Targeting PKC: A Novel Role for Beta-catenin in ER Stress and Apoptotic Signaling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2763-2763
Author(s):  
Marc S Raab ◽  
Iris Breitkreutz ◽  
Giovanni Tonon ◽  
Jing Zhang ◽  
Johannes Fruehauf ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Tumor Cells ◽  
Cell Lines ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Hek 293 ◽  
Pkc Isoforms ◽  
Wide Range

Abstract Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising pre-clinical activity in a wide range of tumor cells. In this study, we further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of b-catenin in regulating growth and survival of tumor cells. Inhibition of PKC leads to rapid accumulation of b-catenin by preventing the phosphorylation required for its proteasomal degradation. Specifically, b-catenin was dephosphorylated at Ser33,37,41 and accumulated in a dose- and time-dependent manner in all cell lines tested (including primary MM cells and 10 MM cell lines, 3 colon cancer, HeLa, as well as HEK 293 cells). Microarray analysis and siRNA-mediated gene silencing in MM cells revealed that accumulated b-catenin activates early ER stress signaling via eIF2a, CHOP and p21, leading to immediate inhibition of proliferation. Conversely, knock-down of components of the ER stress response pathway by siRNA (i.e., CHOP) abrogated the inhibitory effect of enzastaurin on MM cell proliferation. Importantly, accumulated b-catenin also contributes to enzastaurin-induced cell death, since inhibition of b-catenin by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a b-catenin -dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; b-catenin induction by enzastaurin led to p73 (but not p53) activation, which was also abrogated by b-catenin -specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of b-catenin in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. In summary, our data reveal a novel role of b-catenin in ER stress-mediated growth inhibition and a new pro-apoptotic mechanism triggered by b-catenin upon inhibition of PKC isoforms, and further demonstrate that p73 represents a novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies including MM.

scholarly journals Targeting PKC: a novel role for beta-catenin in ER stress and apoptotic signaling

Blood ◽  
2009 ◽  
Vol 113 (7) ◽  
pp. 1513-1521 ◽  
Author(s):  
Marc S. Raab ◽  
Iris Breitkreutz ◽  
Giovanni Tonon ◽  
Jing Zhang ◽  
Patrick J. Hayden ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Inhibition ◽  
Er Stress ◽  
Tumor Cells ◽  
Clinical Investigation ◽  
Hematologic Malignancies ◽  
Beta Catenin ◽  
Pkc Isoforms ◽  
Wide Range

Abstract Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising preclinical activity in a wide range of tumor cells. We further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of β-catenin in regulating growth and survival of tumor cells. Specifically, inhibition of PKC leads to rapid accumulation of β-catenin by preventing the phosphorylation required for its proteasomal degradation. Microarray analysis and small-interfering RNA (siRNA)–mediated gene silencing in MM cells revealed that accumulated β-catenin activates early endoplasmic reticulum stress signaling via eIF2α, C/EBP-homologous protein (CHOP), and p21, leading to immediate growth inhibition. Furthermore, accumulated β-catenin contributes to enzastaurin-induced cell death. Sequential knockdown of β-catenin, c-Jun, and p73, as well as overexpression of β-catenin or p73 confirmed that accumulated β-catenin triggers c-Jun–dependent induction of p73, thereby conferring MM cell apoptosis. Our data reveal a novel role of β-catenin in endoplasmic reticulum (ER) stress-mediated growth inhibition and a new proapoptotic mechanism triggered by β-catenin on inhibition of PKC isoforms. Moreover, we identify p73 as a potential novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies, including MM.

Targeting Proteinkinase C Alters ER-Stress and b-Catenin Signaling in Multiple Myeloma: Therapeutic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 258-258
Author(s):  
Marc S. Raab ◽  
Klaus Podar ◽  
Jing Zhang ◽  
Giovanni Tonon ◽  
Johannes H. Fruehauf ◽  
...  
Keyword(s):  
Cell Death ◽  
Stress Response ◽  
Growth Inhibition ◽  
Er Stress ◽  
Cell Lines ◽  
Wnt Pathway ◽  
Dependent Manner ◽  
P53 Family ◽  
Er Stress Response

Abstract We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a > 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.

