MEK1/2 inhibitors sensitize Bcr/Abl+ human leukemia cells to the dual Abl/Src inhibitor BMS-354/825

Interactions between MEK1/2 inhibitors and the dual Abl/Src kinase inhibitor dasatinib (BMS-354825) were examined in chronic myeloid leukemia (CML) cell lines and primary specimens. Cotreatment of K562 or LAMA cells with subtoxic or marginally toxic concentrations of PD184352 (or U0126) and dasatinib synergistically potentiated mitochondrial damage, caspase activation, and apoptosis. Similar interactions were observed in CD34(+) cells from one CML patient-derived but not in a normal human CD34(+) bone marrow cell specimen. These interactions were associated with multiple perturbations in survival signaling pathways, including inactivation of Bcr/Abl, STAT5, and ERK1/2; down-regulation of Bcl-x(L) and Mcl-1; and dephosphorylation/activation of Bim. They were also associated with BAX/BAK conformational change, mitochondrial dysfunction, and caspase activation. Bim knockdown by shRNA suppressed BAX and BAK conformational change and protected cells from dasatinib/PD184352 lethality. Conversely, K562 cells ectopically expressing Mcl-1 or Bcl-x(L) were significantly less susceptible to dasatinib/PD184352 toxicity. Notably, the dasatinib/PD184352 regimen was active against leukemic cells exhibiting various forms of imatinib mesylate resistance, including Bcr/Abl overexpression, Lyn activation, and several Bcr/Abl kinase domain mutations (eg, E255K, M351T), but not T315I. Together, these findings suggest that strategies combining dasatanib with MEK1/2 inhibitors warrant further investigation in Bcr/Abl(+) malignancies, particularly in the setting of imatinib mesylate-resistant disease.     

MK-0457, a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the T315I BCR-ABL mutation

MK-0457 (VX-680) is a small-molecule aurora kinase (AK) inhibitor with preclinical antileukemia activity. The T315I BCR-ABL mutation mediates resistance to imatinib, nilotinib, and dasatinib. MK-0457 has in vitro activity against cells expressing wild-type or mutated BCR-ABL, including the T315I BCR-ABL mutation. Three patients with T315I abl-mutated chronic myeloid leukemia (CML) or Philadelphia chromosome (Ph)–positive acute lymphocytic leukemia (ALL) have achieved clinical responses to doses of MK-04547 that are not associated with adverse events. Higher MK-0457 dose levels were associated with clinical responses and down-regulation of CrkL phosphorylation in leukemia cells. The possible role of AK inhibition in these clinical responses requires further investigation. The currently reported cases are the first observed clinical activity of a kinase inhibitor against the T315I phenotype. The observation of responses in 3 patients with T315I phenotype–refractory CML or Ph-positive ALL, at doses of MK-0457 associated with no significant extramedullary toxicity, is very encouraging.      

Activation of a novel Bcr/Abl destruction pathway by WP1130 induces apoptosis of chronic myelogenous leukemia cells

Imatinib mesylate (Gleevec) is effective therapy against Philadelphia chromosome-positive leukemia, but resistance develops in all phases of the disease. Bcr/Abl point mutations and other alterations reduce the kinase inhibitory activity of imatinib mesylate; thus, agents that target Bcr/Abl through unique mechanisms may be needed. Here we describe the activity of WP1130, a small molecule that specifically and rapidly down-regulates both wild-type and mutant Bcr/Abl protein without affecting bcr/abl gene expression in chronic myelogenous leukemia (CML) cells. Loss of Bcr/Abl protein correlated with the onset of apoptosis and reduced phosphorylation of Bcr/Abl substrates. WP1130 did not affect Hsp90/Hsp70 ratios within the cells and did not require the participation of the proteasomal pathway for loss of Bcr/Abl protein. WP1130 was more effective in reducing leukemic versus normal hematopoietic colony formation and strongly inhibited colony formation of cells derived from patients with T315I mutant Bcr/Abl-expressing CML in blast crisis. WP1130 suppressed the growth of K562 heterotransplanted tumors as well as both wild-type Bcr/Abl and T315I mutant Bcr/Abl-expressing BaF/3 cells transplanted into nude mice. Collectively, our results demonstrate that WP1130 reduces wild-type and T315I mutant Bcr/Abl protein levels in CML cells through a unique mechanism and may be useful in treating CML.     

