Targeting XIAP and ARC (apoptosis repressor with caspase recruitment domain) Overcomes Imatinib Resitance In Blast Crisis CML Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3415-3415
Author(s):  
Bing Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Marina Konopleva ◽  
Jorge E. Cortes ◽  
...  
Keyword(s):  
Cell Death ◽  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Apoptotic Cell ◽  
Blast Crisis ◽  
Similar Efficacy ◽  
Imatinib Resistance ◽  
Caspase Recruitment Domain ◽  
Abl Tyrosine Kinase ◽  
Induced Apoptosis

Abstract Abstract 3415 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor (TKI) has revolutionized the treatment of patients with CML. Development of resistance and limited activity in blast crisis (BC) CML are evolving problems facing this therapy. We found that XIAP, a potent caspase inhibitor, is highly expressed in CML cells, in both, cell lines and patient samples. Treatment with imatinib deceased XIAP levels in imatinib-sensitive KBM5 but much less so in imatinib-resistant KBM5STI571 cells (harboring T315I mutation) suggesting that XIAP expression in CML is regulated at least in part via Bcr-Abl and that targeting XIAP may promote cell death in CML cells by circumventing imatinib resistance. To test this, we treated BC CML cells with XIAP antisense oligonucleotide (ASO) and with SMAC mimetic ABT-10 and found that inhibition of XIAP induced apoptotic cell death with similar efficacy in KBM5 cells and KBM5STI517 cells (EC50=6.3±0.3 μM and 8.4±0.4 μM at 48 hours, respectively for ABT-10). However, we noted that inhibition of XIAP by ASO induced the expression, in both KBM5 and KBM5STI571 cells, of apoptosis repressor with caspase recruitment domain (ARC) in both mRNA and protein levels but not the expression of Bcl-2 protein. ARC is a unique antiapoptotic protein. It acts through inhibiting caspases and antagonizing the activity and function of p53 and Bax. Therefore, its induction may antagonize the effect of XIAP downregulation. Indeed, inhibition of both XIAP and ARC by ASO induced significantly more cell death than inhibiting either protein alone in both KBM5 and KBM5STI cells. Furthermore, we demonstrated that XIAP inhibition induced-apoptosis was enhanced by imatinib in KBM5, but not in KBM5STI cells. Interestingly, inhibition of Bcr-Abl tyrosine kinase by imatinib not only decreased XIAP, but also suppressed ARC levels in KBM5 but had minimal effects on the levels of these proteins in KBM5STI571 cells and enforced expression of the Bcr-Abl p185 fusion protein (in HL-60 cells) greatly increased both XIAP and ARC levels. This induction was inhibited by imatinib suggesting that ARC is also a downstream target of Bcr-Abl tyrosine kinase. Therefore, imatinib enhancing XIAP inhibition induced-apoptosis in KBM5, not KBM5STI cells can be explained at least in part by its ability to decrease XIAP and ARC levels. In conclusion, XIAP is highly expressed in CML cells and upregulated by Bcr-Abl. Targeting XIAP promotes death of BC and TKI resistant CML cells. Results suggest that XIAP is a potential target in BC and TKI resistant CML cells and that XIAP inhibition-induced apoptosis is enhanced by imatinib in TKI sensitive cells and by ARC inhibition independent of cellular responses to TKIs. Inhibition of XIAP and ARC as a novel therapeutic strategy in CML warrants further investigation. Disclosures: Koller: Isis Pharmaceuticals: Employment.

Regulation and Targeting of Eg5 in Blast Crisis CML: Overcoming Imatinib Resistance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2877-2877
Author(s):  
Bing Z. Carter ◽  
Duncan Mak ◽  
Yue-Xi Shi ◽  
Wendy D. Schober ◽  
Rui-Yu Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Tyrosine Kinase ◽  
Cell Cycle Progression ◽  
Blast Crisis ◽  
Cycle Progression ◽  
Tyrosine Kinase Signaling ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant ◽  
Cell Cycle Block

