemia.

    Funded by: NCI NIH HHS: CA 42551; NIAID NIH HHS: 5T32 AI 07290, AI07290

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;17;10002-7

  • Characterization of a myeloid tyrosine phosphatase, Lyp, and its role in the Bcr-Abl signal transduction pathway.

    Chien W, Tidow N, Williamson EA, Shih LY, Krug U, Kettenbach A, Fermin AC, Roifman CM and Koeffler HP

    Department of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048, USA. chienw@cshs.org

    The Bcr-Abl protein-tyrosine kinase is implicated in the development of chronic myeloid leukemia. The potential role of protein-tyrosine phosphatase in the regulation of Bcr-Abl signaling was explored. First, expression patterns of tyrosine phosphatases in leukemic cell lines were investigated using degenerate primers for reverse transcription-PCR followed by cloning and sequencing of the cDNA. Distinct patterns of distribution of phosphatase were found in erythroid and myeloid leukemic cell lines. Whereas some phosphatases were ubiquitously expressed, others were limited to specific cell types. Surprisingly, a previously cloned "lymphocyte-specific" phosphatase, Lyp, was frequently detected in a number of myeloid cell lines as well as normal granulocytes and monocytes. Lyp was localized to the cytosol, and overexpression of Lyp caused reduction in the phosphorylation levels of multiple proteins in KCL22 chronic myeloid leukemia blast cells including Cbl, Bcr-Abl, Erk1/2, and CrkL. Co-expression of Lyp and Bcr-Abl in Cos-7 cells resulted in decreased levels of Bcr-Abl, Grb2, and Myc. Overexpression of Lyp markedly suppressed anchorage-independent clonal growth of KCL22 cells. Taken together, the data suggest that Lyp may play an antagonistic role in signaling by the Bcr-Abl fusion protein.

    The Journal of biological chemistry 2003;278;30;27413-20

  • The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain.

    Radziwill G, Erdmann RA, Margelisch U and Moelling K

    Institute of Medical Virology, University of Zurich, CH-8028 Zurich, Switzerland.

    The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state.

    Molecular and cellular biology 2003;23;13;4663-72

  • Constitutive and specific activation of STAT3 by BCR-ABL in embryonic stem cells.

    Coppo P, Dusanter-Fourt I, Millot G, Nogueira MM, Dugray A, Bonnet ML, Mitjavila-Garcia MT, Le Pesteur D, Guilhot F, Vainchenker W, Sainteny F and Turhan AG

    INSERM U362, Translational Research-Cell Therapy Laboratory, Villejuif, Cedex, France.

    BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia (CML) arises in a primitive hematopoietic stem cell with both differentiation and self-renewal ability. To study the phenotypic effects of BCR-ABL in a clonal in vitro self-renewal and differentiation model, we have introduced BCR-ABL in the ES cell line CCE. The major effect of BCR-ABL expression was the persistence of primitive morphology of ES cells despite LIF deprivation, correlated with a constitutive activation of STAT3, the major self-renewal factor of ES cells, but no evidence of activation of STAT5. The enforced expression of BCR-ABL in an ES cell line, engineered to express a tetracycline-inducible dominant-negative form of a STAT3, triggered ES cell differentiation with an increased generation of hematopoietic cells expressing erythroid and megakaryocytic phenotypes. RT-PCR analysis for Oct4, Brachyury and beta-globin expression confirmed a delay of differentiation in BCR-ABL expressing clones, which could be entirely reversed upon activation of the dominant-negative form of STAT3. To study the possible relevance of STAT3 activation by BCR-ABL in human CML, Western blot analyses performed on the CD34+ cells, purified from CML patients at different stages of their disease, also demonstrated increased levels of STAT3 proteins phosphorylated both on tyrosine and serine residues. These results represent to our knowledge the first functional link between BCR-ABL oncogene and a self-renewal in the context of ES cells through constitutive activation of STAT3. Thus, the BCR-ABL embryonic stem cell model that we developed as well as the results obtained in human CML samples suggests a role for STAT3 in the pathogenesis of human CML.

    Oncogene 2003;22;26;4102-10

  • Amplification of the BCR/ABL fusion gene clustered on a masked Philadelphia chromosome in a patient with myeloblastic crisis of chronic myelocytic leukemia.

    Gargallo PM, Cuello MT, Aranguren PN and Larripa IB

    Departamento de Genética, Academia Nacional de Medicina de Buenos Aires, Buenos Aires, Argentina.

    Although the chronic phase of chronic myelocytic leukemia (CML) is characterized by the Philadelphia (Ph) chromosome creating a hybrid BCR/ABL gene, additional genetic changes involved in blast crisis are poorly understood. We report a 4-8-fold amplification by tandem duplication of the BCR/ABL fusion gene clustered on a masked Ph chromosome in a 61-year-old male patient with CML in myeloblastic crisis. Our finding suggests that the BCR/ABL amplification may play a role as a novel mechanism in the progression to an aggressive blast transformation in some cases of Ph-positive CML.

    Cancer genetics and cytogenetics 2003;143;2;140-4

  • Prolonged treatment with imatinib mesylate in patients with advanced chronic myeloid leukemia causes a reduction of bcr/abl mRNA levels independent of cytogenetic response.

    Cariani E, Capucci M, Micheletti M, Spalenza F, Zanella I, Albertini A and Rossi G

    III Laboratory, Hospital of Brescia, P.le Spedali Civili 1, 25123, Brescia, Italy. ecariani@hotmail.com

    Bcr/abl mRNA levels were monitored in 13 patients with chronic myeloid leukemia receiving imatinib mesylate over a period of 78 weeks. During treatment median bcr/abl mRNA levels progressively declined from 77.2 normalized dose (nD) at baseline to 11.28 nD after 13 weeks ( P<0.05) and to 1.28 nD after 78 weeks ( P<0.05). After 13 weeks, bcr/abl mRNA levels were significantly lower in cytogenetic responders compared to nonresponders ( P<0.05), but subsequent decrease in the transcript levels caused the loss of any correlation to the cytogenetic status. These results suggest that bcr/abl mRNA levels may reflect cytogenetic response only during the early phases of imatinib therapy.

    Annals of hematology 2003;82;6;333-5

  • The interaction of the Bcr-Abl tyrosine kinase with the Src kinase Hck is mediated by multiple binding domains.

    Stanglmaier M, Warmuth M, Kleinlein I, Reis S and Hallek M

    GSF - National Institute of Health and Environment, Klinische Kooperationsgruppe für Gentherapie, Marchioninistrasse 25, 81377 München, Germany.

    Bcr-Abl is found in more than 95% of cases with CML. The mechanism of Bcr-Abl-induced transformation is not fully understood. Bcr-Abl is a constitutively active tyrosine kinase with transforming capacity for hematopoietic cells. We demonstrated recently that the Src kinase Hck interacts directly with Bcr-Abl by a kinase-independent mechanism. Moreover, the inhibition of the Hck kinase seems to block some of the transforming effects of Bcr-Abl. To identify the binding domains mediating this interaction of Hck with Bcr-Abl, we co-expressed different plasmid and baculovirus vectors containing mutants or single domains of Bcr-Abl and/or Hck in COS7 and Sf9 cells. At least four independent binding regions for Hck were identified in Bcr-Abl, one in Bcr, one in the region comprising the SH3 and SH2 domain of Abl, one in the SH1 domain of Abl, and one in the C-terminal domain of Abl. In the Hck kinase, deletion of the SH2 and/or the SH3 region abolished binding to Bcr-Abl. In contrast, deletion of the Hck SH1 domain enhanced binding of Hck to Abl and Bcr-Abl. In conclusion, the results indicate that the interaction of Bcr-Abl with Hck is mediated by a novel, complex mechanism that involves multiple domains of Bcr-Abl and the SH2 and SH3 domains of Hck.

    Leukemia 2003;17;2;283-9

  • BCR-ABL binds to IRS-1 and IRS-1 phosphorylation is inhibited by imatinib in K562 cells.

    Traina F, Carvalheira JB, Saad MJ, Costa FF and Saad ST

    Hemocentro, Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, P.O. Box 6198, 13083-970 Campinas, SP, Brazil.

    In the present study we used K562 cells to demonstrate that insulin receptor substrate 1 (IRS-1) is expressed and constitutively phosphorylated in BCR-ABL(+) cells. We observed association between BCR-ABL/IRS-1, IRS-1/phosphoinositide 3'-kinase (PI3-kinase), and IRS-1/Grb2 in the K562 cell line. Our findings demonstrate that imatinib treatment resulted in marked attenuation of BCR-ABL/IRS-1 association and of IRS-1-stimulated PI3-kinase activity in K562 cells. We concluded that the IRS-1 protein is involved in the signalling pathway of the BCR-ABL tyrosine kinase.

    FEBS letters 2003;535;1-3;17-22

  • Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.

    Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG and Peters EC

    Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

    The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;2;443-8

  • Bcr and Abl interaction: oncogenic activation of c-Abl by sequestering Bcr.

    Ling X, Ma G, Sun T, Liu J and Arlinghaus RB

    Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. rarlingh@mdanderson.org

    c-Abl tyrosine kinase is under rigorous control because of an unknown cellular inhibitor that maintains c-Abl in a relatively inactive state. Because SH2 domains are positive regulators of the nonreceptor tyrosine kinases, we tested whether this putative inhibitor would bind to an Abl SH2 protein construct and thus activate the c-Abl tyrosine kinase. Expression of a Mr 10,000 Abl SH2 protein in COS-1 and Rat-1 cells activated the tyrosine kinase activity of p145 ABL and induced both morphological transformation and foci formation in Rat-1 cells. Importantly, the R to L mutant of the FLVRES sequence of the Abl SH2 protein also activated the c-Abl tyrosine kinase and induced oncogenic transformation. Addition of the Abl kinase inhibitor STI-571 to ABL SH2-transformed Rat-1 cells inhibited tyrosine phosphorylation of p145 ABL. Overexpression of Bcr has been shown to inhibit the Bcr-Abl oncoprotein, and the endogenous Bcr protein forms a complex with c-Abl in hematopoietic cells and insect cells. Therefore, we determined whether Bcr is the putative c-Abl inhibitor that interacts with the Mr 10,000 Abl SH2 protein. Bcr expression in Rat-1 cells transformed by the Mr 10,000 Abl SH2 protein reduced the activated c-Abl tyrosine kinase activity to near normal levels and reversed the oncogenic effects (morphology changes and foci formation) seen in the Abl SH2-treated cells. We additionally demonstrated that Bcr and the Mr 10,000 Abl SH2 protein are present in a complex. We conclude from these studies that Bcr is a major tyrosine kinase inhibitor of cytoplasmic c-Abl and that procedures that sequester Bcr will release the c-Abl protein from the Bcr/c-Abl complex, which leads to c-Abl oncogenic activation.

    Funded by: NCI NIH HHS: CA16639, CA49639

    Cancer research 2003;63;2;298-303

  • Direct evidence that Bcr-Abl tyrosine kinase activity disrupts normal synergistic interactions between Kit ligand and cytokines in primary primitive progenitor cells.

    Strife A, Wisniewski D, Liu C, Lambek CL, Darzynkiewicz Z, Silver RT and Clarkson B

    Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, New York, NY, USA.

    We previously reported that chronic myelogenous leukemia (CML) primitive granulocyte-monocyte (GM) progenitors have a greatly reduced requirement for kit ligand (KL) to achieve optimal growth with granulocyte colony-stimulating factor (G-CSF) + granulocyte-monocyte colony-stimulating factor (GM-CSF). Conversely, others have demonstrated that unlike normal, CML CD34+ progenitors can proliferate in response to KL as a sole stimulus. To address these seemingly paradoxical findings, we examined the growth responses of CML CD34+ GM progenitors to various cytokines with and without a potent inhibitor of Bcr-Abl tyrosine kinase activity, PD173955. The heightened growth responses of CML GM progenitors to KL alone and to G-CSF + GM-CSF were abrogated by 10 nM PD173955 while having no effect on normal GM progenitors. While normal GM progenitors exhibited the expected synergistic response when KL was added to G-CSF + GM-CSF, CML GM progenitors had a minimal response; however, some synergism was restored by 10 nM PD173955. Normal erythroid progenitors require the synergistic interaction between KL and a saturating amount of erythropoietin (EPO, 1 unit) for optimal growth. In contrast, CML erythroid progenitors had up to 50% of optimal growth in KL alone, and, only a subthreshold amount of EPO (0.1 unit) was needed with KL to achieve 85% of the optimal response; these heightened growth responses were largely abrogated by 10 nM PD173955. Thus, direct evidence is provided that constitutively activated Bcr-Abl kinase pathways in primitive CML progenitors cooperate with single growth factors producing a heightened growth response, and, in so doing, disrupt the normally required synergistic interactions between KL and other cytokines to achieve activation and optimal growth of primitive progenitors. Coupled with our previous findings that a larger than normal proportion of CML primitive progenitors are at a later stage of maturation, we propose that this disruption of normal synergistic responses leads to increased progenitor recruitment into a committed pool by a process of accelerated maturation.

    Funded by: NCI NIH HHS: CA028704-26, CA08748, CA64593, R01 CA028704, R01 CA028704-26

    Molecular cancer research : MCR 2003;1;3;176-85

  • Oncogenic interaction between BCR-ABL and NUP98-HOXA9 demonstrated by the use of an in vitro purging culture system.

    Mayotte N, Roy DC, Yao J, Kroon E and Sauvageau G

    Laboratory of Molecular Genetics of Stem Cells, Clinical Research Institute of Montreal, QC, Canada.

