Imatinib-sensitive K562 Research Articles

Methyl-β-cyclodextrin induces programmed cell death in chronic myeloid leukemia cells and, combined with imatinib, produces a synergistic downregulation of ERK/SPK1 signaling

Lipid rafts mediate several survival signals in the development of chronic myeloid leukemia (CML). Methyl-β-cyclodextrin (MβCD) is an inhibitor specifically designed to disrupt lipid rafts in cells by depleting the cholesterol component. We hypothesize that treatment of CML cells with MβCD and imatinib could reduce imatinib resistance. Apoptotic and autophagic cell death was assayed using annexin V-propidium iodide double staining, immunoblotting, and immunocytochemistry. We next investigated whether MβCD could enhance the cytotoxicity of imatinib in imatinib-sensitive and imatinib-resistant K562 cells. Extracellular signal-regulated kinase/sphingosine kinase 1 signaling downstream of lipid raft-activated signaling pathways was significantly inhibited by treatment of cells with a combination of MβCD and imatinib compared with treatment with either agent alone. MβCD induces programmed cell death in CML cells, and its antileukemia action is synergistic with that of imatinib.

Suppression of STAT5A increases chemotherapeutic sensitivity in imatinib-resistant and imatinib-sensitive K562 cells

STAT proteins are cytoplasmic transcription factors that are involved in the regulation of numerous cellular activities such as cell growth, differentiation, and survival. In this study, we aimed to identify the expression pattern of STAT genes in imatinib-sensitive and -resistant K562 cells, and further, to reveal the effects of STAT5A siRNA knockdown on cell growth and apoptosis induction. The XTT cell proliferation assay showed that both sensitive and resistant K562 cells were sensitized to imatinib upon transfection with STAT5A siRNA. Caspase-3 enzyme activity was increased significantly in both cells. These results may open up new opportunities to overcome chemotherapeutic resistance in leukemia.

579 Cytotoxic effects of resveratrol on imatinib sensitive and resistant K562 chronic myeloid leukemia cells

579 Cytotoxic effects of resveratrol on imatinib sensitive and resistant K562 chronic myeloid leukemia cells

In Vitro and In Vivo Study of Synergistic Effect of Imatinib and Simvastatin in Chronic Myelogenous Leukemia.

Abstract 2202Poster Board II-179 Background:Statins (HMG-CoA reductase inhibitors) are known to show anti-proliferative effects and are anticipated as a potential drug in the treatment of malignancies. To investigate the effect simvastatin on chronic myelogenous leukemia cells, we treated simvastatin on 3 kinds of CML cell lines and CD34+ primary CML cells from patients: erythrocytic lineage (K-562), granulocytic lineage (KCL-22), erythroid-megakaryocytic (LAMA-84) cell line. Also, antiproliferative effect on imatinib-resistant CML cell lines was assessed. Mehtods:Anti-proliferative effect was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Apoptosis was assessed by Annexin V and Western blot study. Killing effect was calculated by CalcuSyn does effect analysis software (Biosoft, Ferguson, MO). CD34+cells for patients with CML were purified using immunomagnetic bead column. Cell cycle analysis was done by flow cytometrc method. Si-RNA transfection study for p27 gene was performed for verification of killing mechanism. Change of intracellular location of BCR/ABL protein was observed by confocal microscopy. Cellular changes of proteins and tyrosin phosphorylation after treatment of simvastain was evaluated by 2 dimensional electrophoresis and MALDI-TOF/TOF mass spectrophotometer. In vivo effect of simvastain was evaluated in BALB/c-nude. Results:Simvastatin inhibited the proliferation of imatinib-sensitive and imatinib-resistant K562 cell line in a dose dependent manner. The IC50 values of simvastatin and imatinib in imatinib-sensitive K562 cells were 14.5 and 0.4 μM. Treatment of simvastain induced apoptosis both in capase-dependent and caspase-independent pathways in all 3 cell lines; apoptosis by Annexin V analysis and increased apoptotic proteins (cytochrome C, AIF, Smac/Diablo, caspase-3, and caspase-9) by western blot. Cell cycle analysis revealed the G1/S arrest on treatment of simvastatin and si-RNA transfection specific for p27 reversed the G1/S arrest, suggesting cell cycle arrest as one of anti-proliferative mechanism. Co-treatment of imatinib and simvastain showed synergistic killing interactions between simvastatin and imatinib in imatinib-resistant K562 cells (mean combination index values were 0.56, P< 0.001). Enhanced killing effect was observed in all 3 imatinib-resistant CML cell lines (K-562: 0.6, KCL-22: 0.42 , LAMA-84; 0.99). Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. In CML cells, simvastatin inhibited tyrosine phosphorylation included protease, cytocrome-c reductase, DNA/RNA processing proteins, oxidoructase protein, chaperones, glycolysis protein, cytoskeleton proteins, microtubule protein. Treatment of simvastain reduced subcutaneous tumor mass in nude mice. Conclusion:We showed that simvastatin killed CML cells in vitro and in vivo animal model and killing effect occurred via the induction of apoptosis, cell cyle arrest via p27 and inhibited BCR/ABL tyrosine kinase (TK) activity. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Disclosures:No relevant conflicts of interest to declare.