scholarly journals Endoplasmic Reticulum Stress Signaling Comprises a G9a Inhibitor Tolerance Pathway and PERK Inhibition Increases Anti-Leukemia Activity of G9a Inhibitor in Leukemia Cells and Leukemia Stem-like Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1360-1360
Author(s):  
Jieun Jang ◽  
Ju-In Eom ◽  
Hoi-kyung Jeung ◽  
So-Young Seol ◽  
Haerim Chung ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Leukemia Cell ◽  
Apoptotic Cell Death ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Eif2α Phosphorylation ◽  
Kg1 Cells

Abstract Background: Histone methyltransferase (HMTase) G9a regulates the transcription of multiple genes by primarily catalyzing dimethylation of histone H3 lysine 9 (H3K9me2), as well as several non-histone lysine sites. Recently, pharmacological and genetic targeting of the G9a was shown to be efficient in slowing down acute myeloid leukemia (AML) cell proliferation in a mouse model and human AML cell lines thus making this HMTase potential target for epigenetic therapy of AML. Activation of adaptive mechanisms to drug plays a crucial role in drug resistance and relapse by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The tolerance mechanism to HMTase regulation in leukemia cell is unclear yet. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Recent previous studies showed that pro-survival ER stress is induced in cancer cells and contributes to development of drug resistance. Methods: We investigated the levels of apoptosis and ER stress by G9a inhibitor BIX-01294 in leukemia cell lines. U937, cytarabine-resistant U937 (U937/AR) and KG1 were used. U937/AR cell line was established in our laboratory by exposing parental U937 cells to stepwise increasing concentrations of cytarabine. Results: We initially examined the expression of G9a in leukemia cell lines and the primary AML cells obtained from a patient at the different time point. In U937/AR cells and primary AML cells obtained at relapse, G9a expression was increased compare to that in U937 cells and primary AML cells obtained at diagnosis, respectively. G9a expression was also increased in KG1 cells. In both of U937 and U937/AR, apoptotic cell death was induced by BIX-01294 in a dose-dependent manner. In contrast, apoptotic cell death was minimal in KG1 cells which are enriched in cells expressing a leukemia stem cell phenotype (CD34+CD38-). To address the activation of ER stress response by BIX-01294 in leukemia cells, we examined the effect of BIX-01294 treatment on PERK and eIF2α protein expression and phosphorylation levels. We found that treatment of U937, U937/AR, KG1 cells with 3μM of BIX-01294 for 24h caused an upregulation of phosphorylated PERK and eIF2α. The upregulation of PERK phosphorylation was associated with a decrease in PERK protein levels after treatment. To further address the role of the PERK-eIF2α phosphorylation in BIX-01294 sensitivity, we examined whether PERK inhibition using small interfering RNA (siRNA) or specific inhibitor could sensitize cells to BIX-01294-mediated death. The siRNA against PERK effectively inhibited BIX-01294-mediated phosphorylation of PERK and eIF2α in U937 and U937/AR cells. The addition of PERK siRNA led to a significant increase in the extent of BIX-01294-induced apoptotic cell death in U937 (P = 0.0003) and U937/AR (P < 0.0001) as compared with that of BIX-01294 treatment alone. PERK inhibitor GSK260641 significantly increased BIX-01294-induced apoptotic cell death in U937 (P < 0.0001) and U937/AR (P = 0.006) cells. To our surprise, addition of PERK siRNA or GSK260641 increased the sensitivity of KG1 cells to BIX-01294-mediated death in a dose-dependent manner (P = 0.0003 for siRNA, P = 0.0053 for GSK260641). Conclusion: These data demonstrated that PERK-eIF2α activation has a pro-survival function to G9a inhibitor in leukemia cells and mediates resistance of AML stem cells to G9a inhibitor treatment. The PERK-eIF2α phosphorylation arm may represent a suitable target for combating resistance to G9a inhibitor in AML. The mechanisms underlying the increased sensitivity of AML cells with PERK inhibition to G9a inhibitor are unclear at present and are needed to define in further studies. Disclosures No relevant conflicts of interest to declare.