Abstract Imatinib (STI571), a tyrosine kinase inhibitor, is becoming the new standard of care for patients with chronic and advanced phase CML. However, the treatment for blast crisis (BC) CML is less effective. Resistance to Imatinib develops in all phases, particularly in BC CML emphasizing the need for alternative therapies. Eg5, a microtubule-associated motor protein plays an important role in establishing a bipolar spindle during mitosis and is essential for cell cycle progression. Eg5 was recently found to be highly expressed in BC CML by microarray analysis (Oncogene22:3952–3963, 2003). In this study, we examined the regulation of Eg5 by Bcr-Abl tyrosine kinase signaling and tested Eg5 as a potential therapeutic target in BC CML and Imatinib resistant CML. We found that Eg5 is expressed in all Philadelphia chromosome positive (Ph+) CML cell lines and in BC CML patient samples. Inhibition of Bcr-Abl activity by Imatinib downregulated Eg5 expression in Imatinib sensitive KBM5 cells (a cell line derived from the blasts of a BC CML patient) and HL-60p185 cells (HL-60 cells transfected with Bcr-Abl fusion protein p185), but not in Imatinib resistant KBM5-STI571 cells and Bcr-Abl negative HL-60 cells suggesting that Eg5 is a downstream effector of Bcr-Abl and is regulated by Bcr-Abl tyrosine kinase signaling in Ph+ cells. Blocking Eg5 expression by its antisense oligonucleotide (Eg5-AS) induced G2/M cell cycle block, and subsequent cell death in both Imatinib sensitive KBM5 cells and Imatinib resistant KBM5-STI571 cells. At 48 hrs, 15.8±5.5% of KBM5 cells and 22.7±10.7% of KBM5-STI571 cells were blocked in G2/M in Eg5-AS treated cells compared to 3.5±1.9% and 7.6±1.4%, respectively, of the mismatched oligonucleotide (Eg5-NS) treated cells. Induction of cell death was observed at 72 hrs (29.1±1.9% in KBM5 and 29.4±1.1 % in KBM5-STI571 cells in Eg5-AS treated compared to 12.5±0.28% and 13.7±1.6% of Eg5-NS treated cells). Metaphase arrest due to disruption of bipolar spindle formation, loss of mitochondrial membrane potential, and caspase activation were observed in both cell lines. Similarly, inhibition of Eg5 activity by a small molecular inhibitor, S-trityl-L-cysteine, induced cell cycle block and cell death indistinguishably in Imatinib sensitive KBM5 and Ba/F3Bcr-Ablwt cells and in Imatinib resistant KBM5-STI571, Ba/F3Bcr-AblE255K, and Ba/F3Bcr-AblT315I cells. Treatment of Scid mice starting 7 days after injection of KBM5 cells with Eg5-AS, 25 mg/kg, 3 times a week for 3 weeks, significantly prolonged the survival of the animals (64 days vs. 49 days of Eg5-NS treated mice, p=0.0344). The effect of Eg5 inhibition on survival of Scid mice harboring Imatinib resistant KBM5-STI571 cells is currently under investigation. Our studies suggest that Eg5 is a downstream target of Bcr-Abl tyrosine kinase. Inhibition of Eg5 expression or its activity blocks cell cycle progression and induces cell death regardless of cell response to Imatinib. Eg5 could be a potential new critical therapeutic target for the treatment of Imatinib resistant CML and BC CML.

Targeting Autophagy Potentiates Imatinib-Induced Cell Death in Philadelphia Positive Cells Including Primary CML Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1070-1070 ◽  
Author(s):  
Cristian Bellodi ◽  
Maria Rosa Lidonnici ◽  
Ashley Hamilton ◽  
Gudmundur V Helgason ◽  
Angela R Soliera ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Competitive Inhibitor ◽  
Myelogenous Leukemia ◽  
Stem Cell Population ◽  
Therapeutic Effects