Targeting Isoprenylcysteine Carboxyl Methyltransferase to Overcome Resistance and Improve Responses in Chronic Myeloid Leukemia.

Abstract 3273Poster Board III-1A strategy to overcome resistance of chronic myeloid leukemia (CML) to Bcr-Abl tyrosine kinase inhibitors (TKI) is by targeting alternative pathways. Increasing evidence suggests that activation of the Ras family of small GTPases is important in leukaemogenic transformation by Bcr-Abl. Ras and Rho GTPases play important roles in the initiation and progression of cancer by disrupting the normal regulation of specific intracellular signalling pathways, promoting cell cycle progression and resistance to apoptosis. The biological function of Ras and Rho GTPases is, to a large extent, dependent on prenylation, a three-step post-translational modification process. The first step is catalysed by farnesyl transferase or geranylgeranyl transferase and the final step is catalysed by isoprenylcysteine carboxyl methyltransferase (Icmt). We have found inhibition of Icmt by a novel selective inhibitor, cysmethynil, enhanced the anti-proliferative effect of imatinib in CML cell lines. The combination of cysmethynil and imatinib was synergistic in inducing apoptosis in the imatinib-sensitive K562 and Bafp210 CML cell lines and apoptosis was further increased when the drugs were given sequentially, with cysmethynil added after imatinib. This combination was also effective in the imatinib-resistant Bcr-Abl- and P-glycoprotein-overexpressing LAMA84 cell line. The synergy was also observed when cysmethynil was combined with dasatinib in both the imatinib-sensitive Bafp210 and imatinib-resistant Bafp210 Y253F mutant but not in the Bafp210 T315I mutant cell lines. The combination therapy was more effective than single agent in inducing apoptosis in treatment-naive primary CML CD34+ cells and was not toxic to cord blood CD34+ cells (Figure 1). [Display omitted] Imatinib decreases p-CrkL through its inhibition of Bcr-Abl kinase. Interestingly, in both K562 and Bafp210, cysmethynil also decreases p-CrkL and when combined with imatinib, p-CrkL levels are reduced further (Figure 2). CrkL activation is dependent on a multimeric protein complex containing PI-3 kinase, docking protein 2, CrkL, Vav and the Rho GTPase, Rac. We hypothesise that inhibition of CrkL phosphorylation in the CML cell lines by cysmethynil is through the inhibition of Rac prenylation. This may be responsible for the synergistic activity observed when cysmethynil is combined with imatinib. In conclusion, our preliminary results suggest that the combination of a Bcr-Abl TKI and an Icmt inhibitor exerts a selective apoptotic effect in CML cells and this combination should be explored further as a useful tool to overcome resistance and improve responses in CML. [Display omitted] Disclosures:Druker:MolecularMD: Equity Ownership; Novartis Pharmaceuticals: ; Bristol-Myers Squibb:.

Effects of Bosutinib (SKI-606) in CML: Kinase Target Profile, Effects on BCR/ABL Mutants, and Synergism with Dasatinib in T315I+ Cells

Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50>1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50<0.5 nM) and most of the ABL mutants tested (H396P, M351T, Q252H, and Y253F) were all completely inhibited by both drugs at 1 μM in the kinase assay, the ABL T315I mutant was inhibited by bosutinib (IC50=26 nM) almost 70 times more potently than by dasatinib. Together, these data show that bosutinib and dasatinib synergize with each other in producing antileukemic effects on CML cells including BCR/ABL T315I+ subclones. These synergistic effects may be explained by differential target kinase profiles and by the fact that bosutinib retains some activity against the BCR/ABL T315I mutant kinase.

Imatinib Inhibits Proteasomal Activity in CML Cells.