The xCT Antiporter Is a Therapeutic Target in the ABC Subtype of DLBCL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3996-3996
Author(s):  
Xiaolei Wei ◽  
Yun Mai ◽  
Ru Feng ◽  
B. Hilda Ye
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Ros Accumulation

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the adult population and can be subdivided into two main subtypes, i.e. GCB-DLBCL and ABC-DLBCL. While both subtypes are derived from normal germinal center (GC) B cells, they differ in B cell maturation stage, transformation pathway, and clinical behavior. When treated with either the combination chemotherapy CHOP or the immuno-chemotherapy R-CHOP, the survival outcome of ABC-DLBCL patients is typically much worse than that of GCB-DLBCL patients. Although the molecular mechanisms underlying this survival disparity remain poorly understood, an attractive hypothesis is that there exist subtype-specific resistance mechanisms directed against the chemo-therapy drugs in the original CHOP formulation. In support of this notion, our previous study has revealed that Doxorubicin (Dox), the main cytotoxic ingredient in CHOP, has subtype-specific mechanisms of cytotoxicity in DLBCLs due to differences in its subcellular distribution pattern. In particular, Dox-induced cytotoxicity in ABC-DLBCLs is largely dependent on oxidative stress rather than DNA damage response. Based on these findings, we hypothesize that agents capable of disturbing the redox balance in ABC-DLBCL cells could potentiate the therapeutic activity of first line lymphoma therapy. As the major route of cystine uptake from extracellular space, the xCT cystine/glutamate antiporter controls the rate-limiting step for glutathione (GSH) synthesis in several types of cancer cells, including CLL. We focused the current study on xCT because its protein stability is known to be positively regulated by a splicing variant of CD44 and we have recently published that expression of CD44 and CD44V6 are poor prognosticators for DLBCL. Indeed, we found that surface CD44 is exclusively expressed in ABC-DLBCL (6/6) but not GCB-DLBCL (0/5) cell lines. In addition, the xCT proteins in two ABC-DLBCL cell lines, Riva and SuDHL2, are extraordinarily stable, with half-lives exceeding 24 hours. As such, transient transfection using siRNA oligos was ineffective in reducing the endogenous xCT protein in ABC-DLBCL cell lines. To circumvent this issue, we turned to a clinically approved anti-inflammatory drug, sulfasalazine (SASP), which is a validated xCT inhibitor in its intact form. When Riva and SuDHL2 cells were treated overnight with the IC50 dose of SASP, the endogenous GSH pool was drastically reduced, leading to significant increase in intracellular ROS, p38 and JNK activation, and progressive apoptosis. Unexpectedly, we found that Dox-treated cells had significantly elevated GSH levels, possibly the result of an antioxidant response to Dox-triggered ROS accumulation. This increase in GSH was completely suppressed when the IC25 dosage of SASP was included in the Dox treatment. As expected, SASP/Dox combination significantly enhanced Dox-triggered ROS accumulation and synergistically promoted cell death in Riva and SuDHL2 cells. Mechanistically, p38 activation and cell death induced by SASP/Dox combination could be markedly attenuated by pretreatment with glutathione monoethyl ester, demonstrating the critical role of oxidative stress. Furthermore, cytotoxicity triggered by SASP/Dox could also be suppressed by the p38 inhibitor, SB203580. We have developed stable cell lines expressing xCT shRNA to confirm the results obtained with SASP. In vivo interactions between SASP and Dox are also being evaluated in xenograft-based ABC-DLBCL models. In summary, we report here for the first time a critical role of xCT in sustaining in vivo GSH production in ABC-DLBCL cells. More importantly, pharmacologic inhibition of xCT function in ABC-DLBCL cells not only prevented Dox-induced endogenous GSH increase, but also potentiated Dox-induced ROS accumulation and cytotoxicity in a p38-dependent manner. With additional evidence from ongoing experiments, our study aims to provide a mechanistic basis for development of novel therapies that target either xCT or redox homeostasis to improve treatment outcomes for ABC-DLBCLs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pan-Sigma Receptor Modulator RC-106 Induces Terminal Unfolded Protein Response In In Vitro Pancreatic Cancer Model

2020 ◽  
Vol 21 (23) ◽  
pp. 9012
Author(s):  
Michela Cortesi ◽  
Alice Zamagni ◽  
Sara Pignatta ◽  
Michele Zanoni ◽  
Chiara Arienti ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Cell Lines ◽  
Transcriptional Gene Silencing ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Protein Response

