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PubMed1989

Unexpected heterogeneity of BCR-ABL fusion mRNA detected by polymerase chain reaction in Philadelphia chromosome-positive acute lymphoblastic leukemia.

The Philadelphia (Ph1) chromosome results in a fusion of portions of the BCR gene from chromosome 22 and the ABL gene from chromosome 9, producing a chimeric BCR-ABL mRNA and protein. In lymphoblastic leukemias, there are two molecular subtypes of the Ph1 chromosome, one with a rearrangement of the breakpoint cluster region (bcr) of the BCR gene, producing the same 8.5-kilobase BCR-ABL fusion mRNA seen in chronic myelogenous leukemia (CML), and the other, without a bcr rearrangement, producing a 7.0-kilobase BCR-ABL fusion mRNA that is seen only in acute lymphoblastic leukemia (ALL). We studied the molecular subtype of the Ph1 chromosome in 11 cases of Ph1-positive ALL, including 2 with a previous diagnosis of CML, using a sensitive method to analyze the mRNA species based on the polymerase chain reaction (PCR). We observed unexpected heterogeneity in BCR-ABL mRNA in this population; in particular, 1 of 6 bcr-rearranged cases and 1 of 5 bcr-unrearranged cases contained none of the known fusion mRNA species, while 1 of the bcr-rearranged cases contained both. This latter case is particularly interesting because it suggests that the acquisition of an additional BCR-ABL fusion species may be a mechanism of disease progression. We conclude that the PCR gives additional information about the Ph1 chromosome gene products that cannot be obtained by genomic analysis, but that it cannot be used as the sole means of detection of this chromosomal abnormality in ALL because of the high incidence of false negative results.

Blotting, Southern↗

PubMed2002

Ectopic expression of protein-tyrosine kinase Bcr-Abl suppresses tumor necrosis factor (TNF)-induced NF-kappa B activation and IkappaBalpha phosphorylation. Relationship with down-regulation of TNF receptors.

Bcr-Abl, the product of the protooncogene bcr-abl, is a constitutively active protein-tyrosine kinase that is highly expressed in chronic myelogenous leukemia and in acute myeloid leukemia cells. Because Bcr-Abl is known to provide mitogenic signals through suppression of apoptosis, we investigated the effect of this oncogene product on signaling by tumor necrosis factor (TNF), a proapoptotic cytokine. We used a bcr-abl-deficient human megakaryocytic leukemia cell line MO7E and an isogenic MBA cell line stably transfected with bcr-abl. Electrophoretic mobility shift assay revealed that TNF activated the nuclear transcription factor NF-kappaB in MO7E cells but not in MBA cells. The impaired NF-kappaB activation in Bcr-Abl-expressing cells was not due to absence of the NF-kappaB proteins p65, p50, or p100 or of IkappaBalpha or IkappaBbeta. Okadaic acid-induced NF-kappaB activation was unaffected by Bcr-Abl expression. TNF induced IkappaBalpha phosphorylation and degradation in MO7E cells but not in MBA cells. The suppression of TNF-induced NF-kappaB activation by Bcr-Abl was not restricted to MBA cells, because ectopic expression of Bcr-Abl in human acute myeloid leukemia HL-60 cells also blocked TNF-induced NF-kappaB activation. When examined for the TNF receptors by the radioreceptor assay, flow cytometry, or Western blot analysis, we found that Bcr-Abl expression down-regulated the expression of the TNF receptors. The RNase protection assay and Northern blot analysis revealed the transcriptional down-regulation of the TNF receptor by Bcr-Abl protein. Overall, these results indicate that ectopic expression of Bcr-Abl interferes with the TNF signaling pathway through the down-regulation of TNF receptors.

Cell Line↗

PubMed2001

The NH(2)-terminal coiled-coil domain and tyrosine 177 play important roles in induction of a myeloproliferative disease in mice by Bcr-Abl.

