Bim Is the Critical, and Bad and Bmf Are the Ancillary BH3-Only Proteins for Imatinib Mesylate-Induced Apoptosis of Bcr/Abl-Positive Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2876-2876
Author(s):  
Junya Kuroda ◽  
Hamsa Puthalakath ◽  
Philippe Bouillet ◽  
Mark S. Cragg ◽  
Priscilla N. Kelly ◽  
...  
Keyword(s):  
Cell Death ◽  
Imatinib Mesylate ◽  
Cell Lines ◽  
Cell Killing ◽  
Leukemic Cells ◽  
Imatinib Treatment ◽  
Killing Activity ◽  
Induced Apoptosis ◽  
Transformed Cell Lines

Abstract Imatinib mesylate (imatinib) exerts the anti-Philadelphia-positive (Ph1+) leukemia activity both by the inhibition of cell proliferation and by the induction of apoptosis. Recent studies demonstrate that the induction of cell death is essential for eradication of Ph1+ leukemic clones in imatinib treatment; however, the molecular mechanisms have not yet been clearly described. By examining the effect of imatinib on parental K562 and subclones overexpressing either Bcl-2, Bcl-XL or a dominant interfering mutant of FADD/MORT1, which blocks death receptor apoptosis signalling, we found that imatinib triggers apoptosis exclusively via the Bcl-2 family-regulated intrinsic apoptotic pathway. We investigated the involvement of BH3-only proteins as apoptotic initiators in imatinib-induced cell death, because the cell life-or-death decision is arbitrated by the balance between pro-apoptotic BH3 only-proteins and anti-apoptotic Bcl-2 proteins. We found that imatinib treatment upregulated Bim in Ph1+ leukemic cell lines and bcr-c-abl transformed murine fetal liver cells (FLCs)-derived cell lines both by transcriptional and post-translational mechanisms. Imatinib also activated Bad through dephosphorylation and upregulated Bmf transcriptionally. To examine the role of Bim in imatinib-induced apoptosis, we examined the cell killing activity of imatinib in subclones of K562 and BV173 Ph1+ cells expressing abnormally reduced levels of Bim using stable RNA interference system. This revealed that the cell killing activity of imatinib largely dependent on Bim expression levels in these cell lines, although significant apoptosis was still evident. To further define the role of Bim, Bad and Bmf in imatinib-induced cell death, we examined the effect of imatinib on retrovirally bcr-c-abl transformed cell lines derived from FLCs from wild type C57BL/6, Bim-/-, Bad-/-, Bim-/-Bad-/- double KO and Bcl-2 transgenic fetuses. The bim-/-bcr-c-abl+ FLCs were shown to be more resistant to imatinib-induced cell death than wt.bcr-c-abl+ FLCs, however, bim-/-bcr-c-abl+ FLCs were eventually induced into cell death, indicating that Bim is not the only initiator of apoptosis. The bad-/-bcr-c-abl+ FLCs were also partially resistant to imatinib-induced cell death. Intriguingly, like in vav.bcl-2.bcr-c-abl+ FLCs, the cell death induction by imatinib (~5.0μM) was largely abrogated in bim-/-bad-/-bcr-c-abl+ FLCs, indicating that Bim collaborates with Bad for the apoptotic induction by imatinib. Importantly, we found that Bim was inducible by ex vivo imatinib treatment in primary Ph1+ leukemic cells only from clinically good responders but not from patients refractory to imatinib treatment. Collectively, these results demonstrate that Bim is the critical but not the only initiator required for imatinib-induced apoptosis of Bcr/Abl-positive hematopoietic cells; Bad and Bmf may be the ancillary BH3-only proteins in this process. Our results provide evidence for the therapeutic significance of regulation of BH3-only proteins, particularly Bim, for the eradication of Ph1+ leukemic cells.