Pancreatic cancer (PC) remains one of the most lethal cancers worldwide. Sigma receptors (SRs) have been proposed as cancer therapeutic targets. Their main localization suggests they play a potential role in ER stress and in the triggering of the unfolded protein response (UPR). Here, we investigated the mechanisms of action of RC-106, a novel pan-SR modulator, to characterize therapeutically exploitable role of SRs in tumors. Two PC cell lines were used in all the experiments. Terminal UPR activation was evaluated by quantifying BiP, ATF4 and CHOP by Real-Time qRT-PCR, Western Blot, immunofluorescence and confocal microscopy. Cell death was studied by flow cytometry. Post-transcriptional gene silencing was performed to study the interactions between SRs and UPR key proteins. RC-106 activated ER stress sensors in a dose- and time-dependent manner. It also induced ROS production accordingly with ATF4 upregulation at the same time reducing cell viability of both cell lines tested. Moreover, RC-106 exerted its effect through the induction of the terminal UPR, as shown by the activation of some of the main transducers of this pathway. Post-transcriptional silencing studies confirmed the connection between SRs and these key proteins. Overall, our data highlighted a key role of SRs in the activation of the terminal UPR pathway, thus indicating pan-SR ligands as candidates for targeting the UPR in pancreatic cancer.

scholarly journals Establishment of Acquired Cisplatin Resistance in Ovarian Cancer Cell Lines Characterized by Enriched Metastatic Properties with Increased Twist Expression

2020 ◽  
Vol 21 (20) ◽  
pp. 7613
Author(s):  
Entaz Bahar ◽  
Ji-Ye Kim ◽  
Hyun-Soo Kim ◽  
Hyonok Yoon
Keyword(s):  
Ovarian Cancer ◽  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Dna Repair ◽  
Er Stress ◽  
Cell Lines ◽  
Cisplatin Resistance ◽  
Cell Models ◽  
Mesenchymal Transition

 Ovarian cancer (OC) is the most lethal of the gynecologic cancers, and platinum-based treatment is a part of the standard first-line chemotherapy regimen. However, rapid development of acquired cisplatin resistance remains the main cause of treatment failure, and the underlying mechanism of resistance in OC treatment remains poorly understood. Faced with this problem, our aim in this study was to generate cisplatin-resistant (CisR) OC cell models in vitro and investigate the role of epithelial–mesenchymal transition (EMT) transcription factor Twist on acquired cisplatin resistance in OC cell models. To achieve this aim, OC cell lines OV-90 and SKOV-3 were exposed to cisplatin using pulse dosing and stepwise dose escalation methods for a duration of eight months, and a total of four CisR sublines were generated, two for each cell line. The acquired cisplatin resistance was confirmed by determination of 50% inhibitory concentration (IC50) and clonogenic survival assay. Furthermore, the CisR cells were studied to assess their respective characteristics of metastasis, EMT phenotype, DNA repair and endoplasmic reticulum stress-mediated cell death. We found the IC50 of CisR cells to cisplatin was 3–5 times higher than parental cells. The expression of Twist and metastatic ability of CisR cells were significantly greater than those of sensitive cells. The CisR cells displayed an EMT phenotype with decreased epithelial cell marker E-cadherin and increased mesenchymal proteins N-cadherin and vimentin. We observed that CisR cells showed significantly higher expression of DNA repair proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly (ADP-ribose) polymerases 1 (PARP1), with significantly reduced endoplasmic reticulum (ER) stress-mediated cell death. Moreover, Twist knockdown reduced metastatic ability of CisR cells by suppressing EMT, DNA repair and inducing ER stress-induced cell death. In conclusion, we highlighted the utilization of an acquired cisplatin resistance model to identify the potential role of Twist as a therapeutic target to reverse acquired cisplatin resistance in OC.

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 Natural Killer Cells Transfer Antimicrobial and Antitumoral Histone H2AZ to Kill Multiple Myeloma Cells Contributing to Transmissible Cytotoxicity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2115-2115
Author(s):  
Beatriz Martin-Antonio ◽  
Guillermo Suñe ◽  
Amer Najjar ◽  
Lorena Perez-Amill ◽  
Maria Velasco-de Andres ◽  
...  
Keyword(s):  
Dna Damage ◽  
Antimicrobial Activity ◽  
Cell Death ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Granzyme B ◽  
Over Expression ◽  
Nk Cytotoxicity