Imatinib mesylate (Gleevec) is a specific tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. In the treatment of CML it has become indispensable and shows few side effects. Nevertheless, imatinib has been described to influence the function and differentiation of APCs, inhibit the effector function of T lymphocytes and decrease the immunogenicity of CML cells. In the present study, we analyzed the possible effects of imatinib on proteasomal activity in CML cells. Proteasomal activity was determined using a biotinylated active site-directed probe which, depending on their activity, covalently binds and labels proteasomal subunits beta-1, beta-2 and beta-5. We show that Imatinib treatment leads to a decreased activity of the proteasome in BCR-ABL positive cells. Imatinib sensitive K-562 cells as well as HLA-A2 transfected K-562 cells displayed a profound downregulation of proteasome activity at sub-therapeutic concentrations of imatinib. This effect was not due to downregulation of the protein expression of proteasome subunits or the induction of apoptosis. In imatinib resistant K-562R cells a higher concentration of imatinib was required for a detectable inhibition of proteasomal activity corresponding to a higher expression of BCR-ABL in these cells. Interestingly, the use of specific siRNA against the BCR-ABL fusion site b3a2 to downregulate the expression of the protein in K-562 and K-562R cells did not influence the activity of the proteasome indicating that this effect is independent of the expression and activity of BCR-ABL. In line with these results transfection of BaF/3 cells with BCR-ABL had no effect on proteasomal activity. To further analyze the mechanisms involved in proteasome inhibition by imatinib we performed activity assays with isolated 20S and 26S proteasomes. Incubation of both proteasome subtypes with imatinib resulted in a reduced acitivity in a concentration dependent manner as shown by reduced proteolytic cleavage of fluorogenic substrates. As the reduction in activity was seen in the ATP-independent 20S proteasome as well as the ATP-dependent 26S proteasome, a competition of imatinib with ATP similar to the mechanism of BCR-ABL inhibition is not responsible for this effect. In order to expand these studies, we additionally examined HLA class I expression on the cell surface to determine the influence of imatinib and subsequent proteasome inhibition on antigen presentation. Correlating with the inhibition of proteasome activity, HLA class I expression was strongly reduced on HLA-A2 transfected K-562 cells after imatinib treatment. Taken together these findings show that imatinib directly affects proteasomal activity in vitro. This effect is more pronounced in BCR-ABL positive cells, which might be due to BCR-ABL mediated deregulation of cellular pathways, but seems to be, however, independent of BCR-ABL.

Synergistic Activity of Nilotinib and Established Chemotherapeutic Agents in Imatinib-Sensitive and -Resistant BCR-ABL-Positive Leukemia Cells.

Resistance towards the tyrosine kinase inhibitor (TK) imatinib frequently emerges in advanced stages of chronic myelogenous leukemia (CML) and complicates disease treatment. Nilotinib (AMN107) represents a novel BCR-ABL TK inhibitor with reported antiproliferative activity against imatinib-resistant cells in vitro. Since combination chemotherapy is a strategy to increase the antileukemic efficacy, we were interested whether combinations of nilotinib and other established chemotherapeutic agents such as mafosfamide, busulfan, treosulfan, daunorubicin or mitoxantrone display synergistic activity in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. Tetrazolium-based MTT assays (48 h incubation) revealed as expected that substantially higher concentrations of single agent nilotinib are required for growth inhibition of the imatinib-resistant K562-R (IC50=19.76±4.94 μM) and LAMA84-R (IC50=0.14±0.03 μM) cell lines as compared to the imatinib-sensitive counterparts (IC50=0.10±0.03 μM for K562 and IC50=0.03±0.01 μM for LAMA84, respectively). Combination treatment of both imatinib-sensitive LAMA84 and -resistant LAMA84-R cells resulted in variable antiproliferative effects. Combination index (CI) values calculated according to the median-effect method of Chou and Talalay denoted mostly additive (CI=1) and antagonistic (CI>1) effects. In both lines a clear synergistic activity with CI<1 could be observed only for the combination nilotinib plus mitoxantrone. By comparison in the imatinib-sensitive K562 and -resistant K562-R lines all applied combinations showed improved antileukemic efficacy with CI values predominantly lower than 1 indicating synergism between nilotinib and most of the substances investigated (see Table I, n=3, results are given as mean CI values at different growth inhibition levels (fraction affected, Fa)). [Display omitted] In BaF3 cells expressing imatinib-resistant mutants of BCR-ABL (E255K and T315I) IC50-values of single agent nilotinib were 0.37±0.04 μM and 23.58±1.92 μM, respectively. Importantly, combination treatment of imatinib-resistant BaF3 cells with both nilotinib plus daunorubicin and nilotinib plus mitoxantrone led to enhanced antiproliferative effects with synergism (CI<1) at high growth inhibition levels even in the T315I resistant mutant cell line (see Table II, n=3). [Display omitted] The results demonstrate the ability of established chemotherapeutic agents to enhance the antileukemic efficacy of nilotinib and indicate the potential of the applied combinations for further study in the clinical setting.