The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 891-891
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Evgenia Rosenberg ◽  
Hila Magen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Cell Lines ◽  
Hdac Inhibitor ◽  
Mtor Inhibitor ◽  
Mtor Inhibition ◽  
Induced Apoptosis

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it. Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis. Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis. Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Jasplakinolide Induces Apoptosis in Various Transformed Cell Lines by a Caspase-3-Like Protease-Dependent Pathway

2000 ◽  
Vol 7 (6) ◽  
pp. 947-952 ◽  
Author(s):  
Chikako Odaka ◽  
Miranda L. Sanders ◽  
Phillip Crews
Keyword(s):  
T Cells ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Dependent Manner ◽  
Jurkat T Cells ◽  
Transformed Cell ◽  
Induced Apoptosis ◽  
Transformed Cell Lines ◽  
Dependent Pathway

ABSTRACT To clarify the mechanisms underlying the antiproliferative effects of jasplakinolide, a cyclic depsipeptide from marine sponges, we examined whether jasplakinolide induces apoptosis in a variety of transformed and nontransformed cells. Jasplakinolide inhibited proliferation of human Jurkat T cells, resulting in cell death. This was accompanied by chromatin condensation and DNA cleavage at the linker regions between the nucleosomes. When caspase-3-like activity in the cytosolic extracts of Jurkat T cells was examined with a fluorescent substrate, DEVD-MAC (N-acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide), the activity in the cells treated with jasplakinolide was remarkably increased in a time-dependent manner. Pretreatment of Jurkat T cells with the caspase inhibitor zVAD [benzyloxycarbonyl(Cbz)-Val-Ala-β-Asp(OMe)-fluoromethylketone] or DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-1-aldehyde) prevented the induction of apoptosis by jasplakinolide. Moreover, exposure of various murine transformed cell lines to jasplakinolide resulted in cell death, which was inhibited by zVAD. Although it has been well established that murine immature thymocytes are sensitive to apoptosis when exposed to various apoptotic stimuli, these cells as well as mature T lymphocytes were resistant to jasplakinolide-induced apoptosis. The results suggest that jasplakinolide induces apoptotic cell death through a caspase-3-like protease-dependent pathway. Another important outcome is that transformed cell lines were more susceptible to jasplakinolide-induced apoptosis than normal nontransformed cells.

Triptolide Sensitizes Leukemic Cells to TRAIL Induced Apoptosis Via Decrease of XIAP and p53-Mediated Increase of DR5.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 528-528
Author(s):  
Bing Z. Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Martin Dietrich ◽  
Clemencia Pinilla ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Chinese Herb ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
U937 Cells ◽  
Apoptosis Resistance ◽  
Protein Levels ◽  
P53 Signaling ◽  
Induced Apoptosis