Bcr-Abl, a fusion protein generated by t(9;22)(q34;q11) translocation, plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It has been shown that Bcr-Abl contains multiple functional domains and motifs and can disrupt regulation of many signaling pathways and cellular functions. However, the role of specific domains and motifs of Bcr-Abl or of specific signaling pathways in the complex in vivo pathogenesis of CML is not completely known. We have previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. We have also shown that the Abl kinase activity within Bcr-Abl is essential for Bcr-Abl leukemogenesis, yet activation of the Abl kinase without Bcr sequences is not sufficient to induce MPD in mice. In this study we investigated the role of Bcr sequences within Bcr-Abl in inducing MPD using this murine model for CML. We found that the NH(2)-terminal coiled-coil (CC) domain was both essential and sufficient, even though not efficient, to activate Abl to induce an MPD in mice. Interestingly, deletion of the Src homology 3 domain complemented the deficiencies of the CC-deleted Bcr-Abl in inducing MPD in mice. We further demonstrated that the Grb2 binding site at Y177 played an important role in efficient induction of MPD. These studies directly demonstrated the important roles of Bcr sequences in induction of MPD by Bcr-Abl.

3T3 Cells↗

PubMed2006

Bcr interacts with components of the endosomal sorting complex required for transport-I and is required for epidermal growth factor receptor turnover.

Virtually all patients with chronic myelogenous leukemia (CML) express an aberrant protein (p210 Bcr-Abl) that contains NH2-terminal sequences from Bcr fused to COOH-terminal sequences from Abl. In a yeast two-hybrid screen, we have identified TSG101 as a binding partner for Bcr. Because TSG101 is a subunit of the mammalian endosomal sorting complex required for transport (ESCRT), which regulates protein sorting during endosomal trafficking, this association suggests that Bcr may have a related cellular function. The docking site for TSG101 has been mapped to the COOH terminus of Bcr, indicating that this interaction may be disrupted in CML. Overexpression studies with full-length TSG101 and Bcr reveal that this interaction can be recapitulated in mammalian cells. The association can also be observed between natively expressed proteins in a panel of hematopoietic and nonhematopoietic cell lines, where a second subunit of the ESCRT complex, vacuolar sorting protein 28 (Vps28), was also found to interact with Bcr. Both Bcr and TSG101 exhibit a punctate cytoplasmic distribution and seem to colocalize in HeLa cells, which would be consistent with an in vivo association. Bacterially purified Bcr and TSG101 also bind, suggesting that the interaction is direct and is not dependent on ubiquitination. Disruption of the endosomal pathway with an ATPase-defective Vps4 mutant results in the cellular redistribution of Bcr, and suppression of Bcr in HeLa cells by small interfering RNA impairs epidermal growth factor receptor turnover. Taken together, these observations suggest that Bcr is a component of the mammalian ESCRT complexes and plays an important role in cellular trafficking of growth factor receptors.

3T3 Cells↗

PubMed2001

BCR-ABL fails to inhibit apoptosis in U937 myelomonocytic cells expressing a carboxyl-terminal truncated STAT5.

Recent experimental data suggest that one of the major effects of BCR-ABL gene expression in hematopoietic cells is the inhibition of apoptosis. Although the exact mechanisms of this phenomenon are not clear, it is thought to be related to the fact that BCR-ABL induces several signalling pathways also activated by growth factors. In order to determine the anti-apoptotic role of BCR-ABL in a hematopoietic cell line and to by-pass the influence of cytokine-dependence, BCR-ABL gene was expressed in the autonomously growing myelomonocytic U937 cell line using retroviral vectors. There was no resistance to apoptosis induced by either serum deprivation or different doses of etoposide in any U937 clones expressing BCR-ABL protein. In addition to serum deprivation and etoposide, BCR-ABL-expressing clones were not protected from apoptosis induced by TNF, ceramide-C2 and FAS-cross-linking. BCL2 expression was absent in U937 cells and BAX levels were identical between Neo and BCR-ABL clones. To further investigate the mechanisms of this phenomenon, band-shift assays were performed to detect activation of STAT molecules. No constitutive activation of STATs was detected in either NeoR or BCR-ABL-U937 cells, although both IFN-gamma and GM-CSF activated STAT1 and STAT5, respectively, with similar kinetics in both NeoR and BCR-ABL-U937 cells. In addition, the GM-CSF-induced-STAT5 activation was found to be weakened in all clones expressing BCR-ABL. In both control NeoR and BCR-ABL-transfected clones, band-shift assays revealed the presence of an abnormal truncated STAT5 recognized only by an anti-N-terminal but not by an anti-C-Terminal STAT5 antibody. These findings suggest a possible link between the absence of anti-apoptotic potential of BCR-ABL and abnormalities of the STAT5 pathway, including, absence of constitutive activation of STAT5, inhibition of GM-CSF-induced STAT5 activation and expression of a carboxyl-terminal-truncated STAT5.

