High Levels Of Bcr-Abl Research Articles

Correlation of C/EBPα expression with response and resistance to imatinib in chronic myeloid leukaemia.

Altered differentiation is a common feature of haematopoietic malignancies with poor prognosis. CAAT/enhancer binding protein alpha (C/EBPα) is a key transcription factor that regulates myeloid differentiation. This study is aimed to know the prognostic value of CAAT/enhancer binding protein alpha expression and correlate its expression with response to imatinib therapy. We quantified the expression of C/EBPα gene in 126 chronic myeloid leukaemia samples (82 from newly diagnosed and 44 from imatinib-resistant patients) and 20 control samples. C/EBPα mRNA level was measured by real-time quantitative polymerase chain reaction using the ΔΔCT method. C/EBPα expression level was significantly lower in the imatinib-resistant group than in the pretreatment and control group (P = 0.0398). Low CAAT/enhancer binding protein alpha levels in the imatinib-resistant group were significantly associated with advanced phase (P = 0.04), with more peripheral blasts (P = 0.0001), high BCR-ABL levels (P = 0.018) and T315I and P-loop mutations (P = 0.0002). In the pretreatment group, low expression showed association with high EUTOS risk score (P = 0.03) and possible partial cytogenetic response (P = 0.010). Our results suggest that low expression of CAAT/enhancer binding protein alpha might have a role in the response to imatinib and progression of disease in patients with chronic myeloid leukaemia.

Velocity of early BCR-ABL transcript elimination as an optimized predictor of outcome in chronic myeloid leukemia (CML) patients in chronic phase on treatment with imatinib.

Early assessment of response at 3 months of tyrosine kinase inhibitor treatment has become an important tool to predict favorable outcome. We sought to investigate the impact of relative changes of BCR-ABL transcript levels within the initial 3 months of therapy. In order to achieve accurate data for high BCR-ABL levels at diagnosis, beta glucuronidase (GUS) was used as a reference gene. Within the German CML-Study IV, samples of 408 imatinib-treated patients were available in a single laboratory for both times, diagnosis and 3 months on treatment. In total, 301 of these were treatment-naïve at sample collection. (i) with regard to absolute transcript levels at diagnosis, no predictive cutoff could be identified; (ii) at 3 months, an individual reduction of BCR-ABL transcripts to the 0.35-fold of baseline level (0.46-log reduction, that is, roughly half-log) separated best (high risk: 16% of patients, 5-year overall survival (OS) 83% vs 98%, hazard ratio (HR) 6.3, P=0.001); (iii) at 3 months, a 6% BCR-ABL(IS) cutoff derived from BCR-ABL/GUS yielded a good and sensitive discrimination (high risk: 22% of patients, 5-year OS 85% vs 98%, HR 6.1, P=0.002). Patients at risk of disease progression can be identified precisely by the lack of a half-log reduction of BCR-ABL transcripts at 3 months.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

High BCR-ABL Levels At Diagnosis Are Associated with Unfavorable Responses to Imatinib Mesylate.