Abstract Triptolide, a recently identified anticancer agent from a Chinese herb, has been shown to synergistically enhance TRAIL-induced cell death in various solid tumor cell lines. We have found that triptolide potently induces apoptosis in leukemic cell lines and blasts from AML patients at least in part by decreasing XIAP levels. XIAP is known to be a resistance factor in TRAIL-induced cell death. XIAP is highly expressed in AML blasts and primary AML cells generally are insensitive to TRAIL. We therefore hypothesize that triptolide will sensitize AML cells to TRAIL-induced apoptosis. We treated OCI-AML3, U937, and Jurkat cells with sub-optimal concentrations of triptolide, TRAIL, and their combination. At concentrations showing no or minimal effects by each agent alone, their combinations significantly promoted cell death with combination indices (CI) <1.0 for all three leukemia cell lines. To ensure that XIAP contributes to TRAIL resistance, we treated U937 cells overexpressing XIAP (U937XIAP) and the control cells (U937neo) with TRAIL. U937XIAP cells were 6-fold more resistant to TRAIL (IC50=455.8 ng/ml) than U937neo cells (IC50=74.3 ng/ml). We then treated these cells with TRAIL and 1396–11, a small molecule XIAP antagonist that binds to BIR2 of XIAP and promotes caspase-dependent apoptosis. At 3μM, 1396–11 had no significant effect on survival of either U937neo or U939XIAP cells. When combined with TRAIL, 1396–11 augmented TRAIL-induced cell death of both U937neo (IC50=45.0 ng/ml with 1396-11 vs. IC50=74.3 ng/ml without) and U939XIAP cells (IC50=318.9 ng/ml with 1396–11 vs. IC50=455.8 ng/ml without). Furthermore, we observed that triptolide decreased MDM2 and increased p53 protein levels in p53 wild type OCI-AML3 but not in p53 null U937 cells. TRAIL receptor DR5 has been shown to be regulated by p53. To elucidate the role of p53 in TRAIL-induced cell death sensitized by triptolide, we treated OCI-AML3 and U937 cells with triptolide and examined DR5 expression. We found that triptolide induced DR5 protein levels in OCI-AML3, but much less so in U937 cells. Treatment of OCI-AML3 cells with Nutlin3a, a MDM2 inhibitor that binds to MDM2 and stabilizes p53, increased DR5 protein levels and sensitized to TRAIL-induced cell death. Knockdown of p53 with retrovirus expressing p53siRNA in OCI-AML3 cells abolished cellular responses to nutlin3a and significantly decreased the sensitization to TRAIL by nutlin3a. Finally, we treated OCI-AML3 cells with 1396–11, nutlin3a, and TRAIL and our results showed that the triple combination (CI=0.045 at 24 hours, average of ED50, ED75, and ED90) was more effective in inducing cell death than either 1396–11 and TRAIL (CI=0.066) or nutlin3a and TRAIL (CI=0.190) combinations supporting our notion that triptolide sensitizes to TRAIL-induced cell death by modulating both XIAP expression and p53 signaling. Collectively, our studies suggest that inhibition of XIAP and induction of DR5 mediated by p53 activation both independently sensitize leukemic cells to TRAIL-induced apoptosis. Triptolide not only inhibits XIAP, which is overexpressed in AML, but also activates p53 signaling, which is intact in the majority of AMLs. Thus, combinations of triptolide and TRAIL may provide a novel strategy for treating AML patients by overcoming critical mechanisms of apoptosis resistance.

ABT-737 Is a Useful Component of Combinatory Chemotherapies for Chronic Myelogenous Leukemias with Diverse Drug Resistance Mechanisms.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 808-808 ◽  
Author(s):  
Junya Kuroda ◽  
Shinya Kimura ◽  
Michael Andreeff ◽  
Eishi Ashihara ◽  
Yuri Kamitsuji ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Resistance Mechanisms ◽  
Cell Killing ◽  
Leukemic Cells ◽  
Killing Effect ◽  
Cell Killing Effect

Abstract Chronic myelogenous leukemia (CML) is characterized by its refractoriness to various apoptotic insults by Bcr-Abl tyrosine kinase (TK)-mediated signalling. Although imatinib mesylate (IM), a Bcr-Abl TK inhibitor, has markedly improved the therapeutic outcomes of CML, additional or alternative molecular targeting strategies are still needed. Since the interplay of anti-apoptotic Bcl-2 proteins and BH3-only proteins, such as Bim and Bad, is crucial for regulating the cellular fate of Bcr-Abl+ leukemic cells (Kuroda J et al, PNAS , 2006; Cell Death Differ, 2007), the direct targeting of anti-apoptotic Bcl-2 proteins by the use of a BH3-only protein mimetic is an attractive approach for treating CML. We here investigated the activity of ABT-737, a mimic of BH3-only proteins that inhibit anti-apoptotic Bcl-2, Bcl-XL and Bcl-w, but not Mcl-1 or A1, against CML. The Annexin-V-staining study, the assay for mitochondrial outer membrane potential and the internucleosomal fragmentation assay revealed that ABT-737 potently induced apoptosis in CML cell lines (BV173, K562, KCL22, KT-1, MEG-01 and MYL) with the IC50 for induction of cell death by 48 h of treatment ranging from 0.04 to 4.06 μM. ABT-737 was also effective in killing primary CML samples in vitro. ABT-737 prolonged the survival of mice xenografted with a CML cell line, BV173, demonstrating its in vivo bioactivity. Higher expression of Bcl-2, Bcl-XL, or Mcl-1 reduced cell killing by ABT-737 in each cell line, but we found no correlation between the sensitivities to ABT-737 and the specific expression patterns of Bcl-2 family proteins among different cell lines. The levels of Bcr-Abl and Lyn, a member of Src kinase family associated with apoptosis resistance in CML, also varied among the cell lines, and we found no consistent relationship between the sensitivity to ABT-737 and the expression levels of these proteins Thus, the cell killing effect of ABT-737 in CML must be determined in part by other drug resistance mechanisms, such as high expression of Bcr-Abl, overexpression of P-glycoprotein (P-gp), a drug-efflux pump, and/or their combination. Importantly, ABT-737 augmented the cell killing effect of IM in CML cell lines with high levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, or even Mcl-1), Bcr-Abl, P-gp, or Lyn, unless leukemic cells harboured IM-insensitive Abl kinase domain (KD) mutations. Moreover, the combination of ABT-737 with homoharringtonine, an herbal-derived anti-CML therapeutic that potently reduces Mcl-1 within 6 hours in vitro, dramatically enhanced the killing by ABT-737 in CML cells with diverse drug resistance mechanisms, including IM-insensitive Abl KD mutations, such as T315I mutation. These results suggest that ABT-737 could be a useful component of combinatory chemotherapies for CML.