Abstract Imatinib mesylate (IM), a potent ATP-competitive inhibitor of the BCR/ABL tyrosine kinase, has become standard therapy for patients with chronic myelogenous leukemia (CML). However, the main limitations of IM- and second generation tyrosine kinase inhibitor (TKI)-based therapy are the insurgence of resistance in patients and the intrinsic refractoriness of primitive Philadelphia-positive stem cells. Therefore, there is the need to develop new therapeutic approaches that, in combination with TKI, might be more effective in targeting the stem cell population and preventing the outgrowth of TKI-resistant CML cells. TKI-induced elimination of BCR/ABL-dependent intracellular signals is known to trigger apoptosis, but it is unclear whether this also activates additional cell death and/or survival pathways. We show that IM treatment induces autophagy in CML blast crisis cell lines, CML primary cells and p210BCR/ABL-expressing 32Dcl3 (32D) myeloid precursor cells, but not in 32D cells expressing v-Src or the IM-resistant T315I p210BCR/ABL mutant. IM-induced autophagy does not involve c-Abl, as it is also observed in cells co-expressing p210BCR/ABL and the IM-resistant T315I c-Abl mutant. Induction of autophagy is associated with endoplasmic reticulum-stress and is suppressed by depletion of intracellular calcium. By contrast, ectopic Bcl-2 expression does not block IM-induced autophagy. Suppression of autophagy by pharmacological inhibitors or siRNA-mediated knockdown of essential autophagy genes enhances cell death induced by IM in cell lines and primary CML cells, demonstrating that induction of autophagy has a pro-survival effect. Critically, the combination of TKI with autophagy inhibitors results in near complete elimination of phenotypically (CD34+38−) and functionally (colony forming cells) defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKI in the treatment of CML.

Overcoming STI571 resistance with the farnesyl transferase inhibitor SCH66336

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1068-1071 ◽  
Author(s):  
Russell R. Hoover ◽  
Francois-Xavier Mahon ◽  
Junia V. Melo ◽  
George Q. Daley
Keyword(s):  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Blood Samples ◽  
Farnesyl Transferase ◽  
Abl Tyrosine Kinase ◽  
Farnesyl Transferase Inhibitor ◽  
Enhanced Expression ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Induced Apoptosis

Abstract The development of chronic myeloid leukemia (CML) is dependent on the deregulated tyrosine kinase of the oncoprotein BCR-ABL. STI571 (imatinib mesylate), an abl tyrosine kinase inhibitor, has proven remarkably effective for the treatment of CML. However, resistance to STI571 because of enhanced expression or mutation of theBCR-ABL gene has been detected in patients. In the current study we show that the farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits the proliferation of STI571-resistant BCR-ABL–positive cell lines and hematopoietic colony formation from peripheral blood samples of STI571-resistant patients with CML. Moreover, SCH66336 enhances STI571-induced apoptosis in STI571-sensitive cells and, in patients with STI571 resistance from gene amplification, cooperates with STI571 to induce apoptosis. Our data provide a rationale for combination clinical trials of STI571 and SCH66336 in CML patients and suggest that combination therapy may be effective in patients with STI571 resistance.

Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome–positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance

Blood ◽  
2007 ◽  
Vol 110 (10) ◽  
pp. 3540-3546 ◽  
Author(s):  
Hagop M. Kantarjian ◽  
Francis Giles ◽  
Norbert Gattermann ◽  
Kapil Bhalla ◽  
Giuliana Alimena ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Philadelphia Chromosome Positive

Abstract Nilotinib, an orally bioavailable, selective Bcr-Abl tyrosine kinase inhibitor, is 30-fold more potent than imatinib in pre-clinical models, and overcomes most imatinib resistant BCR-ABL mutations. In this phase 2 open-label study, 400 mg nilotinib was administered orally twice daily to 280 patients with Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia in chronic phase (CML-CP) after imatinib failure or intolerance. Patients had at least 6 months of follow-up and were evaluated for hematologic and cytogenetic responses, as well as for safety and overall survival. At 6 months, the rate of major cytogenetic response (Ph ≤ 35%) was 48%: complete (Ph = 0%) in 31%, and partial (Ph = 1%-35%) in 16%. The estimated survival at 12 months was 95%. Nilotinib was effective in patients harboring BCR-ABL mutations associated with imatinib resistance (except T315I), and also in patients with a resistance mechanism independent of BCR-ABL mutations. Adverse events were mostly mild to moderate, and there was minimal cross-intolerance with imatinib. Grades 3 to 4 neutropenia and thrombocytopenia were observed in 29% of patients; pleural or pericardial effusions were observed in 1% (none were severe). In summary, nilotinib is highly active and safe in patients with CML-CP after imatinib failure or intolerance. This clinical trial is registered at http://clinicaltrials.gov as ID no. NCT00109707.

Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets

Blood ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 4055-4063 ◽  
Author(s):  
Uwe Rix ◽  
Oliver Hantschel ◽  
Gerhard Dürnberger ◽  
Lily L. Remsing Rix ◽  
Melanie Planyavsky ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
High Specificity ◽  
Imatinib Resistance ◽  
Chemical Proteomics ◽  
Abl Tyrosine Kinase ◽  
Abl Kinases ◽  
Drug Concentrations ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Major Target

Abstract The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia. Because many patients develop imatinib resistance, 2 second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL were developed. To predict potential side effects and novel medical uses, we generated comprehensive drug-protein interaction profiles by chemical proteomics for all 3 drugs. Our studies yielded 4 major findings: (1) The interaction profiles of the 3 drugs displayed strong differences and only a small overlap covering the ABL kinases. (2) Dasatinib bound in excess of 30 Tyr and Ser/Thr kinases, including major regulators of the immune system, suggesting that dasatinib might have a particular impact on immune function. (3) Despite the high specificity of nilotinib, the receptor tyrosine kinase DDR1 was identified and validated as an additional major target. (4) The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations, representing the first nonkinase target of these drugs.

scholarly journals The Interface between BCR-ABL-Dependent and -Independent Resistance Signaling Pathways in Chronic Myeloid Leukemia

2012 ◽  
Vol 2012 ◽  
pp. 1-19 ◽  
Author(s):  
Gabriela Nestal de Moraes ◽  
Paloma Silva Souza ◽  
Fernanda Casal de Faria Costas ◽  
Flavia Cunha Vasconcelos ◽  
Flaviana Ruade Souza Reis ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Resistance Mechanisms ◽  
Imatinib Resistance ◽  
Abl Tyrosine Kinase ◽  
Alternative Drug

Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder characterized by the presence of the Philadelphia chromosome which resulted from the reciprocal translocation between chromosomes 9 and 22. The pathogenesis of CML involves the constitutive activation of the BCR-ABL tyrosine kinase, which governs malignant disease by activating multiple signal transduction pathways. The BCR-ABL kinase inhibitor, imatinib, is the front-line treatment for CML, but the emergence of imatinib resistance and other tyrosine kinase inhibitors (TKIs) has called attention for additional resistance mechanisms and has led to the search for alternative drug treatments. In this paper, we discuss our current understanding of mechanisms, related or unrelated to BCR-ABL, which have been shown to account for chemoresistance and treatment failure. We focus on the potential role of the influx and efflux transporters, the inhibitor of apoptosis proteins, and transcription factor-mediated signals as feasible molecular targets to overcome the development of TKIs resistance in CML.