Overexpression of the heat-shock protein 70 is associated to imatinib resistance in chronic myeloid leukemia

Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Abnormalities in Glucose Uptake and Metabolism in Imatinib-Resistant Bcr-Abl Positive Cells.

The development of imatinib resistance has become a significant therapeutic problem, in which the etiology appears to be multifactoral (Bcr-Abl mutation and/or overexpression, multidrug-resistance, protein overexpression, etc.) and poorly understood. As of today, there are no precise clinical criteria to predict the development of imatinib resistance in CML, other than relapse of the disease. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib. Thus, imatinib-resistant gastrointestinal stromal (GIST) KIT-activated tumors reveal highly elevated glucose uptake in PET scans. Currently, there is no information about the changes in glucose cell metabolism during development of imatinib resistance in CML patients. We used nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS) to assess 13C glucose uptake and metabolism (glycolysis, the TCA cycle, nucleic acid ribose synthesis) during imatinib treatment in cultured human CML cells with different sensitivity to imatinib. Our results demonstrate that imatinib-sensitive K562 and LAMA84 Bcr-Abl positive cells have decreased glucose uptake, decreased lactate production and improved oxidative TCA cycle upon imatinib treatment. The LAMA84-r (resistant to imatinib due to Bcr-Abl overexpression) and the K562-r (unknown mechanism of resistance) cells maintained high glycolytic metabolic phenotype with elevated glucose uptake and lactate production in the presence of imatinib (Figure 1). In addition, oxidative synthesis of RNA ribose from 13C-glucose via glucose-6-phosphate dehydrogenase was decreased and RNA synthesis via the non-oxidative transketolase pathway was increased in imatinib resistant cells. CML cells which exhibited an [oxidative/non-oxidative] flux ratio for nucleic acid ribose synthesis > 1, were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited the [oxidative/non-oxidative] flux ratio < 0.7. We correlated 13C glucose uptake and metabolism with intracellular expression and localization of GLUT-1 transporter protein in these cells. There was no difference in total protein load of GLUT-1 upon imatinib treatment between imatinib-sensitive and resistant cell lines. However, the changes in glucose uptake and metabolism were accompanied by intracellular trans-location of GLUT-1 from the plasma membrane to the cytoplasm in imatinib-sensitive treated cells, while GLUT-1 remained at the membrane surface in LAMA84-r and K562-r cells (Figure 1). In summary, elevated glucose uptake and non-oxidative glycolytic metabolic phenotype can be used as sensitive markers for imatinib resistance in Bcr-Abl positive cells with different origins of resistance. [Display omitted]

Treatment of Imatinib-Sensitive and -Resistant Chronic Myelogenous Leukemia Cells with a Combination of Imatinib and Farnesyltransferase Inhibitors.

Farnesyl protein transferase inhibitors (FTIs) represent a new class of anticancer agents. We were interested whether the farnesyltransferase inhibitors L-744,832 and LB42918 alone and in combination with imatinib are active in imatinib-sensitive and -resistant CML cells. LB42918 was more potent with ED50 = 4 ± 1 μM (mean ± S.D., n = 3) in the BCR-ABL+ imatinib-sensitive cell line EM-3 vs. ED50 = 25 ± 2 μM for L-744,832 after 48 hrs of treatment and assessment by MTT assay. The growth of imatinib-sensitive K562 and LAMA84 cells was not measurably inhibited by L-744,832 doses up to 25 μM. Similarly, the growth of K562 was not affected by 25 μM of LB42918. However, for LB42918 in LAMA84 cells an ED50 value of 30 ± 11 μM could be determined. In the imatinib-sensitive cell line EM-3, combination index values (CI) obtained using the method of Chou and Talalay indicated synergistic effects following simultaneous treatment with imatinib and FTI (CI = 0.5 ± 0.2 and 0.7 ± 0.2 at ED75 for imatinib + L-744,832 and imatinib + LB42918 respectively). In the cell imatinib-sensitive and -resistant lines K562 and LAMA84 a trend to lower ED50 values of imatinib was determined when up to 16 μM of L-744,832 or LB42918 were added, indicating potentiation of imatinib activity. Annexin V / propidium showed a strong increase of the apoptotic cell fraction in all imatinib-sensitive and an increase in the imatinib-resistant cells treated for 48 hrs by the combination imatinib + L-744,832 or imatinib + LB42918 as compared to treatment with each drug alone. Growth inhibition of CFU-GM colonies of primary imatinib-sensitive CML cells obtained from 4 patients is stronger after treatment with different concentrations of the combination of both drugs than after monotherapy with either imatinib or L-744,832. On the basis of the observed potentiation effects FTIs may find their place as supplement for CML patients on imatinib treatment.

The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.

BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.