Expression Of FLT3-ITD Dysregulates The DBC1-Sirt1-p53 Signaling and Promotes Therapy Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3789-3789
Author(s):  
Daniel Sasca ◽  
Patricia Haehnel ◽  
Kaml I.K. Khawaja ◽  
Birgit Enders ◽  
Lars Bullinger ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Expression Analysis ◽  
Myeloid Leukemia ◽  
Cell Activity ◽  
Leukemic Cells ◽  
Genotoxic Agents ◽  
Acute Myeloid

Abstract Background SIRT1 is a NAD+ dependent histone deacetylase, which has been shown to act as an important regulator of apoptosis, DNA-repair and is involved in the maintenance of genetic integrity under conditions of cellular stress. Beside deacetylation of histones H4K16, SIRT1 has numeral other substrates including KU70, FOXO1 or p53. SIRT1 deacetylates p53 at lysine 382 thereby reducing its transcriptional activity followed by loss of p53 dependent apoptosis in response to cell damage. The activity of SIRT1 is negatively regulated by DBC1 (Deleted in Breast Cancer 1) and involves protein–protein interaction (Kim et al., Nature 2008). Recent reports have demonstrated increased expression of SIRT1 in leukemic cells. Of note, SIRT1 seems to be specifically essential to maintain stem cell activity in CML leukemic stem cells (Li et al, Cancer Cell, 2012), however the role of SIRT1 in acute myeloid leukemia (AML) and in the context of other oncogenic tyrosine kinases is poorly understood. Aims To characterize the role of SIRT1 in acute myeloid leukemia in the context of defined genetic backgrounds. Methods To investigate the functional role of SIRT1 we performed expression analysis studies. Inhibition of SIRT1 was achieved by shRNA experiments and pharmacological targeting. Functional consequences were addressed by immunoblotting, co-immunoprecipitation, cell death assays, gamma-H2AX staining and xenotransplantion experiments. Results SIRT1 protein expression analysis in primary human AML samples (n=20) and cell lines (n=13) showed preferential high expression levels in cells harboring FLT3-ITD mutations or oncogenic KRAS/NRAS. Inhibition of FLT3 signaling using shRNA-mediated knockdown of FLT3 or upon treatment with tyrosine kinase inhibitors caused a time and dose dependent suppression of SIRT1 expression accompanied by an increase in p53 acetylation. Vice versa, overexpression of FLT3-ITD in BaF/3 cells or in several murine leukemia models (e.g. MLL/AF9 +/-FLT3-ITD; AML1/ETO +/-FLT3-ITD) induced significant upregulation of SIRT1. Targeting SIRT1 using the SIRT1/2 inhibitor Tenovin-6 (TV-6) or knockdown of SIRT1 resulted in a slight increase in apoptotic cell death in primary AML samples and cell lines. In contrast, inhibition of SIRT1 significantly sensitized to FLT3 inhibitor therapy. Of note, the combination of TV-6 and midostaurin inhibited colony growth and replating activity in MLL-AF9 or AML1-ETO cells harboring FLT3-ITD mutations. Further, knockdown of SIRT1 in the AML cell line MV4-11 expressing a doxycycline-regulated SIRT-shRNA resulted in prolonged survival in a xenotransplantation model. The observed effects prompted us to investigate whether mutated FLT3 is directly involved in the regulation of SIRT1. Inhibition of FLT3-ITD kinase activity caused an increased phosphorylation of the physiological SIRT1-inhibitor DBC1 followed by binding of SIRT1 as revealed by co-immunoprecipitation experiments. We also noticed a slight increase in ATM/ATR phosphorylation and an increase in ATM-DBC1 binding upon FLT3-ITD inhibition indicating SIRT1 inhibition via the ATM-DBC1-SIRT1 axis. These effects were substantially enhanced upon treatment with genotoxic agents (cytarabine; irradiation) in combination with midostaurin compared to either agent alone and were accompanied by increased p53 acetylation and an increased number of gamma-H2AX foci. Conclusion SIRT1 is overexpressed in AML cells and partially regulated by FLT3-ITD or oncogenic RAS. In FLT3-ITD mutated cells SIRT1 is constitutively activated, likely via the inhibition of the physiological inhibitor DBC1. Pharmacologic targeting of SIRT1 sensitizes AML cells to TKI therapy or genotoxic agents and partially restored the FLT3-ITD associated defective stress response pathway ATM-DBC1-SIRT1 resulting in activation of p53. Disclosures: Kindler: Novartis: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Inhibition of WIP1 Phosphatase in Multiple Myeloma Overcomes Bortezomib Resistance and Promotes Cell Death Via ER Stress-Induced Apoptotic JNK/c-Jun Signaling and Downregulation of Inhibitors of Apoptosis Proteins (IAPs)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1366-1366
Author(s):  
Katia Beider ◽  
Evgenia Rosenberg ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Yaarit Sirovsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Potential Role ◽  
Cyclin B1 ◽  
Parp Cleavage ◽  
Induced Apoptosis