    Chronic myelogenous leukemia (CML) is a clonal stem cell disease caused by the BCR-ABL oncoprotein and is characterized, in its early phase, by excessive accumulation of mature myeloid cells, which eventually leads to acute leukemia. The genetic events involved in CML's progression to acute leukemia remain largely unknown. Recent studies have detected the presence of the NUP98-HOXA9 fusion oncogene in acute leukemia derived from CML patients, which suggests that these 2 oncoproteins may interact and influence CML disease progression. Using in vitro purging of BCR-ABL-transduced mouse bone marrow cells, we can now report that recipients of bone marrow cells engineered to coexpress BCR-ABL with NUP98-HOXA9 develop acute leukemia within 7 to 10 days after transplantation. However, no disease is detected for more than 2 months in mice receiving bone marrow cells expressing either BCR-ABL or NUP98-HOXA9. We also provide evidence of high levels of HOXA9 expressed in leukemic blasts from acute-phase CML patients and that it interacts significantly on a genetic level with BCR-ABL in our in vivo CML model. Together, these studies support a causative, as opposed to a consequential, role for NUP98-HOXA9 (and possibly HOXA9) in CML disease progression.

    Blood 2002;100;12;4177-84

  • Jak2 is involved in c-Myc induction by Bcr-Abl.

    Xie S, Lin H, Sun T and Arlinghaus RB

    Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas, TX 77030, USA.

    We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of Bcr-Abl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr-Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by Bcr-Abl. We treated the 32Dp210 Bcr-Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bbeta is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bbeta. Expression of the SH2-Bbeta R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in c-Myc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC RNA expression but also interferes with proteasome-dependent degradation of c-Myc protein.

    Funded by: NCI NIH HHS: CA 16672, CA 49639

    Oncogene 2002;21;47;7137-46

  • e19a2-positive chronic myeloid leukaemia with BCR exon e16-deleted transcripts.

    Wang L, Seale J, Woodcock BE and Clark RE

    Leukemia 2002;16;8;1562-3

  • A 76-kb duplicon maps close to the BCR gene on chromosome 22 and the ABL gene on chromosome 9: possible involvement in the genesis of the Philadelphia chromosome translocation.

    Saglio G, Storlazzi CT, Giugliano E, Surace C, Anelli L, Rege-Cambrin G, Zagaria A, Jimenez Velasco A, Heiniger A, Scaravaglio P, Torres Gomez A, Roman Gomez J, Archidiacono N, Banfi S and Rocchi M

    Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy. saglio@csi.it

    A patient with a typical form of chronic myeloid leukemia was found to carry a large deletion on the derivative chromosome 9q+ and an unusual BCR-ABL transcript characterized by the insertion, between BCR exon 14 and ABL exon 2, of 126 bp derived from a region located on chromosome 9, 1.4 Mb 5' to ABL. This sequence was contained in the bacterial artificial chromosome RP11-65J3, which in fluorescence in situ hybridization experiments on normal metaphases was found to detect, in addition to the predicted clear signal at 9q34, a faint but distinct signal at 22q11.2, where the BCR gene is located, suggesting the presence of a large region of homology between the two chromosomal regions. Indeed, blast analysis of the RP11-65J3 sequence against the entire human genome revealed the presence of a stretch of homology, about 76 kb long, located approximately 150 kb 3' to the BCR gene, and containing the 126-bp insertion sequence. Evolutionary studies using fluorescence in situ hybridization identified the region as a duplicon, which transposed from the region orthologous to human 9q34 to chromosome 22 after the divergence of orangutan from the human-chimpanzee-gorilla common ancestor about 14 million years ago. Recent sequence analyses have disclosed an unpredicted extensive segmental duplication of our genome, and the impact of duplicons in triggering genomic disorders is becoming more and more apparent. The discovery of a large duplicon relatively close to the ABL and BCR genes and the finding that the 126-bp insertion is very close to the duplicon at 9q34 open the question of the possible involvement of the duplicon in the formation of the Philadelphia chromosome translocation.

    Funded by: Telethon: TGM00Z02, TGM06S01

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;15;9882-7

  • p38 MAPK-mediated activation of NF-kappaB by the RhoGEF domain of Bcr.

    Korus M, Mahon GM, Cheng L and Whitehead IP

    Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey, NJ 07103, USA.

    The oncogenic fusion protein p210 Bcr-Abl is causally associated with virtually all cases of chronic myelogenous leukemia. The wild-type Bcr product has several recognizable structural and functional motifs including a domain that contains guanine nucleotide exchange activity for Rho family GTPases (DH/PH domain). Although this domain is retained within p210 Bcr-Abl, it has no known signaling activities in vivo. Here we report that a fragment of Bcr that encodes the isolated DH/PH domain is a potent activator of the NF-kappaB transcription factor. Within the context of full length Bcr, this activity is regulated by proximal flanking sequences that suppress the DH/PH domain encoded guanine nucleotide exchange activity. NF-kappaB activation by Bcr is not mediated by nuclear translocation, but rather by p38 mitogen-activated protein kinase (MAPK)-dependent modification of the RelA/p65 transactivation domain. Although we were able to demonstrate that Bcr can function as an exchange factor for Cdc42 in vivo, NF-kappaB activation appears to occur via a Cdc42-independent mechanism. These studies constitute direct evidence that the Bcr RhoGEF domain can function in vivo, and identify a new signaling activity that may contribute to the transforming potential of p210 Bcr-Abl.

    Funded by: NCI NIH HHS: CA-77493

    Oncogene 2002;21;30;4601-12

  • Clinical significance of dual color-dual fusion translocation fluorescence in situ hybridization in the detection of bcr/abl fusion gene.

    Wu B, Zhou S, Song L and Liu X

    Department of Hematology, South Hospital, First PLA Medical University, Guangzhou 510515, China.

    Objective: To study the expression of bcr/abl hybridized gene in chronic myeloid leukemia (CML), acute lymphatic leukemia (ALL) and polycythemia vera (PV), and its clinical significance.

    Methods: The bcr/abl hybridized gene of interphase metaphase cells of bone marrow in 67 such patients were investigated with a probe of dual color-dual fusion translocation fluorescence in situ hybridization (D-FISH).

    Results: In 38 CML patients, 34 (89.5%) were positive, with one having a typical t (9; 22) at first, which changed into negative after having been treated with interferon for 38 months. In another patient, 60 days after post-allogeneic peripheral blood stem cell transplantation (PBSCT), the cytomorphology and cytogenetics were in completely remission. But 3% cells were bcr/abl positive as detected by D-FISH. Six (25%) of 24 ALL patients were positive for Bcr/abl fusion gene, which was negative in 2 PV patients. Three patients suspected of having CML were also negative and one of these three was finally diagnosed as suffering from primary thrombocythemia and one, acute myeloid leukemia (M(2a)) as detected by ETO/AML(1) gene, though the other one was still not confirmed. Two (67%) of the 3 bcr/abl negative CML patients and 5 (87%) of the 6 bcr/abl positive ALL patients had refractory leukemia.

    Conclusion: bcr/abl hybridized gene is accurately detected by a probe of dual color-dual fusion translocation fluorescence in situ hybridization, which can serve as an effective index for clinical diagnosis, estimation of prognosis and monitor of minimal residual disease in some hematopathies.

    Zhonghua zhong liu za zhi [Chinese journal of oncology] 2002;24;4;364-6

  • The t(4;22)(q12;q11) in atypical chronic myeloid leukaemia fuses BCR to PDGFRA.

    Baxter EJ, Hochhaus A, Bolufer P, Reiter A, Fernandez JM, Senent L, Cervera J, Moscardo F, Sanz MA and Cross NC

    Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury SP2 8BJ, UK.

    Chronic myeloid leukaemia (CML) is characterized by the presence of the BCR-ABL fusion gene, usually in association with the t(9;22)(q34;q11) translocation. We report here the identification and cloning of a rare variant translocation, t(4;22)(q12;q11), in two patients with a CML-like myeloproliferative disease (MPD). RT-PCR indicated that both patients were negative for BCR-ABL, but FISH analysis suggested that the BCR gene was rearranged. Since other translocations in MPDs frequently involve tyrosine kinases, we designed a multiplex PCR to search for mRNA fusions between BCR and three potential partner genes at 4q12: KIT, KDR and PDGFRA. An unusual inframe BCR-PDGFRA fusion mRNA was identified in both patients, with either BCR exon 7 or exon 12 fused to short BCR intron-derived sequences, which were in turn fused to part of PDGFRA exon 12. Sequencing of the genomic breakpoint junctions showed that the chromosome 22 breakpoints fell in BCR introns whereas the chromosome 4 breakpoints were within PDGFRA exon 12. This is the first report of a fusion gene that involves PDGFRA. Our findings indicate that apparently simple cytogenetic variants of t(9;22) do not always mask a cryptic BCR-ABL fusion, even when found in association with clinical and haematological indications of CML.

    Human molecular genetics 2002;11;12;1391-7

  • Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.

    Kim YJ, Kim DW, Lee S, Kim HJ, Kim YL, Hwang JY, Oh IH, Park YH, Lee YK, Min CK, Kim TG, Han TH, Min WS and Kim CC

    Catholic Hemopoietic Stem Cell Transplantation Center, Catholic University of Korea, #62 Youido-dong, Youngdeungpo-gu, Seoul, Korea (South), 150-713.

    The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.

    European journal of haematology 2002;68;5;272-80

  • Association of Bcr-Abl with the proto-oncogene Vav is implicated in activation of the Rac-1 pathway.

    Bassermann F, Jahn T, Miething C, Seipel P, Bai RY, Coutinho S, Tybulewicz VL, Peschel C and Duyster J

    Department of Internal Medicine III, Laboratory of Leukemogenesis, Technical University of Munich, 81675, Germany.

    Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257-265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominant negative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1.

    The Journal of biological chemistry 2002;277;14;12437-45

  • Structure of the Bcr-Abl oncoprotein oligomerization domain.

    Zhao X, Ghaffari S, Lodish H, Malashkevich VN and Kim PS

    Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, Massachusetts 02142-1401, USA.

    The Bcr-Abl oncoprotein is responsible for a wide range of human leukemias, including most cases of Philadelphia chromosome-positive chronic myelogenous leukemia. Oligomerization of Bcr-Abl is essential for oncogenicity. We determined the crystal structure of the N-terminal oligomerization domain of Bcr-Abl (residues 1-72 or Bcr1-72) and found a novel mode of oligomer formation. Two N-shaped monomers dimerize by swapping N-terminal helices and by forming an antiparallel coiled coil between C-terminal helices. Two dimers then stack onto each other to form a tetramer. The Bcr1-72 structure provides a basis for the design of inhibitors of Bcr-Abl transforming activity by disrupting Bcr-Abl oligomerization.

    Nature structural biology 2002;9;2;117-20

  • Fusion of the BCR and the fibroblast growth factor receptor-1 (FGFR1) genes as a result of t(8;22)(p11;q11) in a myeloproliferative disorder: the first fusion gene involving BCR but not ABL.

    Fioretos T, Panagopoulos I, Lassen C, Swedin A, Billström R, Isaksson M, Strömbeck B, Olofsson T, Mitelman F and Johansson B

    Department of Clinical Genetics, Lund University Hospital, Sweden. Thoas.Fioretos@klingen.lu.se

    Constitutive activation of tyrosine kinases as a consequence of chromosomal translocations, forming fusion genes, plays an important role in the development of hematologic malignancies, in particular, myeloproliferative syndromes (MPSs). In this respect, the t(9;22)(q34;q11) that results in the BCR/ABL fusion gene in chronic myeloid leukemia is one of the best-studied examples. The fibroblast growth factor receptor 1 (FGFR1) gene at 8p11 encodes a transmembrane receptor tyrosine kinase and is similarly activated by chromosomal translocations, in which three alternative genes-ZNF198 at 13q12, CEP110 at 9q34, and FOP at 6q27-become fused to the tyrosine kinase domain of FGFR1. These 8p11-translocations are associated with characteristic morphologic and clinical features, referred to as "8p11 MPS." In this study, we report the isolation and characterization of a novel fusion gene in a hematologic malignancy with a t(8;22)(p11;q11) and features suggestive of 8p11 MPS. We show that the breakpoints in the t(8;22) occur within introns 4 and 8 of the BCR and FGFR1 genes, respectively. On the mRNA level, the t(8;22) results in the fusion of BCR exons 1-4 in-frame with the tyrosine kinase domain of FGFR1 as well as in the expression of a reciprocal FGFR1/BCR chimeric transcript. By analogy with data obtained from previously characterized fusion genes involving FGFR1 and BCR/ABL, it is likely that the oligomerization domain contributed by BCR is critical and that its dimerizing properties lead to aberrant FGFR1 signaling and neoplastic transformation.

    Genes, chromosomes & cancer 2001;32;4;302-10

  • Preparation and application of antibodies to phosphoamino acid sequences.

    Sun T, Campbell M, Gordon W and Arlinghaus RB

    Department of Molecular Pathology, Box 89, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

    In this review, we describe methods to generate and characterize sequence-specific phosphoamino acid antibodies. Several of the early contributions regarding the utility of such antibodies are summarized. Three antiphosphopeptide antibodies derived from sequences of the Bcr protein are described. They are anti-Bcr pSer-354, anti-Bcr pTyr-328, and anti-Bcr pTyr-360. These anti-Bcr phosphopeptide antibodies are directed toward phosphorylated sequences encoded by the first exon of the BCR gene, which is the critical portion of the Bcr sequence present in the Bcr-Abl oncoprotein. Using these antibodies, we established/confirmed the in vivo phosphorylation of Ser-354, Tyr-328, and Tyr-360 in Bcr and Bcr-Abl proteins. The cross-reactivity of these antibodies, which is a common problem with antipeptide antibodies, was also investigated and discussed.