AbstractAbstract 2790The approval of second-generation tyrosine kinase inhibitors (TKIs) for the first line treatment of Chronic Myeloid Leukemia (CML) has generated a need for early molecular parameters associated with inadequate responses to Imatinib Mesylate (IM).Recent evidence suggests that CML patients presenting BCR-ABL/ABLIS levels >10% after 3 months of IM or >1% after 6 months of treatment have inferior outcomes in terms of both overall survival (OS) and progression-free survival. We wanted to establish if high BCR-ABL transcripts at diagnosis would also be associated with unfavorable responses to IM. To this end, we correlated quantitative determinations of BCR-ABL measured at diagnosis with the outcome of 230 newly diagnosed CML patients receiving IM 400 mg/die. BCR-ABL transcripts were measured from peripheral blood samples drawn at diagnosis using Real-Time Quantitative PCR (RQ-PCR). All molecular determinations were performed twice (in triplicates) on the same sample using either ABL or glucuronidase-beta (GUS) as reference genes.Median follow-up of the study population was 42 months. Estimated 5-year cumulative incidences of complete hematologic response, complete cytogenetic response (CCyR) and major molecular response were 97.9%, 89.5% and 64.7%. Five-year probabilities of OS, transformation-free survival (TFS: survival without disease transformation to the accelerated phase or blast crisis) and failure-free survival (FFS: survival without IM failure as defined by the 2009 European Leukemia Net recommendations) were 93.8%, 97.8% and 76%. Correlations between high BCR-ABL transcripts at diagnosis and unsatisfactory IM responses were much stronger using GUS in place of ABL as a reference gene. Indeed, while elevated BCR-ABL/GUSIS (p<0.0001) and elevated BCR-ABL/ABLIS (p<0.0001) both correlated with inferior probabilities of optimal response, high BCR-ABL/GUSIS (p<0.0001) but not high BCR-ABL/ABLIS (p=0.18) was associated with lower rates of CCyR after 12 months of IM. Neither BCR-ABL/GUSIS nor BCR-ABL/ABLIS at diagnosis were predictive of OS. However, high BCR-ABL/GUSIS was more accurately associated with lower probabilities of FFS (p<0.0001) and TFS (p=0.01) as compared to BCR-ABL/ABLIS (p=0.02 for FFS; p=0.36 for TFS). When we employed the 2009 European Leukemia Net criteria to subdivide our patient cohort in optimal responders, suboptimal responders and individuals failing IM, we found that elevated BCR-ABL/GUSIS (p<0.0001) was superior to elevated BCR-ABL/ABLIS (p<0.004) in distinguishing patient outcome. Furthermore, while BCR-ABL/ABLIS at diagnosis only discriminated optimal from resistant subjects (p=0.005), BCR-ABL/GUSIS transcripts were significantly different between the three patient groups (optimal vs suboptimal p=0.0002; optimal vs resistant p<0.0001; suboptimal vs resistant p<0.0001). Using receiver operating characteristic curves and the achievement of an optimal response as a specific endpoint, we found that 16.46% BCR-ABL/GUSISat diagnosis defined a threshold distinguishing low risk from high risk patients.High BCR-ABL transcripts at diagnosis measured by RQ-PCR employing GUS as a reference gene allow the identification of CML patients unlikely to benefit from IM that should receive alternative forms of treatment. Disclosures:Turri:Novartis: Consultancy, Novartis Other; Bristol Myers Squibb: Bristol Myers Squibb, Bristol Myers Squibb Other, Consultancy. Morabito:Celgene: Honoraria.

Targeting the CML Stem/Progenitor Cell with JAK2 (BMS-911543)/ABL Inhibitor Combination Therapy