Abstract Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype. Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance. GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells. To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death. Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment. To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction. Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response. To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.

scholarly journals Inhibition of Autophagy Enhances the Antitumor Effect of Thioridazine in Acute Lymphoblastic Leukemia Cells

Life ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 365
Author(s):  
Carina Colturato-Kido ◽  
Rayssa M. Lopes ◽  
Hyllana C. D. Medeiros ◽  
Claudia A. Costa ◽  
Laura F. L. Prado-Souza ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Clinical Problem ◽  
Jurkat Cells ◽  
Leukemia Cells ◽  
Peripheral Mononuclear Blood ◽  
Induced Apoptosis ◽  
Cure Rates

Acute lymphoblastic leukemia (ALL) is an aggressive malignant disorder of lymphoid progenitor cells that affects children and adults. Despite the high cure rates, drug resistance still remains a significant clinical problem, which stimulates the development of new therapeutic strategies and drugs to improve the disease outcome. Antipsychotic phenothiazines have emerged as potential candidates to be repositioned as antitumor drugs. It was previously shown that the anti-histaminic phenothiazine derivative promethazine induced autophagy-associated cell death in chronic myeloid leukemia cells, although autophagy can act as a “double-edged sword” contributing to cell survival or cell death. Here we evaluated the role of autophagy in thioridazine (TR)-induced cell death in the human ALL model. TR induced apoptosis in ALL Jurkat cells and it was not cytotoxic to normal peripheral mononuclear blood cells. TR promoted the activation of caspase-8 and -3, which was associated with increased NOXA/MCL-1 ratio and autophagy triggering. AMPK/PI3K/AKT/mTOR and MAPK/ERK pathways are involved in TR-induced cell death. The inhibition of the autophagic process enhanced the cytotoxicity of TR in Jurkat cells, highlighting autophagy as a targetable process for drug development purposes in ALL.