Apoptosis↗

PubMed1998

The tetramerization domain-independent Ras activation by BCR-ABL oncoprotein in hematopoietic cells.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. We have previously shown that BCR-ABL activates Ras signaling pathways required for transformation. To elucidate the mechanisms whereby BCR-ABL induced transformation in hematopoietic cells, we examined the biological effects of expression of a series of BCR-ABL mutants. We found that the Grb2 binding site-deleted BCR-ABL and the SH2 domain-deleted BCR-ABL, as well as the tetramerization domain-deleted BCR-ABL do not diminish the transforming properties of BCR-ABL in hematopoietic cells, although these mutations were previously shown to drastically reduce the transforming activity of BCR-ABL in fibroblasts. The tetramerization domain-deleted BCR-ABL did not induce tyrosine phosphorylation of CrkL, SHP-2, Vav and the interactions of BCR-ABL and Shc. However, Ras is activated, Shc is tyrosine phosphorylated and binds to Grb2 in the tetramerization domain-deleted BCR-ABL expressing hematopoietic cells. These results suggest that the tetramerization domain-independent Ras activation is mediated by Shc proteins and induces the transformation of hematopoietic cells.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.

Animals↗

PubMed1999

Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia.

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-alpha) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had < or =34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P < 0.0001) or BCR-ABL/ABL ratio (r = 0.86, P < 0.0001) was found. For patients that underwent the procedure in early chronic phase, Ph-negative collections showed different levels of BCR-ABL expression. BCR-ABL transcript numbers varied from a median of 100/microg RNA in the first and second leukaphereses, to 500/microg RNA in the third and fourth leukaphereses, and 1500/microg RNA in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (< or =0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (chi2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.

Adult↗

PubMed1996

Lack of correlation between ABL-BCR expression and response to interferon-alpha in chronic myeloid leukaemia.

Interferon-alpha (IFN-alpha) is useful in the treatment of Philadelphia (Ph)-positive chronic myeloid leukaemia (CML). There is, however, a marked heterogeneity among CML patients in relation to their response to IFN-alpha treatment, the reasons for which are unknown. Since the reciprocal ABL-BCR gene is transcriptionally active in only a proportion of CML patients, it has been suggested that response to IFN-alpha may correlate with ABL-BCR expression. In the present study we have tested 209 Ph-positive CML patients for expression of ABL-BCR, BCR-ABL and the normal BCR and ABL genes by reverse transcriptase/polymerase chain reaction (RT/PCR). Whereas BCR-ABL, BCR and ABL transcripts were detected in all the patients, ABL-BCR expression was observed in 59% of the cases. A group of 105 patients within this series was treated with IFN-alpha; 33% achieved a complete or major cytogenetic response (< 35% Ph-positive metaphases) and the remaining 67% showed minimal or no response to IFN-alpha. The proportions of patients who were ABL-BCR positive (63%) and ABL-BCR negative (37%) were the same for good responders and poor responders, suggesting that there is no correlation between ABL-BCR expression and cytogenetic response to IFN-alpha in CML.

Chimera↗

PubMed1999

Quantification of minimal residual disease in patients with BCR-ABL-positive acute lymphoblastic leukaemia using quantitative competitive polymerase chain reaction.

We analysed 20 patients with BCR-ABL-positive acute lymphoblastic leukaemia (ALL) by quantitative competitive polymerase chain reaction (QC-PCR) to study the kinetics of the leukaemic clone. Consecutive samples of 16 patients (minor-bcr, n = 10; major-bcr, n = 6) were analysed after conventional chemotherapy and/or bone marrow transplantation (BMT). DNA competitor templates co-amplifying with either p210 or p190 BCR-ABL cDNA were used for quantification of leukaemia-specific BCR-ABL mRNA. In all samples, total ABL transcripts were measured as internal control, and the percentage of BCR-ABL/ABL molecules was calculated. Following induction chemotherapy the number of BCR-ABL transcripts was reduced by a maximum of 2-3 logs. In most patients, additional chemotherapy did not lead to further reduction of BCR-ABL mRNA. In two patients, conventional chemotherapy plus autologous BMT in complete haematological remission resulted in a total reduction of the transcript level of more than 3 logs. In two other patients, allogeneic BMT caused a transient reduction of the BCR-ABL transcripts below the detection level of our method (<1 blast cell in 105 normal cells) for a period of 7 and 11 months, respectively. The achievement of PCR negativity did not guarantee sustained remission. Both patients relapsed and BCR-ABL transcript levels rose by more than 1 log prior to frank relapse. Our data demonstrate that quantification of BCR-ABL mRNA allows the evaluation of the dynamics of the leukaemic clone and thus is valuable for the evaluation of minimal residual leukaemia following various therapies and the early detection of increasing BCR-ABL transcripts prior to relapse.