Abstract 2743The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene that originates in hematopoietic stem cells. The BCR-ABL oncoprotein has constitutively elevated tyrosine kinase (TK) activity and drives CML pathogenesis. The first specific BCR-ABL inhibitor, Imatinib Mesylate (IM), has had a major impact on the treatment of chronic phase CML. However, early relapse and molecular evidence of residual disease remain a significant issue in some IM-treated patients. We and others have recently demonstrated that CML stem/progenitor cells are insensitive to IM, genetically unstable, and characterized by elevated BCR-ABL expression and TK activity. The prevention of the development of resistant subclones through the specific targeting of CML stem/progenitor cells consequently represents a key unmet medical need in CML therapy. We recently identified the presence of a physical complex between oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and JAK2. This molecular complex contributes to the transforming activity of BCR-ABL and plays a key role in the IM responsivity/resistance of primary CML stem/progenitor cells. We therefore hypothesized that a combination treatment strategy designed to destabilize the complex might provide a rational approach to the elimination of CML stem/progenitor cells. Several JAK2 inhibitors are currently in various stages of clinical development, but myelosuppressive effects of these inhibitors on normal hematopoietic stem/progenitor cells remains a concern. We have examined a new and selective JAK2 inhibitor (BMS-911543) in combination with a number of different tyrosine kinase inhibitors (TKIs), including IM, dasatinib (DA), and nilotinib (NL) in CML cells. First, Western blot analysis showed that combination treatment with IM and BMS-911543 was more effective at reducing pSTAT5 levels in K562 cells and IM-resistant K562 cells (K562R) than in cells treated with BMS-911543 or IM alone. BMS-911543 treatment alone was not though effective in reducing pSTAT5 levels. Consistent with the Western blot analysis, in colony-forming cell (CFC) assays CML cell lines derived from myeloid or B-lymphoid blast crisis patient samples that expressed high levels of BCR-ABL (K562, K562R, BV173, and UT7 transduced with BCR-ABL) demonstrated reduced colony numbers, especially in medium-sized colonies (50–500 cells per colony) following combination treatment (BMS-911543 and IM) as compared with cells treated with BMS-911543 or IM alone (∼2–3 fold for all cell lines, p<0.05). To determine whether the results obtained following the simultaneous targeting of BCR-ABL and JAK2 in CML cell lines would extend to primary primitive CML cells and whether this combined BCR-ABL-JAK2 targeting approach could also be therapeutically effective for CML patients with inadequate responses to treatment with TKI monotherapy, we investigated CML cells obtained at the time of diagnosis from four CML patients who were classified retrospectively after the initiation of IM therapy as non-responders. To assess the effects on CFCs, CD34+ cells isolated from these samples were plated directly in methylcellose medium containing growth factors and TKIs (5μM IM and NL and 150nM DA) and BMS-911543 (100 and 300nM) alone, or in combination. The number of colonies obtained in cultures containing the respective TKIs and BMS-911543 alone was reduced from control values by about 50%. However, when TKI and BMS-911543 was present, a more significant reduction in colony formation was observed (77–86% inhibition of colony growth, p<0.05). Interestingly, combination treatment significantly inhibits BFU-E colony formation as compared with treatment with TKI alone (92–100% inhibition for the combinations vs 63–66% inhibition for the single agents, p<0.05). Most significantly, our data indicate far less toxicity on normal bone marrow CD34+ cells (n=4) as compared with CML samples treated with either single agent or combination therapy (2–3 fold from 100 to 600nM, p<0.05). The combination approach appears to be effective against treatment-naïve CML stem/progenitor cells derived from patients who were subsequently shown to be clinically resistant to IM therapy, suggesting a rational strategy for improving the clinical outcome of CML patients destined to develop IM resistance that involves the simultaneous targeting of both BCR-ABL and JAK2 in CML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

Minimal residual disease (MRD), both before and after transplantation, is a clinically important yet relatively poorly defined aspect of allogeneic hematopoietic stem cell transplantation (alloHSCT). The clinical relevance of MRD in the context of alloHSCT has been demonstrated by its association with the development of clinical relapse. However, with the possible exception of chronic myeloid leukemia (CML), the specific techniques, timing, frequency, and clinical utility, relative to improvement in patient outcomes, for monitoring MRD in the setting of alloHSCT has yet to be clearly defined. A concise overview of monitoring techniques for detecting MRD, as well as treatment strategies and biological and clinical research initiatives for MRD suggested by the National Cancer Institute First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation, is covered in this article.

Design, synthesis and evaluation of ( E)-α-benzylthio chalcones as novel inhibitors of BCR-ABL kinase

Novel ( E)-α-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells.

Analysis of Molecular Data and the Emergence of Mutations for Chronic-Phase Chronic Myelogenous Leukemia (CML-CP) Patients Treated with Dasatinib After Imatinib Failure.

