Bcl-2 Antagonism Potentiates MEK1/2/Chk1 Inhibitor Lethality in Multiple Myeloma Cells Overexpressing Bcl-2 through a Stat3-Dependent Mechanism

Introduction: Multiple myeloma (MM) is characterized by deregulation of members of the Bcl-2 family of apoptotic regulatory proteins. This has led to the development of BH3-mimetics such as ABT-737 which inhibits Bcl-2/xL but not Mcl-1. Previously, we reported that simultaneous inhibition of Chk1 and MEK1/2 dramatically induced apoptosis in cultured and primary MM cells, including cells resistant to conventional agents, while sparing their normal counterparts (Pei et al., Blood 2007, 2011). Recently, we reported that this strategy circumvented MM cell resistance conferred by overexpression of Mcl-1, an important survival factor in this disease (Pei et al., PLoS One 2014). However, Bcl-2 overexpression confers significant resistance to the Chk1/MEK1/2 inhibition strategy. This raised the possibility that BH3-mimetics targeting Bcl-2 might circumvent this resistance mechanism. The purpose of the present studies was to determine whether BH3-mimetics could overcome such resistance while preserving anti-myeloma selectivity. An additional goal was to employ a new mathematical model to characterize interactions combining three novel agents that coordinately inhibit multiple survival signaling pathways. Methods: Various parental and Bcl-2 or Bcl-xL-over-expressing MM cell lines, as well as primary CD138⁺ MM cells were employed. ABT-737, the MEK1/2 inhibitor PD184352 (PD), and the Chk1/Wee1 inhibitor (Chk1i) were obtained from Abbott, Millipore and Calbiochem, respectively. Cells were exposed to agents alone or in various combinations for 4 -72 h, after which effects on apoptosis and signaling pathways were determined. Results: Co-administration of ABT-737 potentiated PD/Chk1i-mediated lethality in multiple parental MM cell lines, in association with Mcl-1 down-regulation, Bim up-regulation, and increased DNA damage (ΥH2A.X). Consistent with earlier findings, ectopic expression of Bcl-2 or Bcl-xL protected MM cells from the PD/Chk1i regimen. However, co-administration of ABT-737 significantly restored sensitivity towards PD/Chk1i lethality. Mathematical modeling indicated 3-agent synergistic interactions, particularly in Bcl-2 overexpressing cells. PD/Chk1i exposure inhibited phosphorylation (T705 and S727) of Stat3, another important survival factor for MM cells, while cells expressing constitutively active Stat3 (CA-STAT3) exhibited resistance to this regimen. However, the latter event was reversed by co-exposure to ABT-737. Moreover, combining ABT-737 with PD/Chk1i resulted in release of Bim from anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1, accompanied by Bak and Bax conformational change (activation). Knock-down of Bim by shRNA significantly protected cells from apoptosis induced by the 3-agent combination, indicating a functional role for Bim in anti-MM activity of this regimen. Furthermore, similar interactions, together with down-regulation of pStat3, were also observed in bortezomib-resistant MM cells, as well as in patient-derived CD138+ MM cells. In contrast, the regimen was minimally toxic to normal cord blood CD34⁺ cells or CD138^- bone marrow cells. Finally, co-culture of parental or bortezomib-resistant MM cells with HS-5 stromal cells induced up-regulation of pStat3, while treatment with ABT-737 in combination with PD/Chk1i prevented Stat3 activation and robustly induced apoptosis despite the presence of stromal cells. Conclusion: ABT-737 co-administration synergistically potentiates the lethality of the PD/Chk1i regimen in MM cells, including bortezomib-resistant and primary MM cells, but not in normal hematopoietic progenitors. It also overcomes PD/Chk1i resistance conferred by overexpression of Bcl-2 or Bcl-xL, as well as by microenvironmental factors. Mechanisms responsible for these interactions are likely to be multi-factorial, including inactivation of Stat3, up-regulation of Bim, release of Bim from Bcl-2, Bcl-xL, and Mcl-1, and activation of Bak and Bax. Collectively, these findings demonstrate that combining BH3-mimetics with Chk1/MEK1/2 inhibition circumvents multiple forms of drug resistance in MM cells while exhibiting minimal toxicit toward normal hematopoietic cells. They also argue that a strategy targeting three coordinate survival signaling pathways may be highly effective in killing MM cells, particularly those resistant to current anti-MM therapies.