Adult↗

PubMed2000

The influence of the cell cycle, differentiation and irradiation on the nuclear location of the abl, bcr and c-myc genes in human leukemic cells.

abl and bcr genes play an important role in the diagnostics of chronic myelogenous leukemia (CML). The translocation of these genes results in an abnormal chromosome 22 called the Philadelphia chromosome (Ph). The chimeric bcr-abl gene is a fundamental phenomenon in the pathogenesis of CML. Malignant transformation of hematopoietic cells is also accompanied by the c-myc gene changes (translocation, amplification). Nuclear topology of the abl, bcr and c-myc genes was determined in differentiated as well as in irradiated HL-60 cells using dual-colour fluorescence in situ hybridisation and image analysis by means of a high resolution cytometer. After the induction of the granulocytic differentiation of HL-60 cells with all trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO), the abl and bcr homologous genes were repositioned closer to the nuclear periphery and the average distances between homologous abl-abl and bcr-bcr genes as well as between heterologous abl-bcr genes were elongated as compared with untreated human leukemic promyelocytic HL-60 cells. Elongated gene-to-gene and centre-to-gene distances were also found for the c-myc gene during granulocytic differentiation. In the case of the monocytic maturation of HL-60 cells treated with phorbol esters (PMA), the abl and bcr homologous genes were repositioned closer to each other and closer to the nuclear centre. The position of the c-myc gene did not change significantly after the PMA stimulus. The proximity of the abl and bcr genes was also found after gamma irradiation using 60Co (5 Gy). Immediately after the gamma irradiation c-myc was repositioned closer to the nuclear centre, but 24 h after radiation exposure the c-myc position returned back to the pretreatment level. The c-myc gene topology after gamma irradiation (when the cells are blocked in G2 phase) was different from that detected in the G2 sorted control population. We suggest that changes in the abl, bcr and c-myc topology in the case of gamma irradiation are not the effects of the cell cycle. It is possible, that differences in the cell cycle of hematopoietic cells after the gamma irradiation and concurrent proximity of the abl, bcr and c-myc genes could be important from the point of view of contingent gene translocations, that are responsible for malignant transformation of cells.

Cell Cycle↗

PubMed2005

Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia.

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.

Acute-Phase Proteins↗

PubMed2006

Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against Bcr-Abl-expressing human leukemia cells.

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) has potent in vitro and in vivo activity against the unmutated and most common mutant forms of Bcr-Abl. Treatment with the histone deacetylase inhibitor LBH589 (Novartis) depletes Bcr-Abl levels. We determined the effects of AMN107 and/or LBH589 in Bcr-Abl-expressing human K562 and LAMA-84 cells, as well as in primary chronic myelogenous leukemia (CML) cells. AMN107 was more potent than imatinib mesylate (IM) in inhibiting Bcr-Abl tyrosine kinase (TK) activity and attenuating p-STAT5, p-AKT, Bcl-x(L), and c-Myc levels in K562 and LAMA-84 cells. Cotreatment with LBH589 and AMN107 exerted synergistic apoptotic effects with more attenuation of p-STAT5, p-ERK1/2, c-Myc, and Bcl-x(L) and increases in p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of Bcr-Abl or the IM-resistant, point-mutant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis of IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared with either agent alone, cotreatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells. Thus, cotreatment with LBH589 and AMN107 is active against cultured or primary IM-resistant CML cells, including those with expression of Bcr-AblT315I.

Animals↗

PubMed2001

The SH2 domain of bcr-Abl is not required to induce a murine myeloproliferative disease; however, SH2 signaling influences disease latency and phenotype.