    Funded by: NCI NIH HHS: CA16672, CA49639

    Biopolymers 2001;60;1;61-75

  • Cytoplasmic and nuclear localization of the 130 and 160 kDa Bcr proteins.

    Laurent E, Talpaz M, Wetzler M and Kurzrock R

    Department of Bioimmunotherapy, University of Texas MD Anderson Cancer Center, Houston 77030, USA.

    Formation of the Bcr-Abl chimeric protein is the molecular hallmark of Philadelphia-positive leukemia. Normal Bcr is a complex protein which has been found in the cytoplasm, has serine kinase activity, and has been implicated in cellular signal transduction. However, we have recently demonstrated that Bcr can also associate with condensed chromatin. Since two major Bcr proteins have been characterized (p160Bcr and p130Bcr), we sought to determine if different forms of Bcr localized to the nucleus vs the cytoplasm. Metabolic labeling and Western blotting experiments were performed using nuclear and cytoplasmic extracts of three human Philadelphia-negative leukemia/lymphoma cell lines (KG-1, HL-60, and Jurkat). Both methodologies showed that p160Bcr and p130Bcr localized to the cytoplasm, but the p130 form predominated in the nucleus. These results suggest that Bcr serves both nuclear and cytoplasmic functions, and that different forms of Bcr may be preferentially involved in these distinct activities.

    Leukemia 2000;14;11;1892-7

  • The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase.

    Million RP and Van Etten RA

    The Center for Blood Research, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

    The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl-Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins. (Blood. 2000;96:664-670)

    Funded by: NCI NIH HHS: CA09595, CA57593

    Blood 2000;96;2;664-70

  • Characterization of Ggrb4, an adapter protein interacting with Bcr-Abl.

    Coutinho S, Jahn T, Lewitzky M, Feller S, Hutzler P, Peschel C and Duyster J

    Department of Internal Medicine III, Technical University of Munich; Medical Institute for Radiation and Cell Research, Würzburg, Germany.

    We report here the characterization of an adapter protein identified in a yeast 2-hybrid screen with the use of Bcr-Abl as the bait. Grb4 bound to Bcr-Abl in a variety of systems, both in vitro and in vivo, and is an excellent substrate of the Bcr-Abl tyrosine kinase. The association of Grb4 and Bcr-Abl in intact cells was mediated by an src homology (SH)2-mediated phosphotyrosine-dependent interaction as well as an SH3-mediated phosphotyrosine-independent interaction. Grb4 has 68% homology to the adapter protein Nck and has similar but distinct binding specificities in K562 lysates. Subcellular localization studies indicate that Grb4 localizes to both the nucleus and the cytoplasm. Coexpression of kinase-active Bcr-Abl with Grb4 resulted in the translocation of Grb4 from the cytoplasm and the nucleus to the cytoskeleton to colocalize with Bcr-Abl. In addition, expression of Grb4 with kinase-active Bcr-Abl resulted in a redistribution of actin-associated Bcr-Abl. Finally, coexpression of Grb4 and oncogenic v-Abl strongly inhibited v-Abl-induced AP-1 activation. Together, these data indicate that Grb4 in conjunction with Bcr-Abl may be capable of modulating the cytoskeletal structure and negatively interfering with the signaling of oncogenic Abl kinases. Grb4 may therefore play a role in the molecular pathogenesis of chronic myelogenous leukemia. (Blood. 2000;96:618-624) (Blood. 2000;96:618-624)

    Blood 2000;96;2;618-24

  • Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck.

    Lionberger JM, Wilson MB and Smithgall TE

    Department of Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl.

    Funded by: NCI NIH HHS: CA81398

    The Journal of biological chemistry 2000;275;24;18581-5

  • The c-Fes protein-tyrosine kinase suppresses cytokine-independent outgrowth of myeloid leukemia cells induced by Bcr-Abl.

    Lionberger JM and Smithgall TE

    Eppley Institute for Research in Cancer and Department of Pathology and Microbiology University of Nebraska Medical Center, Omaha 68198, USA.

    The c-Fes protein-tyrosine kinase exhibits strong expression in myeloid hematopoietic cells. Previous studies have shown that Fes induces differentiation in the chronic myelogenous leukemia-derived cell line K-562, suggesting that the Fes signal for differentiation is dominant to the Bcr-Abl signal for transformation in these cells. In addition, Fes has been shown to associate with and phosphorylate Bcr on NH2-terminal sequences retained within Bcr-Abl. To determine whether Fes interacts directly with Bcr-Abl, kinase-inactive Bcr-Abl was coexpressed with Fes in 293T cells, and phosphorylation was assessed by anti-phosphotyrosine immunoblotting. Bcr-Abl was strongly phosphorylated by Fes under these conditions, suggestive of direct interaction. Similarly, tyrosine phosphorylation of kinase-inactive Fes was observed after coexpression with active Bcr-Abl. To test for the interaction of Fes with Bcr-Abl under physiological conditions, wild-type and kinase-defective Fes were stably expressed in the cytokine-dependent myeloid leukemia cell line, DAGM. Expression of either form of Fes alone did not affect the proliferation or interleukin 3 dependence of these cells. The DAGM/Fes cells were then infected with Bcr-Abl retroviruses, and their rates of cytokine-independent outgrowth were compared. Fes dramatically suppressed Bcr-Abl-induced DAGM cell outgrowth relative to a cell line expressing beta-galactosidase as a negative control. This effect required Fes tyrosine kinase activity, because the kinase-inactive form of Fes did not affect Bcr-Abl-induced cell outgrowth. The phosphotyrosine content of both wild-type and kinase-inactive Fes was strongly enhanced after coexpression with Bcr-Abl in DAGM cells, similar to the 293T result. Phosphorylation of wild-type Fes correlated with stimulation of Fes tyrosine kinase activity in the presence of Bcr-Abl. These results show that Fes and Bcr-Abl interact in myeloid cells, leading to Fes activation and suppression of Bcr-Abl-induced conversion to cytokine independence.

    Funded by: NCI NIH HHS: CA58667

    Cancer research 2000;60;4;1097-103

  • Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

    Scanlan MJ, Gordan JD, Williamson B, Stockert E, Bander NH, Jongeneel V, Gure AO, Jäger D, Jäger E, Knuth A, Chen YT and Old LJ

    Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. scanlanm@mskcc.org

    The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.

    International journal of cancer 1999;83;4;456-64

  • CRKL binding to BCR-ABL and BCR-ABL transformation.

    Kolibaba KS, Bhat A, Heaney C, Oda T and Druker BJ

    Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland 97201, USA.

    The SH2-SH3 domain-containing adaptor protein CRKL is the predominant tyrosine phosphorylated protein in chronic myelogenous leukemia (CML) neutrophils and BCR-ABL-expressing cell lines. The amino terminal CRKL SH3 domain binds directly to a proline-rich region in the C-terminus of BCR-ABL. BCR-ABL mutants with deletions of this region were constructed to assess biologic effects of eliminating the CRKL binding site. Yeast two-hybrid analysis and gel overlay assays show eradication of the direct interaction of CRKL with BCR-ABL in the proline deletion mutants. However, these BCR-ABL mutants transform myeloid cells to growth factor independence, and in these cells CRKL is tyrosine phosphorylated and associates with BCR-ABL. These findings suggest both direct and indirect interactions of CRKL with BCR-ABL. Thus, disruption of the direct interaction with BCR-ABL has not excluded a role for CRKL in BCR-ABL-mediated transformation.

    Leukemia & lymphoma 1999;33;1-2;119-26

  • GCKR links the Bcr-Abl oncogene and Ras to the stress-activated protein kinase pathway.

    Shi CS, Tuscano JM, Witte ON and Kehrl JH

    B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892-1876, USA.

    The Bcr-Abl oncogene, found in Philadelphia chromosome-positive myelogenous leukemia (CML), activates Ras and triggers the stress-activated protein kinase (SAPK or Jun NH2-terminal kinase [JNK]) pathway. Interruption of Ras or SAPK activation dramatically reduces Bcr-Abl-mediated transformation. Here, we report that Bcr-Abl through a Ras-dependent pathway signals the serine/threonine protein kinase GCKR (Germinal Center Kinase Related) leading to SAPK activation. Either an oncogenic form of Ras or Bcr-Abl enhances GCKR catalytic activity and its activation of SAPK, whereas inhibition of GCKR impairs Bcr-Abl-induced SAPK activation. Bcr-Abl mutants that are impaired for GCKR activation are also unable to activate SAPK. Consistent with GCKR being a functional target in CML, GCKR is constitutively active in CML cell lines and found in association with Bcr-Abl. Our results indicate that GCKR is a downstream target of Bcr-Abl and strongly implicate GCKR as a mediator of Bcr-Abl in its transformation of cells.

    Blood 1999;93;4;1338-45

  • The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein.

    Takeda N, Shibuya M and Maru Y

    Department of Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan.

    The previously uncharacterized CDC24 homology domain of BCR, which is missing in the P185 BCR-ABL oncogene of Philadelphia chromosome (Ph1)-positive acute lymphocytic leukemia but is retained in P210 BCR-ABL of chronic myelogeneous leukemia, was found to bind to the xeroderma pigmentosum group B protein (XPB). The binding appeared to be required for XPB to be tyrosine-phosphorylated by BCR-ABL. The interaction not only reduced both the ATPase and the helicase activities of XPB purified in the baculovirus system but also impaired XPB-mediated cross-complementation of the repair deficiency in rodent UV-sensitive mutants of group 3. The persistent dysfunction of XPB may in part underlie genomic instability in blastic crisis.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;1;203-7

  • Syp Y279, Y304 Can Mediate the Binding of Bcr-Abl to Grb2 and Other Proteins.

    Zhu K, Liu XP, Zeng G and Ye FY

    Clinic Lab./ Research Center, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. zhuk@cesmtp.ccf.org

    In this study we obtained 3 mutants of Syp cDNA Y279F, Y304F and Y546F by using the method of site-directed mutation in vitro. We then inserted them into the pXM mammalian expression vector, and transferred the plasmids into K562 cells. Western Blot has found WT/ 3MT Syp are expressed in the K562 cells. Immunoprecipitation and Immunoblot have found that WT, Y279F, Y304F and Y546F Syp can bind directly to the Bcr-Abl in vivo. However, we found that mutation of Syp Y279F can block the binding of Grb2 SH2 to Syp mutation of Syp Y304F can block the binding of Shc SH2 to Syp in vitro. As a adaptor, Syp can mediate binding of Bcr-Abl to Shc and Grb2.Grb2 bind to Syp pY279, Shc bind to Syp pY304 in vitro.

    Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 1999;31;4;448-450

  • Gads is a novel SH2 and SH3 domain-containing adaptor protein that binds to tyrosine-phosphorylated Shc.

    Liu SK and McGlade CJ

    Department of Medical Biophysics, Ontario Cancer Institute, AMGEN Institute, University of Toronto, Canada.

    Shc proteins are important substrates of receptor and cytoplasmic tyrosine kinases that couple activated receptors to downstream signaling enzymes. Phosphorylation of Shc tyrosine residues 239 and 317 leads to recruitment of the Grb2-Sos complex, thus linking Shc phosphorylation to Ras activation. We have used phosphorylated peptides corresponding to the regions spanning tyrosine 239/240 and 317 of Shc in an expression library screen to identify additional downstream targets of Shc. Here we report the identification of Gads, a novel adaptor protein most similar to Grb2 and Grap that contains amino and carboxy terminal SH3 domains flanking a central SH2 domain and a 120 amino acid unique region. Gads is most highly expressed in the thymus and spleen of adult animals and in human leukemic cell lines. The binding specificity of the Gads SH2 domain is similar to Grb2 and mediates the interaction of Gads with Shc, Bcr-Abl and c-kit. Gads does not interact with Sos, Cbl or Sam68, although the isolated carboxy terminal Gads SH3 domain is able to bind these molecules in vitro. Our results suggest that the unique structure of Gads regulates its interaction with downstream SH3 domain-binding proteins and that Gads may function to couple tyrosine-phosphorylated proteins such as Shc, Bcr-Abl and activated receptor tyrosine kinases to downstream effectors distinct from Sos and Ras.

    Oncogene 1998;17;24;3073-82

  • Regulation of Bcr-Abl-induced SAP kinase activity and transformation by the SHPTP1 protein tyrosine phosphatase.

    Liedtke M, Pandey P, Kumar S, Kharbanda S and Kufe D

    Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The oncogenic Bcr-Abl variant of the c-Abl tyrosine kinase transforms cells by a mechanism dependent on activation of the stress-activated protein kinase (SAPK). Other work has shown that c-Abl interacts with the SHPTP1 protein tyrosine phosphatase in induction of SAPK activity by genotoxic stress. The present studies demonstrate that Bcr-Abl binds constitutively to SHPTP1. We show that Bcr-Abl phosphorylates SHPTP1 on C-terminal Y536 and Y564 sites. The functional significance of the Bcr-Abl/SHPTP1 interaction is supported by the finding that SHPTP1 regulates Bcr-Abl-induced SAPK activity. Importantly, SHPTP1 also decreases Bcr-Abl-dependent transformation of fibroblasts. These findings indicate that SHPTP1 functions as a tumor suppressor in cells transformed by Bcr-Abl.

    Funded by: NCI NIH HHS: CA66996

    Oncogene 1998;17;15;1889-92

  • The SH2-containing adapter protein GRB10 interacts with BCR-ABL.

    Bai RY, Jahn T, Schrem S, Munzert G, Weidner KM, Wang JY and Duyster J

    Department of Internal Medicine III, Technical University of Munich, Germany.