Abstract Natural Killer (NK) cells are antitumoral, antiviral and antimicrobial cells. The first antitumor mechanism described for NK cells was the "missing self" recognition which happens between the inhibitory "Killer Immunoglobulin-like Receptors" (KIRs) on NK cells and the HLA-I present in all nucleated cells. Thus, down-regulation or absence of HLA-I in tumor cells leads to "missing self" recognition activating NK cells. As a consequence, NK cells deliver Granzyme-B activating apoptotic cell death. However, NK cells can mediate Granzyme-B and Caspase-independent cell death against tumor cells expressing HLA-I, such as multiple myeloma (MM) cells. The cytotoxic mediators mediating non-apoptotic cell death remain unknown. Mechanisms mediating NK cell antimicrobial activity have been less studied, being recognized the role of Granulysin mediating Caspase independent cell death. Cord blood (CB) derived NK cells (CB-NK) is a clinically applicable strategy for the generation of highly functional NK cells which can be used to treat MM and potentially other haematological malignancies. We have demonstrated that CB-NK cytotoxicity against MM is Granzyme B and Caspase-independent. This CB-NK cytotoxicity is transmissible among MM cells, meaning that the initial MM recipient cells (termed primary MM cells or 1ºMM cells)-which formed direct contact with CB-NK are able to transfer lipid-protein vesicles to neighbouring unexposed MM cells (termed secondary MM cells or 2ºMM cells) causing a transmissible cytotoxicity to 2ºMM cells, which have not been in direct contact with CB-NK. We aimed to determine the key cytotoxic mediators transferred from CB-NK to 1ºMM and afterwards to 2ºMM cells that mediate secondary cytotoxicity. To do that, we performed TRANS-SILAC proteomics to determine proteome trafficking between CB-NK and MM cells. TRANS-SILAC proteomics demonstrated increased cell-cell communication between MM cells after CB-NK exposure, with secondary communication between MM cells to transfer the previously received cytotoxic CB-NK material. This secondary transfer represents a dilution of the CB-NK cytotoxic material. At the same time, MM cells transferred their own proteome to neighboring MM cells. Histones were among the CB-NK transferred proteins to 1ºMM, and 2ºMM cells. We selected the Histone H2AZ variant 1 (H2AZ) for validation. Time lapse in vitro confocal microscopy with CB-NK over-expressing H2AZ confirmed dynamic transfer of H2AZ from CB-NK to MM cells. Transfer occurred through vesicles and large intercellular structures. Extracellular DNA staining confirmed that these structures with H2AZ were accompanied by DNA. At the extracellular level, Histones exhibit significant antimicrobial activity, being the main antimicrobial effectors of neutrophils. The multiple positive charges and hydrophobic residues present in Histones allow them to bind to and invade negatively-charged cell membranes which are present in both microbial pathogens and tumor cells and interact with the DNA. We determined the cytotoxic role of H2AZ. H2AZ over-expression caused Caspase independent cell death in four different MM cell lines (p<0.0001). No differences were found on K562 cells. H2AZ over-expression increased DNA damage in MM cell lines. Furthermore, MM cell lines over-expressing H2AZ transferred to neighboring MM cells both H2AZ and the DNA damage. H2AZ transfer and DNA damage transfer also translated into a transmissible cytotoxicity between MM cells, as MM cells over-expressing H2AZ transferred cytotoxicity to neighboring MM cells who received this H2AZ. H2AZ involvement in CB-NK cytotoxicity against MM cells was demonstrated as knock-down and over-expression of H2AZ in CB-NK reduced and increased, respectively, CB-NK cytotoxicity against MM cell lines (p<0.05). For K562, no impact was observed confirming again a different CB-NK cytotoxicity mechanism for MM cell lines. Knock-down and over-expression of H2AZ in CB-NK reduced and increased, respectively, CB-NK antimicrobial activity against Candida albicans and Escherichia coli, demonstrating the antimicrobial role of H2AZ. Last, the antitumoral and antimicrobial properties of the recombinant protein H2AZ against MM cell lines and against bacteria were also demonstrated. Taken together, we demonstrate for first time the antitumoral and antimicrobial role of CB-NK Histones, revealing new therapeutic targets Disclosures No relevant conflicts of interest to declare.