A novel mechanism for imatinib mesylate–induced cell death of BCR-ABL–positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2299-2307 ◽  
Author(s):  
Masayuki Okada ◽  
Souichi Adachi ◽  
Tsuyoshi Imai ◽  
Ken-ichiro Watanabe ◽  
Shin-ya Toyokuni ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Serine Protease ◽  
Imatinib Mesylate ◽  
Protease Activity ◽  
Kinase Inhibitor ◽  
Leukemic Cells ◽  
Endonuclease G ◽  
Serine Protease Activity ◽  
Human Leukemic Cells

Abstract Caspase-independent programmed cell death can exhibit either an apoptosis-like or a necrosis-like morphology. The ABL kinase inhibitor, imatinib mesylate, has been reported to induce apoptosis of BCR-ABL–positive cells in a caspase-dependent fashion. We investigated whether caspases alone were the mediators of imatinib mesylate–induced cell death. In contrast to previous reports, we found that a broad caspase inhibitor, zVAD-fmk, failed to prevent the death of imatinib mesylate–treated BCR-ABL–positive human leukemic cells. Moreover, zVAD-fmk–preincubated, imatinib mesylate–treated cells exhibited a necrosis-like morphology characterized by cellular pyknosis, cytoplasmic vacuolization, and the absence of nuclear signs of apoptosis. These cells manifested a loss of the mitochondrial transmembrane potential, indicating the mitochondrial involvement in this caspase-independent necrosis. We excluded the participation of several mitochondrial factors possibly involved in caspase-independent cell death such as apoptosis-inducing factor, endonuclease G, and reactive oxygen species. However, we observed the mitochondrial release of the serine protease Omi/HtrA2 into the cytosol of the cells treated with imatinib mesylate or zVAD-fmk plus imatinib mesylate. Furthermore, serine protease inhibitors prevented the caspase-independent necrosis. Taken together, our results suggest that imatinib mesylate induces a caspase-independent, necrosis-like programmed cell death mediated by the serine protease activity of Omi/HtrA2.

scholarly journals 2',3'-Dideoxyadenosine is selectively toxic for TdT-positive cells

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1601-1608
Author(s):  
Z Spigelman ◽  
R Duff ◽  
GP Beardsley ◽  
S Broder ◽  
D Cooney ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Ex Vivo ◽  
Exposure Period ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Parental Line ◽  
Continuous Exposure ◽  
Hiv Replication

The 2′,3′-dideoxynucleosides (ddNs) are currently undergoing clinical evaluation as antiretroviral agents in HIV-infected individuals. When phosphorylated, the ddNs (ddNTPs) function as chain-terminating substrate analogues with reverse transcriptase, thereby inhibiting HIV replication. These nucleoside analogues can also inhibit, by chain- terminating additions, the primitive lymphoid DNA polymerase, terminal deoxynucleotidyl transferase (TdT). To determine the effect of possible intracellular chain-terminating additions of ddNMPs by TdT, we exposed a series of TdT-positive and TdT-negative cell lines to 2′,3′- dideoxyadenosine (ddA), a representative ddN. At ddA concentrations 25- fold higher than required for inhibition of HIV replication, progressive dose-related cytotoxicity was observed in the TdT-positive cell lines. This was accentuated by the adenosine deaminase inhibitor Coformycin (CF), presumably by enhancing the intracellular generation of ddATP from ddA. A central role of TdT in mediating the ddA/CF cytotoxicity was suggested by studies in a pre-B-cell line rendered TdT positive by infection with a TdT cDNA-containing retroviral vector. After a 48-hour continuous exposure period to 250 mumol/L ddA and 30 mumol/L CF, 30% cell death was observed in the TdT-negative parental line, whereas 90% cell death was observed in the TdT-positive daughter line. Exposure of fresh TdT-positive leukemic cells to ddA/CF for 72 hours ex vivo resulted in cytotoxicity (six cases of acute lymphocytic leukemia [ALL]) while not affecting TdT-negative acute leukemic cells (six cases). We conclude that ddA/CF selectively damages TdT-positive cells, presumably by chain-terminating additions of ddAMP, and that this may have therapeutic relevance in TdT-positive malignant disease.

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