    Bcr-Abl is an oncogenic tyrosine kinase expressed in tumor cells of CML and a subset of ALL which in its unregulated and activated state is thought to cause cell transformation and leukemia. Bcr-Abl contains several autophosphorylation sites which serve as potential docking sites for SH2-containing signaling molecules. Mutational analysis has indicated that these autophosphorylation sites play a critical role in the transforming capability of Bcr-Abl. It has been shown that the SH2-containing adapter protein Grb2 binds to the autophosphorylation site Tyr(p)177 whereby it couples Bcr-Abl to the Ras pathway. The biological consequences of this interaction, however, are presently unclear. A Tyr177-mutated Bcr-Abl which lacks the ability to interact with the Grb2-SH2 domain still transforms myeloid cells and generates tumors in nude mice. We performed a yeast two-hybrid screen to identify signaling proteins which bind to distinct Bcr-Abl autophosphorylation sites. Autophosphorylation of Bcr-Abl in yeast was accomplished by using the DNA binding protein LexA which permits dimerization and crossphosphorylation of the fused bait. Using a LexA-Bcr-Abl full length fusion protein as bait, we identified several SH2-containing proteins. Among them we confirmed molecules already shown by others to interact with Bcr-Abl, in vivo, including Grb2, PI-3-kinase and Crk indicating that dimerization in yeast leads to autophosphorylation of tyrosine residues crucial for Bcr-Abl signaling in vivo. More importantly, we identified the SH2-containing protein Grb10 as a new binding partner for Bcr-Abl. This binding occurs in a phosphotyrosine-dependent manner at Bcr sites of Bcr-Abl. Both Abl and Bcr alone, as well as a kinase-defective Bcr-Abl, failed to interact with Grb10 in yeast. Mutational analysis uncovered a new SH2 binding site in Bcr-Abl located between Bcr aa242-446, which is different from the Grb2 binding site. Binding could be demonstrated in vitro and also in vivo as shown by co-immunoprecipitation analysis in CML cells. Using a temperature sensitive Bcr-Abl stably overexpressed in hematopoetic cells, we demonstrated that complex formation of Grb10 with Bcr-Abl was kinase activation-dependent in vivo. Notably, a Bcr-Abl mutant protein (Bcr/1-242-Abl) which lacks the ability to interact with Grb10 partially alleviated IL-3 dependence of Ba/F3 cells, indicating that the Grb10/Bcr-Abl interaction is important for Bcr-Abl-induced IL-3 independence of Ba/F3 cells. In addition, the Bcr/1-242-Abl mutant has a reduced capacity to induce focus formation in fibroblasts.

    Funded by: NCI NIH HHS: R01 CA043054, R37 CA043054

    Oncogene 1998;17;8;941-8

  • Protein tyrosine phosphatase 1B antagonizes signalling by oncoprotein tyrosine kinase p210 bcr-abl in vivo.

    LaMontagne KR, Flint AJ, Franza BR, Pandergast AM and Tonks NK

    Cold Spring Harbor Laboratory, New York 11724-2208, USA.

    The p210 bcr-abl protein tyrosine kinase (PTK) appears to be directly responsible for the initial manifestations of chronic myelogenous leukemia (CML). In contrast to the extensive characterization of the PTK and its effects on cell function, relatively little is known about the nature of the protein tyrosine phosphatases (PTPs) that may modulate p210 bcr-abl-induced signalling. In this study, we have demonstrated that expression of PTP1B is enhanced specifically in various cells expressing p210 bcr-abl, including a cell line derived from a patient with CML. This effect on expression of PTP1B required the kinase activity of p210 bcr-abl and occurred rapidly, concomitant with maximal activation of a temperature-sensitive mutant of the PTK. The effect is apparently specific for PTP1B since, among several PTPs tested, we detected no change in the levels of TCPTP, the closest relative of PTP1B. We have developed a strategy for identification of physiological substrates of individual PTPs which utilizes substrate-trapping mutant forms of the enzymes that retain the ability to bind to substrate but fail to catalyze efficient dephosphorylation. We have observed association between a substrate-trapping mutant of PTP1B (PTP1B-D181A) and p210 bcr-abl, but not v-Abl, in a cellular context. Consistent with the trapping data, we observed dephosphorylation of p210 bcr-abl, but not v-Abl, by PTP1B in vivo. We have demonstrated that PTP1B inhibited binding of the adapter protein Grb2 to p210 bcr-abl and suppressed p210 bcr-abl-induced transcriptional activation that is dependent on Ras. These results illustrate selectivity in the effects of PTPs in a cellular context and suggest that PTP1B may function as a specific, negative regulator of p210 bcr-abl signalling in vivo.

    Funded by: NCI NIH HHS: CA61033, CA64593, P01 CA064593

    Molecular and cellular biology 1998;18;5;2965-75

  • Interaction of Rac1 with GTPase-activating proteins and putative effectors. A comparison with Cdc42 and RhoA.

    Zhang B, Chernoff J and Zheng Y

    Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.

    The intrinsic GTPase activity of the Rho family GTP-binding protein Rac1 is drastically stimulated upon interaction with its GTPase-activating proteins (GAPs) and is significantly inhibited when coupled to certain effector targets such as the p21-activated kinases (PAKs) and IQGAPs. Here we have characterized the interaction of Rac1 with a panel of mammalian GAPs and putative effectors by measuring the kinetic and binding parameters involved and made comparisons with similar interactions for Cdc42 and RhoA. In contrast with Cdc42 (for which the GAP domain of p50RhoGAP is 50-fold more efficient than those of p190, Bcr, and 3BP-1) and with RhoA (toward which only p50RhoGAP and p190 displayed high efficiencies), the catalytic efficiencies (Kcat/Km) of the GAP domains of p50RhoGAP, p190, Bcr, and 3BP-1 on Rac1 are found to be comparable in a range between 0.9 and 2.6 min-1 microM-1. However, similar to the cases of Cdc42 and RhoA, the Km values of the GAP domains on Rac1 compare well to the binding affinity to the guanylyl imidodiphosphate-bound Rac1, which ranges from 10.5 to 40.5 microM, suggesting a rapid equilibrium reaction mechanism. The dissociation constants of the p21-binding domains of PAK1, PAK2, and the RasGAP-related domain of IQGAP1, which all cause significant reduction of the intrinsic rate of GTP hydrolysis upon binding to Rac1-GTP, are found to be 0.71, 0.26, and 2.13 microM for Rac1-GTP, compared with that determined for Cdc42-GTP at 2.9, 20.5, and 0.39 microM, respectively, under similar conditions. These results suggest that p50RhoGAP, p190, Bcr, and 3BP-1 are all capable of acting as a negative regulator for Rac1-mediated signaling, and that, although PAK1 and IQGAP1 can couple tightly with both Rac1 and Cdc42, PAK2 is likely to be a specific effector for Rac1 instead of Cdc42.

    Funded by: NIGMS NIH HHS: GM53943

    The Journal of biological chemistry 1998;273;15;8776-82

  • Requirement of two specific tyrosine residues for the catalytic activity of Bcr serine/threonine kinase.

    Wu Y, Liu J and Arlinghaus RB

    Department of Molecular Pathology, The University of Texas, MD Anderson Cancer Center, Houston 77030, USA.

    Bcr is a novel serine/threonine protein kinase that is believed to require two cysteine pairs for activity (Maru and Witte, Cell, 67, 459, 1991). Tyrosine phosphorylated Bcr has dramatically reduced kinase activity, and tyrosine 360 of Bcr, which is one of the sites of phosphorylation by the Bcr-Abl oncoprotein, is required for transkinase activity (Liu et al., Mol. Cell Biol., 16, 998, 1996). Results presented here indicate that Bcr tyrosine 328 is also phosphorylated within Bcr-Abl expressing cells and is required for Bcr's serine/threonine kinase activity. Bcr Y328F, like Bcr Y360F, had defective transkinase activity but can autophosphorylate. However, the Y328F/Y360F double mutant of Bcr is defective in both trans- and autokinase activities. Taken together with the kinase inhibitory effects of tyrosine phosphorylation of Bcr by Bcr-Abl, our studies with tyrosine to phenylalanine Bcr mutants indicate that the hydroxyl residues of tyrosines 328 and 360 play crucial roles in Bcr's kinase activity.

    Funded by: NCI NIH HHS: CA16672, CA65611

    Oncogene 1998;16;1;141-6

  • The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr.

    Warmuth M, Bergmann M, Priess A, Häuslmann K, Emmerich B and Hallek M

    Medizinische Klinik, Klinikum Innenstadt, Universität München, D-80336 München, Germany.

    bcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl.

    The Journal of biological chemistry 1997;272;52;33260-70

  • Interaction of growth hormone-activated STATs with SH2-containing phosphotyrosine phosphatase SHP-1 and nuclear JAK2 tyrosine kinase.

    Ram PA and Waxman DJ

    Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215, USA.

    Growth hormone (GH) rapidly stimulates tyrosine phosphorylation followed by serine/threonine phosphorylation of multiple cytoplasmic STAT transcription factors, including one, STAT5b, that is uniquely responsive to the temporal pattern of plasma GH stimulation in rat liver and is proposed to play a central role in the activation of male-expressed liver genes by GH pulses in vivo (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262-13270). We now show that JAK2, the GH receptor-associated tyrosine kinase, is present both in the cytosol and in the nucleus in cultured liver cells and in rat liver in vivo and that GH-activated STAT3 but not STAT5b becomes associated with nuclear JAK2. GH is also shown to activate by 3-4-fold SHP-1, a phosphotyrosine phosphatase that contains two src homology 2 (SH2) domains. GH also induces nuclear translocation and binding of SHP-1 to tyrosine-phosphorylated STAT5b, suggesting that this GH-activated phosphatase may play a role in dephosphorylation leading to deactivation of nuclear STAT5b following the termination of a plasma GH pulse in male rat liver in vivo. No such association of SHP-1 with GH-activated STAT3 was detected, a finding that could help explain the marked desensitization of STAT3, but not STAT5b, to subsequent GH pulses following an initial GH activation event.

    The Journal of biological chemistry 1997;272;28;17694-702

  • Bcr phosphorylated on tyrosine 177 binds Grb2.

    Ma G, Lu D, Wu Y, Liu J and Arlinghaus RB

    Department of Molecular Pathology, The University of Texas, M.D. Anderson Cancer Centre, Houston 77030, USA.

    We and others have shown that the Bcr-Abl oncoprotein binds activators of the Ras pathway such as Grb2 and Shc. Grb2 binding is mediated through a phosphorylated tyrosine residue (Y177) located within a consensus Grb2 binding site encoded by the first exon of the BCR gene. Our results indicate that P160 BCR is tyrosine phosphorylated at the same site by Bcr-Abl in kinase assays (Puil et al., 1994). We performed experiments to determine whether Bcr, which was tyrosine phosphorylated within cells by activated c-Abl, could also bind Grb2, and whether phosphotyrosine 177 was the major binding site. Complexes between Bcr and Abl were detected in a hemopoietic cell line lacking Bcr-Abl and in COS1 cells coexpressing both Bcr and Abl proteins. P160 BCR was tyrosine phosphorylated in COS1 cells coexpressing Abl and Bcr proteins. Similarly, various deletion mutants of Bcr including BCRN553, BCRN413 and BCRN221 were tyrosine phosphorylated by activated c-Abl whereas BCRN159 was not. Wild-type Bcr and Bcr Y177F were examined under these conditions for their ability to co-precipitate with Grb2. The results showed that while wild-type tyrosine phosphorylated Bcr efficiently bound Grb2, tyrosine phosphorylated Bcr Y177F had greatly reduced Grb2-binding ability. Studies with GST-SH2 (Grb2) revealed that tyrosine phosphorylated Bcr was able to bind to GST SH2 (Grb2) but tyrosine phosphorylated Bcr Y177F was deficient in binding. These results indicate that the Bcr protein when phosphorylated at tyrosine 177 binds Grb2, thereby implicating Bcr as a potantial activator of the Ras pathway.

    Funded by: NCI NIH HHS: CA16672, CA65611

    Oncogene 1997;14;19;2367-72

  • P210 Bcr-Abl interacts with the interleukin-3 beta c subunit and constitutively activates Jak2.

    Wilson-Rawls J, Liu J, Laneuville P and Arlinghaus RB

    Department of Molecular Pathology, University of Texas, M. D. Anderson Cancer Center, Houston 77030, USA.

    Chronic myelogenous leukemia is a neoplasm of pluripotent hematopoietic cells. Cytokines such as interleukin-3 and granulocyte-macrophage colony-stimulating factor regulate the growth and differentiation of hematopoietic precursors. These cytokines activate two distinct signals to the nucleus. One signal is through the Ras pathway, and the second involves activation of Jak2. We demonstrated that Bcr-Abl co-immunoprecipitates with and constitutively phosphorylates the common beta c chain of the interleukin-3 (IL-3) and granulocyte-macrophage-macrophage colony-stimulating factor (GM-CSF) receptors. Our data show that formation of this complex leads to the constitutive activation of Jak2. Previously, it has been demonstrated that Bcr-Abl interacts with Grb2 and Shc, which in turn activates the Ras pathway. Thus, Bcr-Abl can activate signalling through both pathways in a factor-independent fashion.

    Leukemia 1997;11 Suppl 3;428-31

  • Tyrosine 207 in CRKL is the BCR/ABL phosphorylation site.

    de Jong R, ten Hoeve J, Heisterkamp N and Groffen J

    Department of Pathology, Childrens Hospital of Los Angeles, CA 90027, USA.