Triptolide Induces Cell Death Independent of Cellular Responses to Imatinib in Blast Crisis CML Cells and Sensitizes Imatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1610-1610
Author(s):  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Wenjing Chen ◽  
Jorge E. Cortes ◽  
Hagop M. Kantarjian ◽  
...  
Keyword(s):  
Cell Death ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Second Generation ◽  
Kinase Inhibitors ◽  
Blast Crisis ◽  
K562 Cells ◽  
Resting Cells ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Abstract Imatinib, a Bcr-Abl tyrosine kinase inhibitor has revolutionized the treatment of patients with CML. However, resistance develops due to Bcr-Abl gene mutations and various other mechanisms. Although second generation Bcr-Abl inhibitors can overcome most of the mutation driven resistance, they cannot overcome other resistance mechanisms. Furthermore, Imatinib has limited effectiveness in patients with blast crisis (BC) CML. We have previously shown that triptolide, an anti-cancer agent isolated from a Chinese herb, potently induces apoptosis in AML cells in part by decreasing the levels of XIAP and Mcl-1, two potent antiapoptotic proteins. Here we investigated its effect on Philadelphia chromosome positive (Ph+) cells and found that at low nM concentrations, triptolide induced significant cell death in K562 (IC50=113.4±3.9 nM) and KBM5 (IC50=30.0±2.1 nM) cells, two cell lines derived from BC CML patients, as well as in ALL-1 cells (IC50=113.8±1.4 nM), a cell line derived from Ph+ ALL. Interestingly, KBM5-STI571 cells, an Imatinib resistant KBM5 subline bearing the T315I mutation which is resistant to most available Bcr-Abl tyrosine kinase inhibitors, were as sensitive as KBM5 cells to triptolide. Likewise, triptolide killed Ba/F3 cells harboring BCR-ABL mutants (E255K and T315I) with similar efficacy as Ba/F3 cells carrying the wild type BCR-ABL gene. We then treated 8 samples from 7 CML patients with blasts ranging from 10–91% with triptolide (up to 100 nM) in vitro. Triptolide induced cell death in all samples tested. Importantly, 6/7 samples were from patients resistant/relapsed after Imatinib. Three were also nonresponsive to Nilotinib and one to neither Nilotinib nor Dasatinib. Next we tried to elucidate the possible apoptosis regulators involved in triptolide-induced cell death. Triptolide decreased antiapoptotic XIAP, Mcl-1 and Bcr-Abl protein levels in K562 cells and in blast cells from CML patients. Based on this observation, we treated CML cells with both triptolide and Imatinib. The combination synergistically induced cell death in K562 cells (CI=0.50±0.14). In KBM5 cells, Imatinib antagonized rather than enhanced triptolide when administrated simultaneously: Triptolide alone induced cell death with IC50=24.3±2.8 nM at 48 hours, while in combination with 1 μM Imatinib, the IC50 increased to 82.9±4.1 nM. This is probably due to the fact that Imatinib primarily blocks KBM5 cells in G0/G1 and that resting cells were less sensitive to triptolide. We therefore pretreated KBM5 cells with triptolide for 24 hrs followed by 1 μM Imatinib for 24 hrs. This sequential treatment was more effective to induce cell death in KBM5 cells (IC50=15.4±0.6 nM). Triptolide did not sensitize Imatinib resistant KBM5-STI571 cells. Conclusion: Results suggest that triptolide potently induces cell death in BC CML cells and that the cell death induced by triptolide is independent of response to Imatinib or other second generation Bcr-Abl kinase inhibitors. Triptolide could be of potential benefit to CML patients in blast crisis and CML patients failing Bcr-Abl tyrosine kinase inhibitors.

scholarly journals Apoptosis in Hydra: function of HyBcl-2 like 4 and proteins of the transmembrane BAX inhibitor motif (TMBIM) containing family

2019 ◽  
Vol 63 (6-7) ◽  
pp. 259-270 ◽  
Author(s):  
Mina Motamedi ◽  
Laura Lindenthal ◽  
Anita Wagner ◽  
Margherita Kemper ◽  
Jasmin Moneer ◽  
...  
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Programmed Cell Death ◽  
Kinase Inhibitor ◽  
Apoptotic Cell ◽  
Family Members ◽  
Sequence Comparisons ◽  
Induced Apoptosis ◽  
Related Proteins

Mechanisms of programmed cell death differ between animals, plants and fungi. In animals, apoptotic cell death depends on caspases and Bcl-2 family proteins. These protein families are only found in multicellular animals, including cnidarians, insects and mammals. In contrast, members of the TMBIM-family of transmembrane proteins are conserved across all eukaryotes. Sequence comparisons of cell death related proteins between phyla indicate strong conservation of the genes involved. However, often it is not known whether this is paralleled by conservation of function. Here we present the first study to support an anti-apoptotic function of Bcl-2 like proteins in the cnidarian Hydra within a physiological context. We used transgenic Hydra expressing GFP-tagged HyBcl-2-like 4 protein in epithelial cells. The protein was localised to mitochondria and able to protect Hydra epithelial cells from apoptosis induced by either the PI(3) kinase inhibitor wortmannin or by starvation. Moreover, we identified members of the TMBIM-family in Hydra including HyBax-Inhibitor-1, HyLifeguard-1a and -1b and HyLifeguard 4. Expressing these TMBIM-family members in Hydra and human HEK cells, we found HyBax-inhibitor-1 protein localised to ER-membranes and HyLifeguard-family members localised to the plasma membrane and Golgi-vesicles. Moreover, HyBax-inhibitor-1 protected human cells from camptothecin induced apoptosis. This work illustrates that the investigated Bcl-2- and TMBIM-family members represent evolutionarily conserved mitochondrial, ER, Golgi and plasma membrane proteins with anti-apoptotic functions. The participation of ER and Golgi proteins in the regulation of programmed cell death might be a very ancient feature.

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