CAL-101, a Potent Selective Inhibitor of the p110d Isoform of Phosphatidylinositol 3-Kinase, Attenuates PI3K Signaling and Inhibits Proliferation and Survival of Acute Lympoblastic Leukemia in Addition to a Range of Other Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 16-16 ◽  
Author(s):  
Brian J Lannutti ◽  
Sarah A. Meadows ◽  
Adam Kashishian ◽  
Bart Steiner ◽  
Sarah May ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
B Cell Lymphoma ◽  
Class I ◽  
Pi3k Signaling ◽  
Wide Range ◽  
Normal Human ◽  
Phosphatidylinositol 3

Abstract The class I phosphatidylinositol 3-kinases (PI3K) regulate a variety of cellular functions including motility, metabolism, proliferation, growth, and survival, depending on cellular milieu. Deregulation of the PI3K/Akt pathway is one of the most frequently observed defects in human malignancies including those of hematological origin and has been shown to play an important role in tumor progression. Therefore, selective targeting of PI3K signaling in hematological tumor cells could provide an effective treatment strategy while limiting potential undesirable effects of pan-inhibitors that broadly block PI3K signaling in all cells. Of the class IA PI3Ks (p110a, p110b, p110d), p110d’s expression is largely restricted to cells of hematopoietic origin and is essential for PI3K signaling in lymphocytes. Here, we report on the characterization of a novel p110d specific inhibitor, CAL-101. This compound is a potent PI3K inhibitor with an IC50 of 1–10 nM against the purified p110d subunit and 30–70 nM cellular potency against p110d-mediated basophil activation in whole blood. Importantly, CAL-101 plasma concentrations of 500–5000 nM that greatly exceed those needed for p110d inhibition in blood were safely maintained in a 7 day multidose normal human volunteer study. CAL-101 demonstrates &gt;30-fold selectivity over other class I, II and III PI3K family members as well as selectivity over other PI3K-related proteins including mTOR and DNA-PK. Furthermore, a genome wide screen of &gt;350 protein kinases did not detect any activity. To investigate the potential role of p110d in hematologic tumors we screened a wide range of leukemia and lymphoma cell lines for p110 isoform expression and constitutive PI3K pathway activation. The expression of p110d was observed in &gt;90% of these cell lines that was in many cases accompanied by constitutive Akt phosphorylation. In this context, CAL-101 was able to reduce p-Akt levels and block additional downstream effectors such as p-p70S6K, p-GSKb, and p-Bad in cells that represent a range of tumor types including acute myeloid leukemia, acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma among others. Recent studies have demonstrated the importance of PTEN loss and enhanced PI3K signaling in primary T-ALL cells. We report high levels of p110d protein and activated Akt in 6 of 6 ALL cell lines evaluated. Inhibition of p110d with CAL-101 treatment of both T-ALL and B-ALL cell lines resulted in a reduction of Akt and GSK-3b phosphorylation and a decrease in cellular proliferation that was accompanied by cell death demonstrating an essential role of PI3K signaling independent of PTEN status. Treatment of T-ALL cell lines with CAL-101 induced processing of pro-caspase-3 and cleavage of PARP supporting a role for caspase mediated cell death. These studies have now been extended to the analysis of primary patient blast samples to further establish preclinical proof of concept for therapeutic application of CAL-101 for the treatment of ALL. In summary, CAL-101 is a highly potent and selective p110d kinase inhibiter with broad anti-tumor activity against cancer cells of hematologic origin. Clinical studies in normal human volunteers demonstrated good tolerability with high drug exposure and favorable steady-state pharmacokinetic properties. Taken together, these data support the on going Phase 1 clinical trial that includes a wide range of hematological malignancies.

The BCR-ABL Fusion Protein Sensitizes Acute Lymphoblastic Leukemia to Apoptotic Death Induced by the Dual Glycolytic and Glycosylation Inhibitor 2-Deoxy-D-Glucose.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2439-2439
Author(s):  
Guy J Leclerc ◽  
Jianfeng Du ◽  
Joanna DeSalvo ◽  
Gilles M. Leclerc ◽  
Julio C. Barredo
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Er Stress ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
K562 Cells ◽  
Single Drug ◽  
Glycosylation Inhibitor ◽  
Gsk 3Β