    BCR/ABL has a causal role in Philadelphia (Ph)-chromosome positive leukemia. The SH2/SH3 adapter protein CRKL is a major substrate of the deregulated BCR/ABL tyrosine kinase and is aberrantly tyrosine-phosphorylated in Ph-positive leukemia cells. In this study, experiments were pursued to identify and analyse the CRKL phosphorylation site(s). In an in vitro kinase assay, CRKL phosphorylation by the abl kinase was limited to a small region between the two CRKL SH3 domains. Within this region, mutation of tyrosine residue 207 yielded a mutant CRKL which could not be phosphorylated by BCR/ABL. Stable overexpression of CRKL or CRKL-Y207F did not transform NIH3T3 cells, while the Y207F mutation eliminated tyrosine-phosphorylation of CRKL. These studies indicate that Y207 in CRKL represents the major in vivo phosphorylation site. Phosphorylation of Y207 provides a binding site for the CRKL SH2 domain and potentially for other SH2-containing proteins. The Y207F mutation in CRKL did not enhance or decrease association with various target signalling proteins, including SOS or C3G, which interact specifically with the CRKL N-SH3 domain. These findings suggest that complex formation with cellular targets is not modulated by CRKL tyrosine-phosphorylation.

    Funded by: NCI NIH HHS: CA 47456

    Oncogene 1997;14;5;507-13

  • Interaction of BCR-ABL with the retinoblastoma protein in Philadelphia chromosome-positive cell lines.

    Miyamura T, Nishimura J, Yufu Y and Nawata H

    Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.

    The tyrosine kinase activity of BCR-ABL fusion proteins plays an important role in the pathogenesis of leukemia that is for the Philadelphia chromosome (Ph1). Because nuclear c-ABL is regulated during the cell cycle through a specific interaction with the retinoblastoma protein (pRB), the possible interaction of BCR-ABL with pRB in Ph1-positive cell lines was investigated. P145 c-ABL as well as P190 and P210 BCR-ABL proteins interacted with pRB. Furthermore, c-ABL and BCR-ABL associated with both phosphorylated and nonphosphorylated forms of pRB. These findings suggest that BCR-ABL interferes with pRB function and thereby regulates cell growth.

    International journal of hematology 1997;65;2;115-21

  • Direct binding of CRKL to BCR-ABL is not required for BCR-ABL transformation.

    Heaney C, Kolibaba K, Bhat A, Oda T, Ohno S, Fanning S and Druker BJ

    Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland 97201, USA.

    CRKL has previously been shown to be a major tyrosine phosphorylated protein in neutrophils of patients with BCR-ABL+ chronic myelogenous leukemia and in cell lines expressing BCR-ABL CRKL and BCR-ABL form a complex as demonstrated by coimmunoprecipitation and are capable of a direct interaction in a yeast two-hybrid assay. We have mapped the site of interaction of CRKL and BCR-ABL to the amino terminal SH3 domain of CRKL with a proline rich region in the C-terminus of ABL. The proline-rich region was mutated and the effect of this deletion on BCR-ABL transforming function was assayed. Our data show that this deletion does not impair the ability of BCR-ABL to render myeloid cells factor independent for growth. In cells expressing the proline deletion mutation of BCR-ABL, CRKL is still tyrosine phosphorylated and forms a complex with BCR-ABL as demonstrated by coimmunoprecipitation. Our data suggest that the interaction between CRKL and the proline deletion mutant of BCR-ABL is an indirect interaction as CRKL does not interact directly with the proline deletion mutant of BCR-ABL in a gel overlay assay or in a yeast two-hybrid assay. Thus, a direct interaction of CRKL and BCR-ABL is not required for CRKL to become tyrosine phosphorylated by BCR-ABL and suggests that CRKL function may still be required for BCR-ABL function through an indirect interaction.

    Funded by: NCI NIH HHS: CA65823

    Blood 1997;89;1;297-306

  • Co-expression with BCR induces activation of the FES tyrosine kinase and phosphorylation of specific N-terminal BCR tyrosine residues.

    Li J and Smithgall TE

    Eppley Institute for Research in Cancer and Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA. tsmithga@unmc.edu

    The human BCR gene encodes a protein with serine/threonine kinase activity and regulatory domains for the small G-proteins RAC and CDC42. Previous work in our laboratory has established that BCR is a substrate for c-FES, a non-receptor tyrosine kinase linked to myeloid growth and differentiation. Tyrosine phosphorylation led to the association of BCR with the RAS guanine nucleotide exchange complex GRB2-SOS in vivo via the GRB2 SH2 domain, linking BCR to RAS signaling (Maru, Y., Peters, K. L., Afar, D. E. H., Shibuya, M., Witte, O. N., and Smithgall, T. E. (1995) Mol. Cell. Biol. 15, 835-842). In the present study, we demonstrate that BCR Tyr-246 and at least one of the closely spaced tyrosine residues, Tyr-279, Tyr-283, and Tyr-289 (3Y cluster), are phosphorylated by FES both in vitro and in 32Pi-labeled cells. Mutagenesis of BCR Tyr-177 to Phe completely abolished FES-induced BCR binding to the GRB2 SH2 domain, identifying Tyr-177 as an additional phosphorylation site for FES. Co-expression of BCR and FES in human 293T cells stimulated the tyrosine autophosphorylation of FES. By contrast, tyrosine phosphorylation of BCR by FES suppressed BCR serine/threonine kinase activity toward the 14-3-3 protein and BCR substrate, BAP-1. These data show that tyrosine phosphorylation by FES affects the interaction of BCR with multiple signaling partners and suggest a general role for BCR in non-receptor protein-tyrosine kinase regulation and signal transduction.

    Funded by: NCI NIH HHS: CA58667, P30 CA36727

    The Journal of biological chemistry 1996;271;51;32930-6

  • In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

    Bewarder N, Weinrich V, Budde P, Hartmann D, Flaswinkel H, Reth M and Frey J

    Biochemie II, Fakultät für Chemie, Universität Bielefeld, Germany.

    Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.

    Molecular and cellular biology 1996;16;9;4735-43

  • Interaction of the receptor tyrosine kinase p145c-kit with the p210bcr/abl kinase in myeloid cells.

    Hallek M, Danhauser-Riedl S, Herbst R, Warmuth M, Winkler A, Kolb HJ, Druker B, Griffin JD, Emmerich B and Ullrich A

    Medizinische Klinik, Klinikum Innenstadt, Universität München, Germany.

    The chimaeric bcr/abl oncogene is detected in virtually all cases of chronic myelogenous leukaemia (CML). It encodes a constitutively active tyrosine kinase of 210 kDalton, p210bcr/abl, which stimulates a variety of cytosolic signalling intermediates. The effects of bcr/abl on the activity of growth factor receptors are less well known. In order to investigate interaction of p210bcr/abl with the receptor tyrosine kinase p145c-kit, we used two myeloid, factor-dependent cell lines, MO7 and 32D, to generate bcr/abl positive sublines, MO7p210 and 32Dp210, by transfection with the bcr/abl gene. Since 32D and 32Dp210 cells did not express p145c-kit, a c-kit retrovirus was used to generate c-kit positive cell lines (32Dkit, 32Dp210kit). In contrast to MO7 and 32Dkit cells, MO7p210 and 32Dp210kit cells were factor independent and did not respond to the growth-promoting effects of recombinant human Steel factor (rhSF). Preincubation with a monoclonal antibody (MAb) neutralizing the binding of SF to p145c-kit did not affect the growth of MO7p210 cells, thus eliminating the possibility of an autocrine SF secretion. 32Dkit cells transfected with bcr/abl containing an inactivating point mutation (Lys-->Arg271) in the Abl kinase domain (32Dp210(Arg271)kit) retained their responsiveness to the effects of rhSF. Immune complex kinase assays showed that the kinase activity of p145c-kit was several-fold higher in MO7p210 and 32Dp210kit cells than in MO7, 32Dkit and 32Dp210(Arg271)kit cells, suggesting that Abl kinase activity was necessary to activate p145c-kit. Co-immunoprecipitation experiments with anti-Kit and anti-Abl MAbs demonstrated that p145c-kit and p210bcr/abl were associated in an intracellular complex in human bcr/abl positive, c-kit positive cell lines (MO7p210; GM/SO). Finally, colony assays with bone marrow from bcr/abl positive CML patients showed that the haemopoietic progenitors of three of four patients did not respond to rhSF. Taken together, the results suggest that p145c-kit can be activated by p210bcr/abl via an Abl-kinase dependent mechanism involving the complex formation of both proteins. These findings could explain some clinical features (basophilia, increase of immature myeloid cells) of chronic-phase CML.

    British journal of haematology 1996;94;1;5-16

  • Inhibition of Bcr serine kinase by tyrosine phosphorylation.

    Liu J, Wu Y, Ma GZ, Lu D, Haataja L, Heisterkamp N, Groffen J and Arlinghaus RB

    Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, USA.

    The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired.

    Funded by: NCI NIH HHS: CA16672, CA50248, CA65611

    Molecular and cellular biology 1996;16;3;998-1005

  • p210BCR/ABL induces formation of complexes containing focal adhesion proteins and the protooncogene product p120c-Cbl.

    Salgia R, Sattler M, Pisick E, Li JL and Griffin JD

    Division of Hematologic Malignacies, Dana-Farber Cancer Institute, Boston, MA 02115.

    Chronic myelogenous leukemia (CML) is a myeloproliferative disorder caused by the t(9;22) translocation. This translocation creates a unique tyrosine kinase oncogene, bcr/abl, whose product, p210BCR/ABL, is localized to the actin cytoskeleton. One of the major tyrosine phosphoproteins in cells transformed by p210BCR/ABL is the protooncoprotein p120c-Cbl. We have previously shown that p210BCR/ABL induces formation of a multimeric complex of proteins which include p120c-Cbl, phosphotidylinositol-3' kinase, and p210BCR/ABL itself. Here we show that certain focal adhesion proteins are also part of this complex, including paxillin and talin. The sites in paxillin required to bind to p120c-Cbl in this complex have been partially mapped. The interaction of pl20c-Cbl with paxillin is specific, since other focal adhesion proteins, such as p125FAK, vinculin, and alpha-actinin, are not in this complex. The binding of p120c-Cbl to the focal adhesion protein paxillin could contribute to the known adhesive defects of CML cells.

    Funded by: NCI NIH HHS: CA36167, CA60821

    Experimental hematology 1996;24;2;310-3

  • Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells.

    Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ, Wen SC, Zon G, Gewirtz AM, Perussia B and Calabretta B

    Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

    The BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukemic phenotype of Philadelphia chromosome (Ph1)-positive cells. Phosphatidylinositol-3 (PI-3) kinase is known to interact with and be activated by receptor and nonreceptor tyrosine kinases. We investigated whether PI-3 kinase associates with and/or is regulated by BCR/ABL, whether this interaction is functionally significant for Ph1 cell proliferation, and, if so, whether inhibition of PI-3 kinase activity can be exploited to eliminate Ph1-positive cells from bone marrow. We show that the p85 alpha subunit of PI-3 kinase associates with BCR/ABL and that transient expression of BCR/ABL in fibroblasts and down-regulation of BCR/ABL expression using antisense oligodeoxynucleotides (ODNs) in Ph1 cells activates and inhibits, respectively, PI-3 kinase enzymatic activity. The use of specific ODNs or antisense constructs to downregulate p85 alpha expression showed a requirement for p85 alpha subunit in the proliferation of BCR/ABL-dependent cell lines and chronic myelogenous leukemia (CML) primary cells. Similarly, wortmannin, a specific inhibitor of the enzymatic activity of the p110 subunit of PI-3 kinase, inhibited growth of these cells. The growth of normal bone marrow and erythromyeloid, but not megakaryocyte, progenitors was inhibited by p85 alpha antisense [S]ODNs, but wortmannin, at the concentrations tested, did not affect normal hematopoiesis. The proliferation of two BCR/ABL- and growth factor-independent cell lines was not affected by downregulation of the expression of the p85 alpha subunit or inhibition of p110 enzymatic activity, confirming the specificity of the observed effects on Ph1 cells. Thus, PI-3 kinase is one of the downstream effectors of BCR/ABL tyrosine kinase in CML cells. Moreover, reverse transcriptase-polymerase chain reaction performed on single colonies to detect BCR-ABL transcripts showed that wortmannin was able to eliminate selectively CML-blast crisis cells from a mixture of normal bone marrow and Ph1 cells.

    Funded by: NCI NIH HHS: CA-37155, CA-45284, CA-46782

    Blood 1995;86;2;726-36

  • Sequence and analysis of the human ABL gene, the BCR gene, and regions involved in the Philadelphia chromosomal translocation.

    Chissoe SL, Bodenteich A, Wang YF, Wang YP, Burian D, Clifton SW, Crabtree J, Freeman A, Iyer K, Jian L et al.

    University of Oklahoma, Department of Chemistry and Biochemistry, Norman 73019, USA.

    The complete human BCR gene (152-141 nt) on chromosome 22 and greater than 80% of the human ABL gene (179-512 nt) on chromosome 9 have been sequenced from mapped cosmid and plasmid clones via a shotgun strategy. Because these two chromosomes are translocated with breakpoints within the BCR and ABL genes in Philadelphia chromosome-positive leukemias, knowledge of these sequences also might provide insight into the validity of various theories of chromosomal rearrangements. Comparison of these genes with their cDNA sequences reveal the positions of 23 BCR exons and putative alternative BCR first and second exons, as well as the common ABL exons 2-11, respectively. Additionally, these regions include the alternative ABL first exons 1b and 1a, a new gene 5' to the first ABL exon, and an open reading frame with homology to an EST within the BCR fourth intron. Further analysis reveals an Alu homology of 38.83 and 39.35% for the BCR and ABL genes, respectively, with other repeat elements present to a lesser extent. Four new Philadelphia chromosome translocation breakpoints from chronic myelogenous leukemia patients also were sequenced, and the positions of these and several other previously sequenced breakpoints now have been mapped precisely, although no consistent breakpoint features immediately were apparent. Comparative analysis of genomic sequences encompassing the murine homologues to the human ABL exons 1b and 1a, as well as regions encompassing the ABL exons 2 and 3, reveals that although there is a high degree of homology in their corresponding exons and promoter regions, these two vertebrate species show a striking lack of homology outside these regions.