Abstract 3282Poster Board III-1The kinase inhibitor dasatinib has demonstrated efficacy in patients with CML-CP who fail imatinib due to resistance or intolerance. Imatinib failure is often associated with the acquisition of resistant mutations within BCR-ABL. Failure of dasatinib is associated with a limited spectrum of mutations, which are T315I/A, V299L, and F317L/I/V, suggesting these mutations are dasatinib resistant (DR). Only a fraction of patients with T315I respond to dasatinib, most patients with F317L have hematologic responses but few have cytogenetic responses, and V299L is uncommon in imatinib-resistant patients but is mostly seen after dasatinib failure. In vitro data suggests Q252H and E255K/V also confer a degree of dasatinib resistance. However, their clinical association with dasatinib resistance or inferior response is unclear. This retrospective analysis seeks to determine if the mutation status at the start of dasatinib impacts the initial molecular response, and to examine the type of mutations lost and gained during therapy. To assess the significance of intermediate sensitivity (DI) mutations (Q252H and E255K/V), BCR-ABL levels were measured against the International Scale (IS), and mutation status was assayed at initiation of dasatinib (baseline), at progression, or study termination or completion (progression/completion). Data were taken from 479 patients with CML-CP treated with dasatinib 70 mg twice daily, after imatinib failure for the phase II START-C or -R studies. Patients were grouped according to their baseline mutation status: DR, DI, dasatinib sensitive (DS, all other mutations not DR or DI), and no mutations. Patients with more than one mutation at baseline that qualified for more than one grouping were rare, and classified according to the most resistant mutation. By 6 months of dasatinib therapy, patients with DR mutations at baseline had the highest levels of BCR-ABL transcripts (Table 1). By comparison, those with DI, DS, or no mutations had a reduced proportion with high transcript levels. In addition, no patients with DR at baseline achieved a reduction of BCR-ABL by 6 months to IS ratio < 1, whereas significant reductions were identified in patients with DI, DS, or no mutations. A subset of 267 patients had mutation analysis performed at progression/completion. Of these, 115 had mutations at baseline and 152 had no mutations. The patients with baseline imatinib resistance were more likely than those with imatinib intolerance to acquire new mutations. Of the resistant patients with no mutations at baseline, 7% (9/124) developed new mutations and the majority (7/9) was DR. Among resistant patients with baseline mutations, 23% (26/112) had new mutations and the majority was DR (20/26). Although 36 patients gained new mutations (including 16 T315I/A, 8 F317L, and 6 V299L), many baseline mutations were no longer detectable at progression/completion. DS mutations were lost in 68/102 patients. In total, 76 patients had detectable mutations at progression/completion, with 51% (39/76) having DR mutations but only 5% (4/76) with DI mutations. Patients with baseline DR mutations were more likely to retain their DR mutations (11/13). Of 8 patients with DI mutations at baseline, only 3 retained their DI mutations and 3 gained DR mutations. Of 94 patients with only DS mutations at baseline, 36 (38%) retained their DS mutations, and 17 (18%) developed DR mutations. Patients with high levels of BCR-ABL at 3 months had the highest incidence of new DR mutations at progression/completion (18/145 evaluable patients; 12%) compared to patients with lower levels (5/79; 6%). In conclusion, the development of new mutations during second-line dasatinib is rare (36/267). Patients who harbored DI mutations at baseline were also rare, and an expanded prospective analysis of dasatinib efficacy in this cohort may be necessary to clarify their significance. However, from the current analysis there is no compelling clinical evidence to suggest that these patients have an inferior molecular response. Relative to DR mutations, the DI mutations (Q252H and E255K/V) were rarely present at progression/completion.Patients with baseline mutations and 6-month BCR-ABL transcript level after second-line dasatinibIS ratio ≤ 1.01 < IS ratio ' 10IS ratio > 10DR0% (0/13)23% (3/13)77% (10/13)DI56% (5/9)0% (0/9)44% (4/9)DS36% (46/128)20% (25/128)45% (57/128)No mutations42% (92/221)18% (40/221)40% (89/221) Disclosures:Branford:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Hochhaus:Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Bahceci:Bristol-Myers Squibb: Employment. Ploughman:Bristol-Myers Squibb: Employment. Mukhopadhyay:Bristol-Myers Squibb: Employment. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding.