Efficacy of MK-0457 and in combination with vorinostat against Philadelphia chromosome positive acute lymphoblastic leukemia cells

Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division and Aurora kinases are overexpressed in a number of human cancers. In this study, we examined the intracellular signaling of Aurora kinases inhibitor, MK-0457 (VX-680), in BCR-ABL positive cell lines and in primary samples. MK-0457 induced apoptosis. Caspase 3 and poly (ADP-ribose) polymerase (PARP) were activated and heat shock proteins were reduced. A combination of MK-0457 and histone deacetylase inhibitor, vorinostat, increased apoptosis. Caspase 3 and PARP were activated and phosphorylation of BCR-ABL, Lyn, and Crk-L were reduced. BCR-ABL and Aurora A and B were reduced after vorinostat treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the extent of apoptosis in primary acute lymphoblastic leukemia cells with T315I mutation. Our study increases insight into how MK-0457 may mediate its effects on BCR-ABL positive leukemia cells with T315I mutation, and information of potential therapeutic relevance.     

Growth inhibitory effect of dihydroartemisinin on Bcr/Abl+ chronic myeloid leukemia K562 cells involve AKT, ERK and NF-κB modulation

Purpose

In our previous publication, we have shown that dihydroartemisinin could significantly inhibit the growth of CML K562 cells by its anti-proliferative and inducing apoptotic effects. Given the pivotal effect of Bcr/Abl tyrosine kinase and its downstream signal factors on CML cell proliferation and survival, we extend our study to investigate the effect of DHA on Bcr/Abl and related signal factors to further illuminate the possible mechanisms of the effect of DHA on CML cells.

Methods

The expression of Bcr/Abl was analyzed with PCR and Western blotting methods at both mRNA and protein levels. Measurement of protein expression and tyrosine phosphorylation activity of Bcr/Abl, AKT, ERK1/2, NF-κB and cytochrome c were performed with Western blotting and immunoprecipitation methods. Using the activity kits analyzed the activity of caspase 9 and caspase 3.

Results

The treatment with DHA results in a significant suppression on Bcr/Abl expression and leads to a concentration-dependent reduction on the Bcr/Abl tyrosine activity. Moreover, it also results in a strong influence on the downstream signal factors of Bcr/Abl, which includes inhibition of tyrosine kinase activity of AKT and ERK1/2, suppression of NF-κB protein expression, promotion of the cytochrome c release and the consequential activation of caspase 3/9 in CML K562 cells.

Conclusions

Together with our previous report, our data show that the growth inhibitory effect of DHA on CML cells might be due to the influence on Bcr/Abl expression and its downstream signal factors. DHA might be a potential novel anti-CML drug candidate and worthy of further study.     

Induction of autophagy by Imatinib sequesters Bcr‐Abl in autophagosomes and down‐regulates Bcr‐Abl protein

Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr‐Abl. Expression levels of this constitutively active tyrosine kinase are critical for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr‐Abl expressing cells. Autophagy has been associated with the clearance of large macromolecular signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr‐Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr‐Abl is sequestered into vesicular structures that co‐localize with the autophagy marker LC3 or GABARAP. This association is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacological inhibition of autophagy also reduced Bcr‐Abl/LC3 co‐localization in both K562 and CML patient cells. Bcr‐Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr‐Abl protein levels to those of untreated cells. This ability to down‐regulate Bcr‐Abl protein levels through the induction of autophagy may be an additional and important feature of the activity of imatinib. 88:455–462, 2013. © 2013 Wiley Periodicals, Inc.     

Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance

Resistance to the Ableson protein tyrosine (Abl) kinase inhibitor imatinib mesylate has become a critical issue for patients in advanced phases of chronic myelogenous leukemia. Imatinib-resistant tumor cells develop, in part, as a result of point mutations within the Abl kinase domain. As protein kinase B (Akt) plays a pivotal role in Abl oncogene-mediated cell survival, we hypothesize that concurrent inhibition of Akt will sensitize resistant cells to the residual apoptotic activity of imatinib mesylate, thereby overcoming the resistance. Here, we examined the effect of OSU-03012, a celecoxib-derived phosphoinositide-dependent kinase-1 (PDK-1) inhibitor, on imatinib mesylate-induced apoptosis in 2 clinically relevant breakpoint cluster region (Bcr)-Abl mutant cell lines, Ba/F3p210(E255K) and Ba/F3p210(T315I). The 50% inhibitory concentration (IC50) values of imatinib mesylate to inhibit the proliferation of Ba/F3p210(E255K) and Ba/F3p210(T315I) were 14 +/- 4 and 30 +/- 2 microM, respectively. There was no cross-resistance to OSU-03012 in these mutant cells with an IC50 of 5 microM irrespective of mutations. Nevertheless, in the presence of OSU-03012 the susceptibility of these mutant cells to imatinib-induced apoptosis was significantly enhanced. This synergistic action was, at least in part, mediated through the concerted effect on phospho-Akt. Together these data provide a novel therapeutic strategy to overcome imatinib mesylate resistance, especially with the Abl mutant T315I.     

The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571

Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor kappa B (NF-kappa B) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies.     

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.     

Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines

The tyrosine kinase activity of the Bcr/Abl oncogene is required for transformation of hematopoietic cells. The tyrosine kinase inhibitor STI571 (formerly called CGP57148B, Novartis Pharmaceuticals) inhibits BCR/ABL, TEL/ABL, and v-ABL kinase activity and inhibits growth and viability of cells transformed by any of these ABL oncogenes. Here we report the generation of 2 BCR/ABL-positive cell lines that have developed partial resistance to STI571. BCR/ABL-transformed Ba/F3 hematopoietic cells and Philadelphia-positive human K562 cells were cultured in gradually increasing concentrations of STI571 over a period of several months to generate resistant lines. Resistant Ba/F3.p210 cells were found to have an increase in Bcr/Abl messenger RNA, amplification of the Bcr/Abl transgene, and a greater than tenfold increase in the level of BCR/ABL protein. In contrast to Ba/F3.p210 cells, drug-resistant K562 cells did not undergo detectable amplification of the BCR/ABL gene, although they displayed a 2-fold to 3-fold increase in p210BCR/ABL protein. The addition of STI571 to both resistant Ba/F3. p210 and K562 cells resulted in a rapid reduction of tyrosine phosphorylation of cellular proteins, similar to that observed for nonresistant cells. However, the inhibition of kinase activity was transient and partial and was not accompanied by apoptosis. The results suggest that resistance to STI571 may be multifactorial. Increased expression of the target protein BCR/ABL was observed in both lines, and resulted from oncogene amplification in one line. However, altered drug metabolism, transport, or other related mechanisms may also contribute to drug resistance.     

Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique

Recently, various mutations within the Abl sequence have been described that negatively affect imatinib binding to Bcr/Abl resulting in cellular resistance of chronic myeloid leukemia (CML) cells. So far, little is known as to whether these mutations are preexisting or develop under imatinib therapy as current mutation analyses are limited by a low sensitivity of approximately 1:2 (50%) to 1:5 (20%). By combining peptide nucleic acid (PNA)-based DNA clamping with a fluorescence hybridization probe assay, we developed a new and highly sensitive technique for the detection of known mutations within the Bcr/Abl kinase domain. With this approach we investigated 19 cases of CML refractory to imatinib treatment before and during therapy. By clamping of wild-type Abl through PNA we could effectively enhance the detection sensitivity for the Bcr/Abl mutations Thr315Ile, Glu255Lys, and Tyr253His such that 1 mutant cDNA molecule could be detected in 500 negatives (0.2%). We observed in one case that a Gly255Lys mutation was detectable before treatment. By DNA analysis of buccal swaps, a genetic polymorphism could be excluded. In two cases clonal evolution of known mutations developed gradually under treatment. In another case an initially detectable Tyr253His mutation disappeared after therapy onset but was again observed after 6 weeks of imatinib treatment. Preexisting and evolving Bcr/Abl mutations associated with an unfavorable prognosis could be safely detected by the presented technique. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.     

Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: implications for CML

The deregulated, oncogenic tyrosine kinase Bcr-Abl causes chronic myeloid leukemia (CML). Imatinib mesylate (Gleevec, STI571), a Bcr-Abl kinase inhibitor, selectively inhibits proliferation and promotes apoptosis of CML cells. Despite the success of imatinib mesylate in the treatment of CML, resistance is observed, particularly in advanced disease. The most common imatinib mesylate resistance mechanism involves Bcr-Abl kinase domain mutations that impart varying degrees of drug insensitivity. AP23464, a potent adenosine 5'-triphosphate (ATP)-based inhibitor of Src and Abl kinases, displays antiproliferative activity against a human CML cell line and Bcr-Abl-transduced Ba/F3 cells (IC(50) = 14 nM; imatinib mesylate IC(50) = 350 nM). AP23464 ablates Bcr-Abl tyrosine phosphorylation, blocks cell cycle progression, and promotes apoptosis of Bcr-Abl-expressing cells. Biochemical assays with purified glutathione S transferase (GST)-Abl kinase domain confirmed that AP23464 directly inhibits Abl activity. Importantly, the low nanomolar cellular and biochemical inhibitory properties of AP23464 extend to frequently observed imatinib mesylate-resistant Bcr-Abl mutants, including nucleotide binding P-loop mutants Q252H, Y253F, E255K, C-terminal loop mutant M351T, and activation loop mutant H396P. AP23464 was ineffective against mutant T315I, an imatinib mesylate contact residue. The potency of AP23464 against imatinib mesylate-refractory Bcr-Abl and its distinct binding mode relative to imatinib mesylate warrant further investigation of AP23464 for the treatment of CML.     

Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.     

Targeting of heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) in leukemic cells in chronic myeloid leukemia: a novel approach to overcome resistance against imatinib

Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1-targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.     

In vitro anti-leukaemia activity of sphingosine kinase inhibitor

Compelling evidence indicates the role of sphingosine kinase 1 (SPHK1) deregulation in the processes of carcinogenesis and acquisition of drug resistance, providing the rationale for an effective anti-cancer therapy. However, no highly selective inhibitors of SPHK1 are available for in vitro and in vivo studies, except for the newly discovered 'SK inhibitor' (SKI). The present study showed that, in a panel of myeloid leukaemia cell lines, basal level of SPHK1 correlated with the degree of kinase inhibition by SKI. Exposure to SKI caused variable anti-proliferative, cytotoxic effects in all cell lines. In particular, SKI induced an early, significant inhibition of SPHK1 activity, impaired cell cycle progression and triggered apoptosis in K562 cells. Moreover, SKI acted synergistically with imatinib mesylate (IM) to inhibit cell growth and survival. Finally, the inhibitor affected the clonogenic potential and viability of primary cells from chronic myeloid leukaemia (CML) patients, including one harbouring the IM-insensitive Abl kinase domain mutation T315I. Due to the fact that the phenomenon of resistance to IM remains a major issue in the treatment of patients with CML, the identification of alternative targets and new drugs may be of clinical relevance.     

Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia

Mutations in the kinase domain (KD) of BCR-ABL are the most prevalent mechanism of acquired imatinib resistance in patients with chronic myeloid leukemia (CML). Here we examine predisposing factors underlying acquisition of KD mutations, evidence for acquisition of mutations before and during therapy, and whether the detection of a KD mutation universally implies resistance. We also provide a perspective on how the second-line Abl inhibitors dasatinib and nilotinib are faring in the treatment of imatinib-resistant CML, especially in relation to specific KD mutations. We discuss the growing importance of the multi-inhibitor-resistant 315T>I mutant and the therapeutic potential that a 315T>I inhibitor would have. Last, we assess the potential of Abl kinase inhibitor combinations to induce stable responses even in advanced CML and interpret the emerging data in the context of CML pathogenesis.