PF562 BLOCKING OF WIP1 PHOSPHATASE OVERCOMES BORTEZOMIB RESISTANCE AND PROMOTES CELL DEATH VIA ER STRESS‐INDUCED APOPTOTIC JNK/C‐JUN SIGNALING: NOVEL THERAPEUTIC TARGET IN MULTIPLE MYELOMA

Abstract

Background:Acquired or de novo resistance to the traditional and novel anti‐multiple myeloma (MM) agents remains a major treatment obstacle. 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.Aims:Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype.Methods:We utilized in vivo xenograft model of BM‐disseminated human myeloma, as well as in vitro analysis using MM cell lines, stromal components and primary samples from patients with MM.Results:Increased expression of WIP1 was detected in MM cell lines (n = 8) and primary samples (n = 18) as compared with normal PBMCs (n = 5). WIP1 expression levels were significantly up‐regulated following bortezomib (Bort) exposure and in Bort‐resistant MM cell lines, suggesting a role for WIP1 in the acquisition of 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 and caspase 3 activation. 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, mitochondrial destabilization and extensive DNA damage. 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 cIAP1, cIAP2, XIAP and Survivin. IAPs expression maintenance is part of an adaptive UPR that promotes MM survival upon Bort‐induced 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, combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro‐apoptotic factors ATF4, CHOP and GADD34. Concomitantly, cell cycle progression factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment. Furthermore, 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. These results identify pro‐apoptotic JNK/c‐Jun signaling being preferential target of WIP1 in the process of dampening Bort‐induced stress response. Finally, in xenograft model of systemic MM with BM involvement in vivo addition potentiated the Bort/GSK combination effectively reduced tumor burden, decreasing the number of MM cells in BM and M protein levels in blood.Summary/Conclusion: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.image