Persistence of Leukemia Stem Cells in Chronic Myelogenous Leukemia Patients in Complete Cytogenetic Remission on Imatinib Treatment for 5 Years

Imatinib mesylate (IM) treatment results in marked reduction in burden of leukemia cells in chronic myelogenous leukemia (CML) patients, as indicated by achievement of complete cytogenetic remission and major reduction in Bcr-Abl transcript levels on Q-PCR analysis. However patients treated with IM alone without prior interferon treatment appear to invariably relapse on discontinuation of IM treatment. In addition we and others have shown that residual Bcr-Abl+ progenitors persist in IM-treated CML patients following achievement of CCR. These observations suggest that despite its remarkable activity in CML, IM fails to eliminate all malignant stem and progenitor cells in CML patients. However our previous studies were conducted on patients within the first year or two of IM treatment, whereas recent studies have indicated that Bcr-Abl levels continue to decline on Q-PCR analysis with continued IM treatment. This together with the decreasing rate of disease relapse observed after 3 years of IM treatment raises the possibility that prolonged IM treatment may cause depletion of residual CML stem cells. In this study we investigated whether prolonged IM treatment was associated with a reduction in Bcr- Abl+ stem and progenitor cells. We evaluated 14 CML patients followed at our center who were in CCR, had been treated with IM for at least 4 years, and from whom multiple cryopreserved bone marrow samples were available for study. Bone marrow mononuclear cells (MNC) were thawed, CD34+ cells were selected by immunomagnetic columns, and CD34+CD38+ (38+) committed progenitors and CD34+CD38− (38−) stem/primitive progenitor cells were isolated by flow cytometry sorting. Q-PCR analysis of Bcr-Abl and Bcr transcript levels was performed on RNA isolated from MNC, 38+ and 38− cells and Bcr-Abl levels were reported as the ratio of Bcr-Abl to Bcr. Bcr-Abl levels in MNC were 0.010±0.005, 0.011± 0.005 and 0.013±0.005 at 3, 4 and 5 years. We observed that Bcr-Abl levels were higher in both 38+ and 38− cells in comparison with levels in MNC. A gradual decline in Bcr-Abl levels in 38+ cells was seen (0.285±0.185 at 3 years, 0.121±0.056 at 4 years, and 0.071±0.028 at 5 years). In contrast high Bcr-Abl levels were maintained in the 38− fraction despite continued IM treatment (0.162±0.086 at 3 years, 0.116±0.041 at 4 years, and 0.361±0.107 at 5 years). In contrast to IM-treated patients, Bcr-Abl transcripts were not detected in MNC and CD34+ cells from BM of CML patients who had received allogeneic hematopoietic cell transplants (n=5). To further investigate whether malignant stem cells persisted after prolonged IM treatment, MNC from 5 of the patients described above were transplanted by tail vein injection into sublethally irradiated NOD/SCID-IL2Rγ-chain knockout (NSG) mice. High levels of human cell engraftment were observed 4–5 weeks after injection, and Q-PCR analysis revealed high levels of Bcr-Abl expression in engrafted cells from 4 of 5 patients, confirming the presence of Bcr-Abl+ cells with NOD/SCID mouse repopulating capacity. In conclusion, our results clearly demonstrate the persistence of Bcr-Abl+ stem cells in the BM of CML patients in prolonged remission after 5 years of IM treatment. The observed persistence of leukemia stem cells raises the concern that patients remain at risk for relapse on drug discontinuation or through acquisition of IM resistance. The assays described here may have considerable utility for evaluating and monitoring the effects of experimental treatment strategies directed against residual CML stem cells.

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

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.

Effects of Elevated BCR/ABL Expression Level on Cellular Transformation and Sensitivity to Imatinib in Primitive Human Hematopoietic Cells.