    Funded by: NCI NIH HHS: CA47456, CA50248; PHS HHS: H00313

    Genomics 1995;27;1;67-82

  • Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

    Wetzler M, Talpaz M, Yee G, Stass SA, Van Etten RA, Andreeff M, Goodacre AM, Kleine HD, Mahadevia RK and Kurzrock R

    Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.

    The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.

    Funded by: NCI NIH HHS: CA-16672

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;8;3488-92

  • Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL.

    Salgia R, Li JL, Lo SH, Brunkhorst B, Kansas GS, Sobhany ES, Sun Y, Pisick E, Hallek M, Ernst T et al.

    Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.

    Paxillin is a 68-kDa focal adhesion protein that is phosphorylated on tyrosine residues in fibroblasts in response to transformation by v-src, treatment with platelet-derived growth factor, or cross-linking of integrins. Paxillin has been shown to have binding sites for the SH3 domain of Src and the SH2 domain of Crk in vitro and to coprecipitate with two other focal adhesion proteins, vinculin and focal adhesion kinase (p125fak). After preliminary studies showed that paxillin was a substrate for the hematopoietic oncogene p210BCR/ABL, we investigated the role of this protein in hematopoietic cell transformation and signal transduction. A full-length length cDNA encoding human paxillin was cloned, revealing multiple protein domains, including four tandem LIM domains, a proline-rich domain containing a consensus SH3 binding site, and three potential Crk-SH2 binding sites. The paxillin gene was localized to chromosome 12q24 by fluorescence in situ hybridization analysis. A chicken paxillin cDNA was also cloned and is predicted to encode a protein approximately 90% identical to human paxil-lin. Paxillin coprecipitated with p210BCR/ABL and multiple other cellular proteins in myeloid cell lines, suggesting the formation of multimeric complexes. In normal hematopoietic cells and myeloid cell lines, tyrosine phosphorylation of paxillin and coprecipitation with other cellular proteins was rapidly and transiently induced by interleukin-3 and several other hematopoietic growth factors. The predicted structure of paxillin implicates this molecule in protein-protein interactions involved in signal transduction from growth factor receptors and the BCR/ABL oncogene fusion protein to the cytoskeleton.

    Funded by: NCI NIH HHS: CA 60821, CA36167; NIGMS NIH HHS: GM 3818

    The Journal of biological chemistry 1995;270;10;5039-47

  • Tyrosine phosphorylation of BCR by FPS/FES protein-tyrosine kinases induces association of BCR with GRB-2/SOS.

    Maru Y, Peters KL, Afar DE, Shibuya M, Witte ON and Smithgall TE

    Department of Genetics, University of Tokyo, Japan.

    The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.

    Funded by: NCI NIH HHS: CA 53867, CA 58667, P30 CA 36727-08

    Molecular and cellular biology 1995;15;2;835-42

  • Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family.

    Reuther GW, Fu H, Cripe LD, Collier RJ and Pendergast AM

    Department of Pharmacology, Duke University Medical Center, Durham, NC 27710.

    In this study, a protein that interacts with sequences encoded by the first exon of the protein kinase Bcr was cloned. The Bcr-associated protein 1 (Bap-1) is a member of the 14-3-3 family of proteins. Bap-1 interacts with full-length c-Bcr and with the chimeric Bcr-Abl tyrosine kinase of Philadelphia chromosome (Ph1)-positive human leukemias. Bap-1 is a substrate for the Bcr serine-threonine kinase and is also phosphorylated on tyrosine by Bcr-Abl but not by c-Abl. Bap-1 may function in the regulation of c-Bcr and may contribute to the transforming activity of Bcr-Abl in vivo. 14-3-3 proteins are essential for cell proliferation and have a role in determining the timing of mitosis in yeast. Through direct binding to sequences present in Bcr and in other proteins implicated in signaling, the mammalian 14-3-3 proteins may link specific signaling protein components to mitogenic and cell-cycle control pathways.

    Funded by: NCI NIH HHS: CA61033; NIDDK NIH HHS: DK01965; NIGMS NIH HHS: GM07184; ...

    Science (New York, N.Y.) 1994;266;5182;129-33

  • No evidence for genomic imprinting of the human BCR gene.

    Fioretos T, Heisterkamp N and Groffen J

    Department of Pathology, Childrens Hospital of Los Angeles, CA 90027.

    Chronic myeloid leukemias and 5% to 20% of acute lymphoid leukemias are characterized by the Philadelphia chromosome, a reciprocal chromosomal translocation, t(9;22)(q34;q11), generating BCR-ABL and ABL-BCR fusion genes. Cytogenetic studies have recently shown a preferential involvement of the paternally derived chromosome 9 and the maternally derived chromosome 22 in this translocation, indicating that imprinting might be involved in the formation or selection of the translocation. In this study, we have identified a BamHI polymorphism in the coding region of BCR exon 1, allowing us to investigate whether both BCR alleles are transcribed. By using a reverse transcriptase-polymerase chain reaction assay, we show that both BCR alleles are expressed in the peripheral blood cells of normal individuals.

    Funded by: NCI NIH HHS: R0I CA47456, R0I CA50248

    Blood 1994;83;12;3441-4

  • SH2-containing phosphotyrosine phosphatase Syp is a target of p210bcr-abl tyrosine kinase.

    Tauchi T, Feng GS, Shen R, Song HY, Donner D, Pawson T and Broxmeyer HE

    Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202.

    The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction and neoplastic transformation. These mechanisms are regulated by the activities of both protein-tyrosine kinases and protein-tyrosine phosphatases. Recent studies have identified a novel protein-tyrosine phosphatase, termed Syp, that is widely expressed in various tissues. Syp encodes a cytoplasmic phosphatase that contains two Src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules to activated tyrosine kinases, experiments were performed to determine whether Syp might form specific complexes with p210bcr-abl, a fusion protein believed to be involved in the pathogenesis of chronic myelogenous leukemia and, thus, possibly alter or mediate p210bcr-abl tyrosine kinase activity. We found that Syp was highly and constitutively tyrosine phosphorylated in three different murine cell lines transfected with a p210bcr-abl expression vector. Furthermore, p210bcr-abl, Syp, and Grb2 formed stable complexes in BCR-ABL-expressing cells. Complex formation between p210bcr-abl and Syp was mediated in vitro by the NH2-terminal SH2 domain of Syp. Last, p210bcr-abl tyrosine kinase was effectively dephosphorylated by Syp in vitro. These results suggest an interaction between Syp and BCR-ABL protein, which might play a role in cellular transformation of BCR-ABL.

    Funded by: NCI NIH HHS: R37 CA36464; NHLBI NIH HHS: R01 HL46549, R01 HL49202

    The Journal of biological chemistry 1994;269;21;15381-7

  • Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

    Puil L, Liu J, Gish G, Mbamalu G, Bowtell D, Pelicci PG, Arlinghaus R and Pawson T

    Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

    The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interactions with cytoplasmic proteins that mediate Ras activation. Such polypeptides include Grb2, which comprises a single Src homology 2 (SH2) domain flanked by two SH3 domains, and the 66, 52 and 46 kDa Shc proteins which possess an SH2 domain in their carboxy-terminus. Grb2 associates with tyrosine phosphorylated proteins through its SH2 domain, and with the Ras guanine nucleotide releasing protein mSos1 through its SH3 domains. mSos1 stimulates conversion of the inactive GDP-bound form of Ras to the active GTP-bound state. In bcr-abl-transformed cells, Grb2 and mSos1 formed a physical complex with Bcr-Abl. In vitro, the Grb2 SH2 domain bound Bcr-Abl through recognition of a tyrosine phosphorylation site within the amino-terminal bcr-encoded sequence (p.Tyr177-Val-Asn-Val), that is common to both Bcr-Abl proteins. These results suggest that autophosphorylation within the Bcr element of Bcr-Abl creates a direct physical link to Grb2-mSos1, and potentially to the Ras pathway, and thereby modifies the target specificity of the Abl tyrosine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NCI NIH HHS: CA16672

    The EMBO journal 1994;13;4;764-73

  • A molecular inventory of human pancreatic islets: sequence analysis of 1000 cDNA clones.

    Takeda J, Yano H, Eng S, Zeng Y and Bell GI

    Howard Hughes Medical Institute, University of Chicago, IL 60637.

    The islets of Langerhans play a central role in glucose homeostasis by secreting the polypeptide hormones insulin and glucagon. They are comprised primarily of four endocrine cell types: insulin-secreting beta-cells which represent about 70% of the cells in the islet along with smaller number of cells secreting glucagon, somatostatin and pancreatic polypeptide. Diabetes mellitus results from the specific loss or dysfunction of the beta-cells. Because of the central role of the islets of Langerhans in the regulation of glucose homeostasis, we are preparing a database of genes expressed in this tissue. One thousand cDNA clones randomly isolated from a human pancreatic islet library were partially sequenced yielding 280 kilobases of sequence. Database searches indicated that 397 of the cDNAs represented known human genes or human homologs of genes identified in other species and a further 58 sequences corresponded to expressed sequence tags identified in other tissues or cells (contamination by exocrine pancreatic tissue was estimated to be less than 10%). 545 of the cDNAs were not related to any other sequences in the databases. The islet cDNA collection provides a unique source of genes for genetic studies of diabetes as well as for molecular studies of islet function in normal and diabetic states.

    Human molecular genetics 1993;2;11;1793-8

  • DNA sequence analysis of the major breakpoint cluster region of the BCR gene rearranged in Philadelphia-positive human leukemias.

    Sowerby SJ, Kennedy MA, Fitzgerald PH and Morris CM

    Department of Pathology, Christchurch School of Medicine, Christchurch Hospital, New Zealand.

    We sought sequence characteristics that might explain the apparent high recombination frequency of the 5-kb BglII segment containing M-bcr exons 1, 2 and 3, and the intron to exon 4. An Alu sequence (subfamily Sx), in 5'-->3' orientation, lay in the middle of a 3-kb region that contains the great majority of Philadelphia chromosome breakpoint sites. The breakpoint of only one out of five chronic myeloid leukemia patients, for whom the BCR breakpoint site had been sequenced, was located within this Alu. Other features of interest for recombination were a 51-bp AT-rich region close to the 3' end, six hypervariable minisatellite consensus octamers, GC[A/T]GG[A/T]GG, six lymphoid recombinase heptamer signal sequences, one nonamer and a 16-bp inverted repeat. Dot matrix comparisons of the 5-kb M-bcr sequence with a 3-kb m-bcr2 segment showed significant homology only in corresponding Alu sequences.

    Oncogene 1993;8;6;1679-83

  • Duplication of small segments within the major breakpoint cluster region in chronic myelogenous leukemia.

    Litz CE, McClure JS, Copenhaver CM and Brunning RD

    Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis.

    The t(9;22) in chronic myelogenous leukemia (CML) may be reciprocal or, in a minority of cases, may result in an extensive deletion of a portion of the major breakpoint cluster region (M-bcr) of the BCR. This report provides evidence of the duplication of small segments within the M-bcr in a small group of patients with CML. Southern blots of Bgl II and Bgl II/BamHI double-digested DNA from the blood or bone marrow of 46 patients with CML were probed with a 5' 1.4-kb Taq I/HindIII M-bcr probe and a 3' 2-kb HindIII/BamHI M-bcr probe. In three patients, rearrangements were noted with both probes in Bgl II-digested DNA, but were not present in Bgl II/BamHI-digested DNA with either probe. Southern analysis of DNA samples double-digested with Bgl II and BspHI from two of these three cases showed no rearrangements with either probe; the M-bcr BspHI site is located 26 bp 3' of the BamHI site in the second intron of the M-bcr. The presence of a rearranged M-bcr with both probes in Bgl II-digested DNA and the lack of rearrangement in Bgl II/BamHI and Bgl II/BspHI double-digested DNA suggest the presence of M-bcr BamHI and BspHI sites on both 9q+ chromosome (9q+) and the Philadelphia chromosome (Ph). This implies a duplication of at least the 26-bp M-bcr BamHI/BspHI fragment in these two samples. Sequence data from one of these two cases confirmed the M-bcr breakpoints to be staggered; the Ph M-bcr breakpoint occurred 258 bp downstream from the 9q+ M-bcr breakpoint. It is concluded that a duplication of small segments within the M-bcr occurs in a small group of patients with CML, which may lead to pseudogermline patterns on Southern blot. Such a duplication may provide insight into the mechanism of some chromosomal translocations in neoplasia.

    Blood 1993;81;6;1567-72

  • A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

    Muller AJ, Pendergast AM, Havlik MH, Puil L, Pawson T and Witte ON

    Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.

    SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.

    Molecular and cellular biology 1992;12;11;5087-93

  • Amplification and sequencing of genomic breakpoints located within the M-bcr region by Vectorette-mediated polymerase chain reaction.

    Mills KI, Sproul AM, Ogilvie D, Elvin P, Leibowitz D and Burnett AK

    Department of Haematology, Glasgow Royal Infirmary, Scotland, UK.

    The polymerase chain reaction (PCR) cannot be used to amplify the breakpoint in the chimaeric BCR-ABL gene in CML and acute leukaemias due to the large variation in the sites of breakpoint in the BCR gene (within a 5.8 kb region) and in the ABL gene (within a 150 kb region). The disease state is usually monitored using RNA-PCR to monitor abnormal transcripts. We have used a new modification of the PCR to amplify breakpoints within zone 3 of the M-bcr. A synthetic oligonucleotide linker, the Vectorette, is ligated to restriction digested DNA, and amplification is carried out between primers for a known target sequence and the Vectorette linker. Three Philadelphia chromosome Ph1-positive CML patients with breakpoints within the ALU region of zone 3 have been amplified and the sequence immediately around the breakpoint determined. The breaks occurred within 70 bp and two were only 14 bp apart. The Vectorette-PCR technique has the potential to rapidly identify and sequence breakpoints, and will enable the design of patient-specific primers to monitor disease progression, particularly following bone marrow transplantation.