Abstract Abstract 2439 Tumor metabolism has emerged as a hallmark of cancer by which the oncogenic profile of cancer cells pairs energy availability with cell growth and survival. BCR-ABL positive acute lymphoblastic leukemia (BCR-ABL+ ALL) is a highly resistant phenotype, and the BCR-ABL fusion protein has been correlated with alterations in glucose metabolism. The glucose analogue 2-deoxy-D-glucose (2DG) has been found to be an effective antitumor agent in both animal models and human clinical trials for solid tumors based on the dependency of hypoxic tumor cells on anaerobic glycolysis to generate ATP. 2DG inhibits two key glycolytic enzymes, hexokinase (HK) and phosphoglucose isomerase (PGI), and interfers with N-linked glycosylation by its incorporation in place of mannose into lipid-linked oligosaccharide (LLO) chains which leads to premature termination of LLO synthesis. Unlike hypoxic solid tumors, we recently demonstrated that 2DG induces cell death in ALL under normoxia preferentially by inhibiting N-linked glycosylation resulting in ER stress/UPR-mediated apoptosis, although inhibition of glycolysis also contributed to cell death in a phenotype specific-manner (Mol Cancer Res 10:969, 2012). Among ALL subtypes, BCR-ABL+ ALL t(9;22) cell lines SupB15 and TOM1 exhibited the highest sensitivity to 2-DG suggesting that BCR-ABL expression may be linked to sensitivity to 2DG. To investigate the role of BCR-ABL expression in this process, we constructed NALM6 (Bp-ALL) stable cell lines expressing BCR-ABL p190 fusion, and assessed their level of sensitivity to 2DG under normoxia. Expression of the BCR-ABL p190 fusion in NALM6 cells significantly increased 2DG-induced apoptosis compared to mock transfected NALM6 cells, indicating that BCR-ABL expression results in higher sensitivity to 2DG in this experimental system. Western blot analysis of BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL (NALM6) cell lines demonstrated that BCR-ABL expression induced alterations in the PI3K/Akt/p70S6K, GSK-3β/Mcl-1, p-STAT3 (Ser727), p-PKCδ (Thr505), and GRP78/p-eIF2α (Ser51) signaling pathways. Treatment with 2DG resulted in upregulation of p-AMPK (Thr172) and p-GSK-3β (Ser9), and downregulation of p-mTOR (Ser2448), p-p70S6K (Thr389), p-STAT3 (Ser727) and Mcl-1 in both BCR-ABL+ ALL (SUPB15 and TOM1) and Bp-ALL cells (NALM6). As we have previously described, 2DG induces ER stress and the expression of the unfolded protein response (UPR) markers (Mol Cancer Res 10:969, 2012). In BCR-ABL+ ALL cells, 2DG led to upregulation of IRE1α, GRP78 and CHOP (a marker of UPR induced cell death) and resulted in apoptotic cell death (>60%), which correlated with lower level of Mcl-1 expression. To further investigate the role of Mcl-1 on 2DG's sensitivity we used pharmacological inhibitors and shRNA. We found that Mcl-1 down-regulation sensitized Bp-ALL cells to 2DG (36% ± 4% cell death; p<0.001 for NALM6/shMcl-1 vs. NALM6/shScramble treated with 2DG 0.5mM). We then examined the role of GSK-3β/Mcl-1 signaling in K562, a highly 2DG resistant CML cell line expressing BCR-ABL p210 fusion. We found that K562 cells expressed high levels of Mcl-1. Inhibition of PI3K/Akt/GSK-3β pathway using LY294002 sensitized K562 cells to 2DG, suggesting that downregulation of Mcl-1 by GSK-3β activation leads to 2DG sensitivity. Finally, we co-targeted BCR-ABL+ ALL cells with TKIs (Imatinib) plus the glycolytic/glycosylation inhibitor 2DG and found significant increase in cell death for the combination compared to single drug (62.6% ± 0.8% cell death; p<0.001 for 2DG 4mM + Imatinib 0.5μM vs. single drug). Further, no cross-resistance was detected between 2DG and Imatinib in TKI-resistant BCR-ABL+ ALL cells. Taken together, our data demonstrate that BCR-ABL p190 expression sensitizes Bp-ALL cells to 2DG by altering the GSK-3β/Mcl-1 signaling pathway and inducing ER stress/UPR mediated cell death. This study supports further consideration of strategies combining inhibitors of glucose metabolism such as 2DG with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL. Disclosures: No relevant conflicts of interest to declare.

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