Increased levels of BCR/ABL (BA) expression in CML hematopoietic cells have been associated with disease progression and resistance to the tyrosine kinase inhibitor imatinib mesylate (IM). Although cell lines with varying levels of BA expression have been studied, the role of elevated BA expression in cell transformation and drug resistance has not been directly evaluated in the primitive human hematopoietic cells in which the disease arises. Here we have used a human transduction model of CML to determine the effects of varying BA expression levels on cellular transformation (proliferation, apoptosis and differentiation) as well as responsiveness to IM. Cord blood (CB) CD34+ cells were transduced with MSCV vectors expressing BA and GFP, or control vectors expressing GFP alone followed by CD34+GFP+ cells selection by flow cytometry sorting. For BA expressing cells, two separate populations were selected based on low or high GFP expression (BAlo and BAhi). Quantitative RT-PCR analysis confirmed increased expression of BA in high GFP expressing cells (5.9±1.5 fold increase in BA:B2M levels in BAhi compared with the BAlo cells, n=3). The proliferation rate of BAhi cells, measured by fold expansion after 3 days of growth factor (GF) culture, was 2.0±0.2 fold higher than BAlo cells, and 6.7±0.1 fold higher than control (n=3). Upon GF deprivation, BAhi cells demonstrated increased resistance to apoptosis (24.4±11.4%, n=3) compared with BAlo (42±12%, n=3) or control cells (45±12%, n=3). BA transduced CD34+ cells generated higher numbers of glycophorin A+ cells than control (35.6%) following GF culture for 7 days. This effect was enhanced in BAhi (91.5%) compared with BAlo cells (77.1%). This was accompanied by an increase in CD33+ myeloid cells and a decrease in CD11b+ cells (36.3, 60.1 and 65.2% CD33+ cells and 6.0, 1.8 and 0.5% CD11b+ cells for control, BAlo and BAhi cells respectively). In addition the frequency of CD41a+ megakaryocytic cells was higher in BAhi (7.0%) relative to BAlo (3.1%) and control cells (1.0%). Next, we asked whether elevated expression of BA resulted in altered sensitivity to IM (0.025μM to 1μM) in an MTS assay. We observed that BAhi cells were more sensitive (83% inhibition at 1μM) to the IM compared with BAlo cells (14% inhibition at 1μM). We also investigated whether elevated levels of expression of two BA kinase mutants, M351T and E255K, were associated with altered IM sensitivity in CD34+ cells. The M351T mutation leads to intermediate level of IM resistance in cell lines. As was observed for wild type BA, CD34+ cells expressing higher levels of M351T demonstrated increased sensitivity to IM. On the other hand, cells expressing high and low levels of the E255K mutant, which is associated with high levels of IM resistance, demonstrated similar levels of IM sensitivity. In conclusion, increased levels of BA expression in human CD34+ cells results in enhanced proliferation; increased resistance to apoptosis following GF withdrawal, and altered differentiation with increased expression of erythroid, megakaryocytic and early myeloid markers and reduced expression of mature myeloid markers. Interestingly, expression of high levels of BA was associated with enhanced rather than reduced sensitivity to IM. Taken together these observations suggest that the effects of varying levels of BA expression on imatinib sensitivity in primitive human hematopoietic cells are determined primarily by increased proliferation rather than reduced apoptosis resulting from enhanced BA expression.

Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia

Aberrant genome-wide hypomethylation is thought to be related to tumorigenesis by promoting genomic instability. Since DNA methylation is considered an important mechanism for the silencing of retroelements, hypomethylation in human tumors may lead to their reactivation. However, the role of DNA hypomethylation in chronic myeloid leukemia (CML) remains to be elucidated. In this study, the methylation status of the LINE-1 (L1) retrotransposon promoter was analysed in CML samples from the chronic-phase (CP, n=140) and the blast crisis (BC, n=47). L1 hypomethylation was significantly more frequent in BC (74.5%) than in CP (38%) (P<0.0001). Furthermore, L1 hypomethylation led to activation of both ORF1 sense transcription (P<0.0001) and c-MET gene antisense transcription (P<0.0001), and was significantly associated with high levels of BCR-ABL (P=0.02) and DNMT3b4 (P=0.001) transcripts. Interestingly, in CP-CML, extensive L1 hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (P=0.004) or imatinib (P=0.034) and progression-free survival (P=0.005). The above results strongly suggest that activation of both sense and antisense transcriptions by aberrant promoter hypomethylation of the L1 elements plays a role in the progression and clinical behavior of the CML.