    Leukemia 1992;6;5;481-3

  • Locating protein-coding regions in human DNA sequences by a multiple sensor-neural network approach.

    Uberbacher EC and Mural RJ

    Biology Division, Oak Ridge National Laboratory, TN.

    Genes in higher eukaryotes may span tens or hundreds of kilobases with the protein-coding regions accounting for only a few percent of the total sequence. Identifying genes within large regions of uncharacterized DNA is a difficult undertaking and is currently the focus of many research efforts. We describe a reliable computational approach for locating protein-coding portions of genes in anonymous DNA sequence. Using a concept suggested by robotic environmental sensing, our method combines a set of sensor algorithms and a neural network to localize the coding regions. Several algorithms that report local characteristics of the DNA sequence, and therefore act as sensors, are also described. In its current configuration the "coding recognition module" identifies 90% of coding exons of length 100 bases or greater with less than one false positive coding exon indicated per five coding exons indicated. This is a significantly lower false positive rate than any method of which we are aware. This module demonstrates a method with general applicability to sequence-pattern recognition problems and is available for current research efforts.

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;24;11261-5

  • The BCR gene encodes a novel serine/threonine kinase activity within a single exon.

    Maru Y and Witte ON

    Howard Hughes Medical Institute, University of California, Los Angeles 90024-1570.

    Sequences encoded by the first exon of BCR that bind to the ABL SH2 domain are essential for the activation of the ABL tyrosine kinase and transforming potential of the chimeric BCR-ABL oncogene. The normal cellular BCR gene encodes a 160,000 dalton phosphoprotein associated with a serine/threonine kinase activity, but it shows only weak dispersed homologies to protein kinases. p160c-BCR was purified to apparent homogeneity as an oligomer of greater than 600,000 daltons that contains autophosphorylation activity and transphosphorylation activity for several protein substrates. A region containing paired cysteine residues within the 426 amino acids encoded by the first exon of BCR is essential for its novel phosphotransferase activity, which overlaps with the strong SH2-binding regions. The recent demonstration of a GTPase-activating function within the C-terminal portion of BCR suggests that the protein kinase and SH2-binding domains may work in concert with other regions of the molecule in intracellular signalling processes.

    Funded by: NCI NIH HHS: R01 CA27507, R35 CA53867

    Cell 1991;67;3;459-68

  • Entire ABL gene is joined with 5'-BCR in some patients with Philadelphia-positive leukemia.

    Morris CM, Heisterkamp N, Groffen J and Fitzgerald PH

    Cytogenetic and Molecular Oncology Unit, Christchurch School of Medicine, New Zealand.

    In four patients, the chromosome 9 breakpoint of the t(9; 22)(q34;q11) had occurred at different sites within an 8.25-kilobase (kb) region situated 5' of ABL exon 1B. Chromosome in situ hybridization and field inversion gel electrophoresis (FIGE) studies showed that ABL exons 1A and 1B were present on the Ph chromosome. Yet this large fusion gene produced an mRNA conventional for chronic myelogenous leukemia (CML). Splicing from BCR exon 3 to ABL exon 2 crossed more than 200 kb and deleted exons 1A and 1B. This breakpoint site may occur in about 10% of all CML patients. Three of our patients have pronounced thrombocytosis, and two had been diagnosed as having Ph-positive essential thrombocythemia. The platelet count of the other patient was not available.

    Funded by: NCI NIH HHS: CA 47073, CA 47456

    Blood 1991;78;4;1078-84

  • BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner.

    Pendergast AM, Muller AJ, Havlik MH, Maru Y and Witte ON

    Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024-1570.

    BCR-ABL is a chimeric oncogene implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL. We have tested the hypothesis that activation of BCR-ABL may involve direct interaction between BCR sequences and the tyrosine kinase regulatory domains of ABL. Full-length c-BCR as well as BCR sequences retained in BCR-ABL bind specifically to the SH2 domain of ABL. The binding domain has been localized within the first exon of BCR and consists of at least two SH2-binding sites. This domain is essential for BCR-ABL-mediated transformation. Phosphoserine/phosphothreonine but not phosphotyrosine residues on BCR are required for interaction with the ABL SH2 domain. These findings extend the range of potential SH2-protein interactions in growth control pathways and suggest a function for SH2 domains in the activation of the BCR-ABL oncogene as well as a role for BCR in cellular signaling pathways.

    Cell 1991;66;1;161-71

  • Bcr encodes a GTPase-activating protein for p21rac.

    Diekmann D, Brill S, Garrett MD, Totty N, Hsuan J, Monfries C, Hall C, Lim L and Hall A

    Chester Beatty Beatty Laboratories, Institute of Cancer Research, London, UK.

    More than thirty small guanine nucleotide-binding proteins related to the ras-encoded oncoprotein, termed Ras or p21ras, are known. They regulate many fundamental processes in all eukaryotic cells, such as growth, vesicle traffic and cytoskeletal organization. GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis of Ras-related proteins, leading to down-regulation of the active GTP-bound form. For p21ras, two GAP proteins are known, rasGAP and the neurofibromatosis (NF1) gene product. There is evidence that rasGAP may also be a target protein for regulation by Ras and be involved in downstream signalling. We have purified a GAP protein for p21rho, which is involved in the regulation of the actin cytoskeleton. Partial sequencing of rhoGAP reveals significant homology with the product of the bcr (breakpoint cluster region) gene, the translocation breakpoint in Philadelphia chromosome-positive chronic myeloid leukaemias. We show here that the carboxy-terminal domains of the bcr-encoded protein (Bcr) and of a Bcr-related protein, n-chimaerin, are both GAP proteins for the Ras-related GTP-binding protein, p21rac. This result suggest that Bcr could be a target for regulation by Rac and has important new implications for the role of bcr translocations in leukaemia.

    Nature 1991;351;6325;400-2

  • Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines.

    Shah NP, Witte ON and Denny CT

    Department of Microbiology and Molecular Genetics, University of California-Los Angeles 90024.

    The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.

    Funded by: NCI NIH HHS: CA-50433, T32 CA-09056

    Molecular and cellular biology 1991;11;4;1854-60

  • Reconstruction and analysis of human Alu genes.

    Jurka J and Milosavljevic A

    Linus Pauling Institute of Science and Medicine, Palo Alto, CA 94306.

    The existing classification of human Alu sequences is revised and expanded using a novel methodology and a larger set of sequence data. Our study confirms that there are two major Alu subfamilies, Alu-J and Alu-S. The Alu-S subfamily consists of at least five distinct subfamilies referred to as Alu-Sx, Alu-Sq, Alu-Sp, Alu-Sc, and Alu-Sb. The Alu-Sp and Alu-Sq subfamilies have been revealed by this study. Alu subfamilies differ from one another in a number of positions called diagnostic. In this paper the diagnostic positions are defined in quantitative terms and are used to evaluate statistical significance of the observed subfamilies. Each Alu subfamily most likely represents pseudogenes retroposed from evolving functional source Alu genes. Evidence presented in this paper indicates that Alu-Sp and Alu-Sc pseudogenes were retroposed from different source genes, during overlapping periods of time, and at different rates. Our analysis also indicates that the previously identified Alu-type transcript BC200 comes from an active Alu gene that might have existed even before the origin of dimeric Alu sequences. The source genes for Alu pseudogene families are reconstructed. It is assumed that diagnostic differences between reconstructed source genes reflect mutations that have occurred in true source Alu genes under natural selection. Some of these mutations are compensatory and are used to reconstruct a common secondary structure of Alu RNAs transcribed from the source genes. The biological function of Alu RNA is discussed in the context of its homology to the elongation-arresting domain of 7SL RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

    Journal of molecular evolution 1991;32;2;105-21

  • Unique organization of the human BCR gene promoter.

    Zhu QS, Heisterkamp N and Groffen J

    Department of Pathology, Childrens Hospital of Los Angeles, CA 90027.

    The promoter of the human BCR gene, regulating the transcription of the chimeric BCR/ABL mRNA in leukemia, has been isolated and characterized. A region of 1.1 kb immediately 5' to the transcription start site was analyzed in detail by sequencing, DNase 1 footprinting, gel retardation and functional studies. These experiments localized a minimal promoter to a 650 bp sequence, composed of 270 bp of 5' flanking sequences and 380 bp of exon 1 transcribed sequences. The promoter region includes a TTTAA box, one Sp1 site and a novel protein-binding sequence absolutely necessary for efficient transcription in vivo. Six additional protein-binding regions were identified more to the 5'. Of these, one is found in an inverted repeat in the 3' coding and splice donor region of BCR exon 1.

    Funded by: NCI NIH HHS: CA 47073, CA 47456

    Nucleic acids research 1990;18;23;7119-25

  • A novel variant of the bcr-abl fusion product in Philadelphia chromosome-positive acute lymphoblastic leukemia.

    Soekarman D, van Denderen J, Hoefsloot L, Moret M, Meeuwsen T, van Baal J, Hagemeijer A and Grosveld G

    Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.

    Two patients with Philadelphia chromosome-positive acute lymphoblastic leukemia showed novel variants of the chimeric bcr-abl mRNA. The bcr-abl breakpoint region on cDNA derived from the chimeric mRNA was amplified using the polymerase chain reaction (PCR). Sequence analysis of the breakpoint-containing fragment showed that in both patients exon a2 of the abl gene was deleted, giving rise to an in-frame joining at the mRNA level of 5' bcr sequences to the abl exon a3. These findings were confirmed by Southern blot analysis and cloning of chromosomal DNA. Protein studies showed a bcr-abl protein with heightened tyrosine kinase activity in blast cells of both patients: one of the P190 type, the other of the P210 type. The significance of these findings and the role of this new type of translocation in the disregulation of the abl gene are discussed.

    Leukemia 1990;4;6;397-403

  • Chronic myeloid leukemia may be associated with several bcr-abl transcripts including the acute lymphoid leukemia-type 7 kb transcript.

    Selleri L, von Lindern M, Hermans A, Meijer D, Torelli G and Grosveld G

    Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.

    In the majority of Philadelphia (Ph)-positive chronic myeloid leukemia (CML) patients, the c-abl gene is fused to the bcr gene, resulting in the transcription of an 8.5 kb chimeric bcr-abl mRNA, which is translated into a p210bcr-abl fusion protein. In about 50% of the Ph-positive acute lymphoid leukemias (ALL), the bcr-abl gene fusion is identical to CML, while in 50% an alternative fusion between these two genes occurs, in which the central bcr-sequences are absent. This results in transcription of a 7 kb bcr-abl mRNA, encoding a P190bcr-abl fusion protein. Cloning and sequencing of the chimeric part of bcr-abl cDNAs from two Ph-positive CML patients in chronic phase showed that in one patient, as in the Ph-positive ALL, all central bcr sequences are absent, while in the other patient, part of the bcr central sequences are deleted. Therefore, we speculate that the presence of the 7 kb chimeric ALL type mRNA in one of the patients is not sufficient to drive an acute rather than a chronic leukemic process in this case. The deletions of the central bcr-sequences described here define the minimal sequence requirement of the bcr-abl fusion gene in CML patients so far.

    Blood 1990;75;5;1146-53

  • Structural alterations of the BCR and ABL genes in Ph1 positive acute leukemias with rearrangements in the BCR gene first intron: further evidence implicating Alu sequences in the chromosome translocation.

    Chen SJ, Chen Z, Font MP, d'Auriol L, Larsen CJ and Berger R

    Unité INSERM U 301 and SDI CNRS no. 159541, CNRS, Hôpital Saint-Louis, Paris, France.

    In the Philadelphia positive bcr negative acute leukemias (Ph1+bcr- AL), the chromosomal breakpoints on chromosome 22 have been shown clustered within 10.8kb (bcr2) and 5kb (bcr3) fragments of the first intron of the BCR gene. We previously reported that the breakpoints were localized in Alu repeats on chromosomes 9 and 22 in a Ph1+bcr- acute lymphoblastic leukemia with a rearrangement involving bcr2. Molecular data of two other Ph1 translocations, one a Ph1+bcr- acute myeloblastic leukemia in the bcr2 region, and the other an acute lymphoblastic leukemia in the bcr3 region are presented. In the former, the breakpoints on chromosomes 9 and 22 are localized in Alu repeats, in regions with two inverted Alu sequences, as in our previously reported case. In the second leukemia, the breakpoints are not located in Alu sequences, but such repeats are found in their vicinity. The implications of these findings are discussed.

    Nucleic acids research 1989;17;19;7631-42

  • Alternative 5' end of the bcr-abl transcript in chronic myelogenous leukemia.

    Romero P, Beran M, Shtalrid M, Andersson B, Talpaz M and Blick M

    Department of Clinical Immunology, University of Texas M.D. Anderson Cancer Center, Houston 77030.

    Philadelphia chromosome positive acute lymphocytic leukemia and chronic myelogenous leukemia are strongly associated with two distinct forms of bcr-abl chimeric protein, known as P190 and P210, respectively. By studying cDNA clones obtained from the cell line KBM-5, we identified two new bcr-abl transcripts. These are formed by alternative splicing of at least two exons to the known bcr exon 2. One novel transcript can encode a protein kinase of approximately 190 kd, while the other can direct the synthesis of a larger protein whose amino terminus remains to be defined. The alternative exons can be spliced also to the two normal bcr transcripts, reflecting the activation of a cryptic promoter. These messages were present at low abundance in two cases of blastic crisis but were not detected in the chronic phase. It is conceivable that the proteins encoded by the new bcr-abl mRNAs are involved in the transformation to the acute phase in some cases of chronic myelogenous leukemia.

    Oncogene 1989;4;1;93-8

  • The first BCR gene intron contains breakpoints in Philadelphia chromosome positive leukemia.

    Heisterkamp N, Knoppel E and Groffen J

    Section of Molecular Genetics, Childrens Hospital of Los Angeles, CA 90027.

    The hallmark of chronic myelogenous leukemia (CML) is a translocation between chromosomes 9 and 22 - the Philadelphia (Ph') translocation. The translocation is also found in acute lymphocytic leukemia (ALL) albeit in a lower percentage of patients. The breakpoint on chromosome 22 is located within the BCR gene: in CML, breakpoints are clustered within 5.8 kb of DNA, the major breakpoint cluster region (Mbcr). In ALL, breakpoints have been reported within the Mbcr but also in more 5' regions encompassing the BCR gene. To characterize the latter breakpoints, we have molecularly cloned and mapped the entire gene, which encompasses approximately 130 kb of DNA. Mbcr negative, Ph'-positive ALL breakpoints were not distributed at random within the gene but rather were found exclusively within the 3' half of the first BCR gene intron. In contrast to the Mbcr, which is limited to a region of 5.8 kb, this part of the intron has a size of 35 kb. Translocation breakpoints in this region appear to be specific for ALL, since it was not rearranged in clinically well-defined CML specimens nor in any other tumor DNA samples examined.

    Nucleic acids research 1988;16;21;10069-81

  • Expression of a distinctive BCR-ABL oncogene in Ph1-positive acute lymphocytic leukemia (ALL).

    Clark SS, McLaughlin J, Timmons M, Pendergast AM, Ben-Neriah Y, Dow LW, Crist W, Rovera G, Smith SD and Witte ON

    Department of Microbiology, UCLA 90024.

    The Philadelphia chromosome (Ph1) is a translocation between chromosomes 9 and 22 that is found in chronic myelogenous leukemia (CML) and a subset of acute lymphocytic leukemia patients (ALL). In CML, this results in the expression of a chimeric 8.5-kilobase BCR-ABL transcript that encodes the P210BCR-ABL tyrosine kinase. The Ph1 chromosome in ALL expresses a distinct ABL-derived 7-kilobase messenger RNA that encodes the P185ALL-ABL protein. Since the expression of different oncogene products may play a role in the distinctive presentation of Ph1-positive ALL versus CML, it is necessary to understand the molecular basis for the expression of P185ALL-ABL. Both P210BCR-ABL and P185ALL-ABL are recognized by an antiserum directed to BCR determinants in the amino-terminal region of both proteins. Antisera to BCR determinants proximal to the BCR-ABL junction in CML immunoprecipitated P210BCR-ABL but not P185ALL-ABL. Nucleotide sequence analysis of complementary DNA clones made from RNA from the Ph1-positive ALL SUP-B15 cell line, and S1 nuclease protection analysis confirmed the presence of BCR-ABL chimeric transcripts in Ph1-positive ALL cells. In Ph1-positive ALL, ABL sequences were joined to BCR sequences approximately 1.5 kilobases 5' of the CML junction. P185ALL-ABL represents the product of a BCR-ABL fusion gene in Ph1-positive ALL that is distinct from the BCR-ABL fusion gene of CML.

    Funded by: NCI NIH HHS: CA-20180, CA-21765, CA-34233

    Science (New York, N.Y.) 1988;239;4841 Pt 1;775-7

  • bcr genes and transcripts.

    Lifshitz B, Fainstein E, Marcelle C, Shtivelman E, Amson R, Gale RP and Canaani E

    Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.

    Human chronic myelogenous leukemia (CML) is a clonal hematologic disorder. CML is characterized by the t(9:22) chromosome translocation which results in translocation of the oncogene abl from chromosome 9 into the breakpoint cluster region (bcr) gene on chromosome 22. We cloned and characterized the cDNA of the normal bcr gene. The bcr gene codes for a protein of 1271 amino acids. The open reading frame is preceded by a region high in GC. At the 5' of this region we identified several GC motifs which are probably involved in the initiation of bcr transcription. bcr transcripts of 7.0 and 4.5 kb are expressed in all cell types examined. These transcripts share all cDNA sequences analysed, including the 5' untranslated region. The latter as well as 902 or 927 amino acids are included within the CML-specific bcr-abl mRNA transcribed from the chimeric bcr-abl gene on chromosome 22. In addition to the complete bcr gene, the human genome contains 3 bcr-related genes containing the last seven exons of the intact bcr gene. One of these genes was analysed in detail and showed high sequence homology with the latter. The three bcr-related genes were probably derived from the intact gene by subsequent steps of duplication.

    Funded by: NCI NIH HHS: CA-38569

    Oncogene 1988;2;2;113-7

  • Mapping of four distinct BCR-related loci to chromosome region 22q11: order of BCR loci relative to chronic myelogenous leukemia and acute lymphoblastic leukemia breakpoints.

    Croce CM, Huebner K, Isobe M, Fainstain E, Lifshitz B, Shtivelman E and Canaani E

    Wistar Institute, Philadelphia, PA 19104.

    A probe derived from the 3' region of the BCR gene (breakpoint cluster region gene) detects four distinct loci in the human genome. One of the loci corresponds to the complete BCR gene, whereas the others contain a 3' segment of the gene. After HindIII cleavage of human DNA, these four loci are detected as 23-, 19-, 13-, and 9-kilobase-pair fragments, designated BCR4, BCR3, BCR2, and BCR1, respectively, with BCR1 deriving from the original complete BCR gene. All four BCR loci segregate 100% concordantly with human chromosome 22 in a rodent-human somatic cell hybrid panel and are located at chromosome region 22q11.2 by chromosomal in situ hybridization. The BCR2 and BCR4 loci are amplified in leukemia cell line K562 cells, indicating that they fall within the amplification unit that includes immunoglobulin lambda light chain locus (IGL) and ABL locus on the K562 Philadelphia chromosome (Ph1); additionally, in chronic myelogenous leukemia-derived mouse-human hybrids retaining a Ph1 chromosome in the absence of the 9q+ and normal chromosome 22, BCR2 and BCR4 loci are retained, whereas the 3' region of BCR1 and the BCR3 locus are lost, indicating that BCR3 is distal to BCR1 on chromosome 22. Similarly, in mouse-human hybrids retaining a Ph1 chromosome derived from an acute lymphoblastic leukemia-in the absence of the 9q+ and 22, only BCR2 and BCR4 loci are retained, indicating that the breakpoint in this acute lymphoblastic leukemia, as in chronic myelogenous leukemia, is proximal to the BCR1 3' region, but distal to the IGLC locus and the BCR2 and BCR4 3' loci. Thus, the order of loci on chromosome 22 is centromere----BCR2, BCR4, and IGL----BCR1----BCR3----SIS, possibly eliminating BCR2 and BCR4 loci as candidate targets for juxtaposition to the ABL gene in the acute lymphoblastic leukemia Ph1 chromosome.

    Funded by: NCI NIH HHS: CA-10805, CA-21124, CA-39860

    Proceedings of the National Academy of Sciences of the United States of America 1987;84;20;7174-8

  • cDNA sequence for human bcr, the gene that translocates to the abl oncogene in chronic myeloid leukaemia.

    Hariharan IK and Adams JM

    The hallmark of human chronic myeloid leukaemia is a 9;22 chromosome translocation that fuses most of the c-abl oncogene to the 5' portion of the breakpoint cluster region (bcr) gene, such that a hybrid bcr-abl mRNA and polypeptide are generated. To clarify further the nature of this translocation, we have analysed the structure of normal human bcr mRNA by isolating large cDNA clones that collectively span the entire coding region and extend 2.6 kb upstream of those previously described. The 3150-bp nucleotide sequence reported here includes 534 bp of a GC-rich 5' non-coding segment and indicates, in conjunction with published sequences, that the bcr polypeptide comprises 1271 amino acid residues. The predicted polypeptide is unrelated to serine or tyrosine kinases, or indeed to any previously published sequence; its structure provides no evidence of a transmembrane region. Since probes from throughout the 4.8-kb cloned region hybridized to both the 4.5 and 6.7 kb normal bcr transcripts, both RNAs contain most or all of that region.

    Funded by: NCI NIH HHS: CA 12421

    The EMBO journal 1987;6;1;115-9

  • Overlapping cDNA clones define the complete coding region for the P210c-abl gene product associated with chronic myelogenous leukemia cells containing the Philadelphia chromosome.

    Mes-Masson AM, McLaughlin J, Daley GQ, Paskind M and Witte ON

    The Philadelphia chromosome, observed in greater than 90% of patients with chronic myelogenous leukemia, results from a reciprocal translocation between chromosomes 9 and 22. The translocation breakpoint on chromosome 9 occurs near the ABL gene and correlates with the production of a chronic myelogenous leukemia-specific 8.5-kilobase ABL-related mRNA species accompanied by a structurally altered ABL protein (P210c-abl). The N-terminal sequence of the protein is derived from the BCR gene on chromosome 22. We have isolated overlapping cDNA clones from the K-562 cell line corresponding to approximately 8.5 kilobases of mRNA and have sequenced 2550 nucleotides at the 5' end. Our results indicate that the 5' end of the 8.5-kilobase mRNA consists of greater than 400 nucleotides of noncoding sequence that are greater than 80% G + C rich. Based on our sequence analysis, we propose that initiation of translation occurs at nucleotide 471, such that the initial 927 amino acids of P210c-abl are derived from BCR sequences. Our cDNA clones thus define the complete coding sequences for the P210c-abl gene product.

    Proceedings of the National Academy of Sciences of the United States of America 1986;83;24;9768-72

  • Molecular analysis of both translocation products of a Philadelphia-positive CML patient.

    de Klein A, van Agthoven T, Groffen C, Heisterkamp N, Groffen J and Grosveld G

    The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.

    Nucleic acids research 1986;14;17;7071-82

  • The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

    Grosveld G, Verwoerd T, van Agthoven T, de Klein A, Ramachandran KL, Heisterkamp N, Stam K and Groffen J

    In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

    Molecular and cellular biology 1986;6;2;607-16

  • Fused transcript of abl and bcr genes in chronic myelogenous leukaemia.

    Shtivelman E, Lifshitz B, Gale RP and Canaani E

    Human chronic myelogenous leukaemia is characterized by a reciprocal translocation between chromosomes 9 and 22 resulting in an abbreviated form of chromosome 22 and the transfer of the abl cellular oncogene from chromosome 9 into the bcr gene of chromosome 22. Characterization of an 8-kilobase RNA specific to chronic myelogenous leukaemia shows it to be a fused transcript of the two genes. The fused protein that would be produced is probably involved in the malignant process.

    Nature 1985;315;6020;550-4

  • High resolution chromosomes of the t(9;22) positive leukemias.

    Prakash O and Yunis JJ

    A combined bromodeoxyuridine (BrdU) and actinomycin D (AMD) treatment of methotrexate (MTX) synchronized bone marrow cells from four patients with chronic myelogenous leukemia (CML), and two each with acute lymphocytic (ALL) and acute nonlymphocytic (ANLL) leukemia were used to define the breakpoints involved in the t(9;22) found in subgroups of these three disorders. An additional 20 patients with CML were studied with the MTX technique alone. All cases showed the same breakpoints at subbands 9q34.1 and 22q11.2. In CML, it was possible to further define the breakpoint of chromosome 22 to subband q11.21.

    Funded by: NCI NIH HHS: CA-31024

    Cancer genetics and cytogenetics 1984;11;4;361-7

  • Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22.

    Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR and Grosveld G

    We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.

    Funded by: NCI NIH HHS: N01-CP-75380

    Cell 1984;36;1;93-9

  • Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia.

    Heisterkamp N, Stephenson JR, Groffen J, Hansen PF, de Klein A, Bartram CR and Grosveld G

    The human c-ab1 oncogene maps within the region (q34-qter) of chromosome 9 which is translocated to chromosome 22, the Philadelphia (Ph') chromosome, in chronic myelocytic leukaemia (CML). The position of the Ph' chromosomal break point is shown to be variable and, in one CML patient, has been localized immediately 5' of, or within, the c-ab1 oncogene. A DNA restriction fragment corresponding to this site has been molecularly cloned and shown to represent a chimaeric fragment of DNA from chromosomes 9 and 22.

    Funded by: NCI NIH HHS: N0I-CP-75380

    Nature 1983;306;5940;239-42

  • Isolation of human oncogene sequences (v-fes homolog) from a cosmid library.

    Groffen J, Heisterkamp N, Grosveld F, Van de Ven W and Stephenson JR

    To define the human homolog (or homologs) of transforming sequences (v-fes gene) common to Gardner (GA) and Snyder Theilen (ST) isolates of feline sarcoma virus (FeSV), a representative library of human lung carcinoma DNA in a cosmid vector system was constructed. Three cosmid clones were isolated containing GA/ST FeSV v-fes homologous cellular sequences, within 32- to 42-kilobase cellular inserts representing 56 kilobases of contiguous human cellular DNA. Sequences both homologous to, and colinear with, GA or ST FeSV v-fes are distributed discontinuously over a region of up to 9.5 kilobases and contain a minimum of three regions of nonhomology representing probable introns. A thymidine kinase selection system was used to show that, upon transfection to RAT-2 cells, the human c-fes sequence lacked detectable transforming activity.

    Funded by: NCI NIH HHS: N0I-CO-75380

    Science (New York, N.Y.) 1982;216;4550;1136-8

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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