Bcr-Abl plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It was previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. This in vivo experimental system allows the direct determination of the effect of specific domains of Bcr-Abl, or specific signaling pathways, on the complex in vivo pathogenesis of CML. In this report, the function of the SH2 domain of Bcr-Abl in the pathogenesis of CML is examined using this murine model. It was found that the Bcr-Abl SH2 mutants retain the ability to induce a fatal MPD but with an extended latency compared with wild type (wt) Bcr-Abl. Interestingly, in contrast to wt Bcr-Abl-induced disease, which is rapid and monophasic, the disease caused by the Bcr-Abl SH2 mutants is biphasic, consisting of an initial B-lymphocyte expansion followed by a fatal myeloid proliferation. The B-lymphoid expansion was diminished in mixing experiments with bcr-abl/DeltaSH2 and wt bcr-abl cells, suggesting that the Bcr-Abl-induced MPD suppresses B-lymphoid expansion.

3T3 Cells↗

PubMed2003

Inhibition of wild-type and mutant Bcr-Abl by pyrido-pyrimidine-type small molecule kinase inhibitors.

Imatinib mesylate (STI571, Glivec), a 2-phenylaminopyrimidine small-molecule ATP competitor-type kinase inhibitor, proved to be active in Philadelphia-positive leukemias. Resistance toward imatinib develops frequently in advanced-stage Philadelphia-positive leukemia, and is even observed in chronic-phase chronic myelogenous leukemia. Point mutations within the BCR-ABL kinase domain emerged as a major mechanism of resistance toward imatinib. Mutations occur at positions that determine specific contacts of imatinib to the ATP-binding site. We aimed to examine whether pyrido-pyrimidine-type kinase inhibitors were capable of inhibiting both wild-type and mutant forms of BCR-ABL. We screened 13 different pyrido-pyrimidine with cells expressing wild-type and mutant BCR-ABL. All of the substances specifically suppressed the Bcr-Abl dependent phenotype and inhibited Bcr-Abl kinase activity with higher potency than imatinib. Two of the most active compounds were PD166326 and SKI DV-M016. Interestingly, these compounds suppressed the activation loop mutant Bcr-Abl H396P as effectively as wild-type Bcr-Abl. In addition, nucleotide-binding loop mutations (Y253H, E255K, and E255V) were selectively and potently inhibited. In contrast, T315I, a mutant located at a position that makes a direct contact with imatinib, was not affected. This observation is consistent with the hypothesis that unlike imatinib, pyrido-pyrimidine inhibitors bind Bcr-Abl regardless of the conformation of the activation loop. We conclude that pyrido-pyrimidine-type kinase inhibitors are active against different frequently observed kinase domain mutations of BCR-ABL that cause resistance toward imatinib. Resistance as a consequence of selection of mutant BCR-ABL by imatinib may be overcome using second-generation kinase inhibitors because of their higher potency and their ability to bind Bcr-Abl irrespective of the conformation of the activation loop.

Animals↗

PubMed1988

Identification of two normal bcr gene products in the cytoplasm.

A monoclonal antibody (7C6) has been derived against a synthetic bcr peptide and used to study normal bcr gene products. The expression of a bcr phosphoprotein of 130 kd was demonstrated, in addition to the previously identified bcr phosphoprotein of 160 kd. Sequential immunoprecipitation demonstrated that both p160 and p130 had determinants from two separate regions of the putative bcr translated sequence. The synthesis of bcr products in Philadelphia positive and negative cells was examined by metabolic labelling and it was shown that the rate of synthesis of the p210 bcr-abl product was comparable with that of the normal bcr products. The in vivo phosphorylation of the p160 exceeded that of the p130 and both normal products were unaffected by the increased phosphorylation of the p210 bcr-abl. There was no evidence with the 7C6 antibody of any normal bcr products larger than 160 kilodaltons. Immunofluorescence analysis by conventional and confocal microscopy identified normal bcr products as cytoplasmic proteins with relatively high expression in the myeloid cell line KGl.

Amino Acid Sequence↗

PubMed1998

Detection of bcr-abl transcript in chronic myelogenous leukemia patients by reverse-transcription-polymerase chain reaction and capillary electrophoresis.

BACKGROUND AND OBJECTIVE: Capillary electrophoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis). DESIGN AND METHODS: The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 microgram of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per microgram of total RNA examined). RESULTS: CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two types of bcr-abl junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (SD) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/microgram total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value 128,859 vs. 331,722 in patients with 0% blast cells and > 1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score < 0.8 and > 1.2, respectively; p = 0.003). INTERPRETATION AND CONCLUSIONS: Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.

Adolescent↗

PubMed1994

Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

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)

Adaptor Proteins, Signal Transducing↗