Dose-dependent effects of Bcr-Abl in cell line models of different stages of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is caused by Bcr-Abl, an activated tyrosine kinase. The amounts of Bcr-Abl mRNA and protein in cells from patients in blast crisis (BC) are higher than in those chronic phase (CP), indicating that their expression rises with disease progression. In order to study this phenomenon on cells with the same genetic background, we transfected the 32D cell line with the BCR-ABL transgene and selected clones with graded expression of Bcr-Abl within the range found in cells from CP to BC. In vitro, we found that Bcr-Abl exerted dose-dependent effects upon growth factor dependence, clonogenicity and migration. However, the relationship between Bcr-Abl expression and cellular adhesion to fibronectin was more complex: rather than a direct positive correlation, clones that expressed low, but not high, levels of Bcr-Abl were less adhesive than growth factor stimulated, parental BCR-ABL-negative 32D cells. This finding parallels the situation with normal and CML-CP progenitors where the latter exhibit defective adhesion to fibronectin. Treatment with the tyrosine kinase inhibitor imatinib mesylate reversed the adhesion deficient phenotype of clones expressing low levels of Bcr-Abl. When injected subcutaneously into syngeneic mice, cell lines expressing high levels of Bcr-Abl rapidly induced tumors, whereas low-expressing clones led to tumor formation only after a prolonged latency. These findings suggest that the level of Bcr-Abl may be essential in determining the phenotype of the leukemic clone at different stages of the disease.

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

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.

Kinase-addiction and bi-phasic sensitivity-resistance of Bcr-Abl- and Raf-1-expressing cells to imatinib and geldanamycin

By activating anti-apoptotic factors (e.g., Hsp70, Raf-1, Bcl-xL), Bcr-Abl blocks apoptotic pathways at multiple levels, thus rendering leukemia cells resistant to chemotherapeutic agents such as doxorubicin (DOX). In Bcr-Abl-transfected HL60 (HL/Bcr-Abl) cells, pro-caspase-9 was increased and partially processed. The Bcr-Abl inhibitor imatinib (Gleevec, STI-571) released the apoptotic stream. Also, HL/Bcr-Abl cells were hyper-sensitive to geldanamycin (GA), which depletes Bcr-Abl and Raf-1. Raf-1 and Bcr-Abl-transfected FDC-P1 hematopoietic cells were selectively sensitive to GA and imatinib, respectively. Remarkably, cell clones with high levels of Bcr-Abl that could not be depleted by GA were relatively resistant to both GA and imatinib. GA and flavopiridol sensitized such resistant cells to imatinib. These data suggest bi-phasic sensitivity to mechanism-based therapeutic agents. Although Bcr-Abl renders cells hyper-sensitive, an excess of Bcr-Abl results in resistance (due to the remaining activity). We discuss therapeutic approaches to overcome bi-phasic resistance to mechanisms-based agents.

Bcr-Abl upregulates cytosolic p21WAF-1/CIP-1 by a phosphoinositide-3-kinase (PI3K)-independent pathway.

Chronic myeloid leukaemia invariably progresses from a drug-sensitive to a drug-resistant, aggressive acute leukaemia. The mechanisms responsible for this are unknown, although loss of p53 has been reported in approximately 25% of cases. Elevated expression of Bcr-Abl is also associated with disease progression. We have shown that cells expressing high levels of Bcr-Abl also express elevated levels of p53 and the cell cycle inhibitor, p21WAF-1. Despite this, cells continue to cycle and are drug resistant. As p21WAF-1 inhibitory activity is associated with nuclear localization, we investigated its localization in Bcr-Abl-expressing cells, and found that it is predominantly cytoplasmic. We have also shown that it associates physically with the serine/threonine kinase AKT, but this association and the cytosolic location of p21WAF-1 are phosphinositide-3-kinase (PI3K) independent. Cytosolic p21WAF-1 has been reported to have a prosurvival role in other transformed cells. In Bcr-Abl-expressing cells, p21WAF-1 rapidly diminishes as the cells are sensitized to apoptosis, using the inhibitor STI571. It is possible therefore that p21WAF-1 could also have a positive, prosurvival role in these cells. This study suggests that, by retaining p21WAF-1 in a cytosolic location, Bcr-Abl can evade the cell cycle arrest normally induced by nuclear p21WAF-1 and therefore also enable the cells to negate an important feature of a tumour suppressor response.

Down-regulation of BRCA1 in BCR-ABL–expressing hematopoietic cells

AbstractBCR-ABL fusion oncogene is the molecular hallmark of chronic myelogenous leukemia (CML), a condition characterized by a progression from a chronic to acute phase leukemia because of secondary genetic events, the nature of which remains largely unknown. Here, we report that the expression of the p210 BCR-ABL fusion protein leads to a down-regulation of BRCA1 protein, a gene product involved in the maintenance of genome integrity. BRCA1 protein is nearly undetectable in leukemia cells from patients with CML, both during the chronic phase and in blast crisis. Similarly, stable transfection-enforced expression of p210 protein in established hematopoietic cell lines leads to severe BRCA1 depletion. The lack of significant change in BRCA1 mRNA level in cells expressing p210 supports the hypothesis that the regulation of BRCA1 protein level occurs after transcription. It is abolished on exposure of the cells to STI571 and by mutation in the adenosine triphosphate (ATP) pocket of p210 and thus seems to require the tyrosine kinase activity of BCR-ABL. Cell lines expressing high levels of BCR-ABL display an increased rate of sister chromatid exchange and chromosome aberrations after ionizing radiation. These findings reveal a novel link between the oncoprotein BCR-ABL and the tumor-suppressor protein BRCA1.

Estimations of BCR-ABL/ABL transcripts by quantitative PCR in chronic myeloid leukaemia after allogeneic bone marrow transplantation and donor lymphocyte infusion

Serial assays of qualitative (multiplex and nested) and quantitative PCR were carried out for detecting and estimating the level of BCR-ABL transcripts in 39 CML patients following bone marrow transplantation. Seven of these patients, who received donor lymphocyte infusions (DLIs) following to relapse, were also monitored. Quantitative estimates of BCR-ABL transcripts were obtained by co-amplification with a competitor sequence. Estimates of ABL transcripts were used, an internal control and the ratio BCR-ABL/ABL was thus estimated for evaluating the kinetics of residual clones. Twenty four patients were followed shortly after BMT; two of these patients were in cytogenetic relapse coexisting with very high BCR-ABL levels while other 22 were in clinical, haematologic and cytogenetic remission 2–42 months after BMT. In this latter group, seven patients showed a favourable clinical-haematological progression in association with molecular remission while in 14 patients quantitative PCR assays indicated molecular relapse that was not associated with an early cytogenetic–haematologic relapse. BCR-ABL/ABL levels could not be correlated with presence of GVHD in 24 patients after BMT. In all seven patients treated with DLI, high levels of transcripts were detected at least 4 months before the appearance of clinical haematological relapse. Following DLI, five of these patients showed decreasing transcript levels from 2 to 5 logs between 4 and 12 months. In eight other patients studied long after BMT, five showed molecular relapse up to 117 months post-BMT and only one showed cytogenetic relapse. Our findings indicated that quantitative estimates of BCR-ABL transcripts were valuable for monitoring minimal residual disease in each patient.

Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype.

A characteristic feature of chronic myeloid leukaemia (CML) is the inevitable advancement from a treatable chronic phase to a fatal, drug-resistant stage referred to as blast crisis. The molecular mechanisms responsible for this disease transition remain unknown. As increased expression of Bcr-Abl has been associated with blast crisis CML, we have established transfectants in 32D cells that express low and high levels of Bcr-Abl, and assessed their drug sensitivity. Cells with high Bcr-Abl expression levels are resistant to conventional cytotoxic drugs, and also require higher levels of STI571 (an inhibitor of Bcr-Abl), to induce cell death. Co-treatment with cytotoxic drugs and STI571 increased the sensitivity of the drug-resistant cells. Despite the drug-resistant phenotype, high Bcr-Abl levels concomitantly increased the expression of p53, p21, Bax and down-regulated Bcl-2. These cells maintain a survival advantage irrespective of a reduced proportion of cycling cells and the pro-apoptotic shift in gene expression. In addition, the level of Bcr-Abl expression (high or low) does not alter the growth factor independence and elevated Bcl-xL expression observed. Our study indicates that drug resistance can be primarily attained by increased Bcr-Abl expression, and highlights the potential of therapy which combines STI571 with conventional cytotoxic drugs.

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7

There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors.