Abstract 3390: Simultaneous Chk1 and MEK1/2 inhibition circumvents Mcl-1-mediated anti-apoptotic functions and bortezomib resistance in human multiple myeloma cells.
Authors: Pei Xin-Yan , Dai Yun , Felthousen Jessica , Chen Shuang , Takabatake Yukie , Youssefian Leena E. , Bodie Wesley W. , Kramer Lora B. , Orlowski Robert Z. , Grant Steven 
Published: August 2014

Abstract
Mcl-1 is a critical survival factor for human multiple myeloma (MM) cells, and also confers resistance to various anti-MM agents, including bortezomib (btz). We have previously reported that exposure of MM cells to Chk1 inhibitors elicits activation of the MEK1/2/ERK1/2 pathway, and that prevention of that process e.g., by MEK1/2 inhibitors resulted in a dramatic increase in apoptosis (Pei et al., Blood 118:5189, 2011). The goal of the present study was to determine whether a strategy combining Chk1 (e.g., CEP3891; CEP) with MEK1/2 inhibitors (e.g., PD184352; PD) might circumvent Mcl-1 related resistance, and if so, by what mechanism(s). In various MM cell lines (e.g., U266, 8226, MM.1S), blockade of CEP-induced ERK1/2 activation by PD sharply down-regulated Mcl-1 in association with Stat3 inactivation, events accompanied by pronounced apoptosis. Ectopic expression of constitutively active (CA)-Stat3 up-regulated Mcl-1 expression and significantly attenuated PD/CEP lethality. While Mcl-1 over-expression conferred marked btz resistance, no cross resistance to PD/CEP occurred. Interestingly, btz-resistant U266 (PS-R) and OPM-2 (O/V10R) cells displayed both increased Stat3 activation and elevated Mcl-1 levels, but were fully sensitive to PD/CEP. Notably, in Mcl-1-overexpressing cells and drug-naïve or -resistant cells exposed to PD/CEP, co-IP demonstrated a marked increase in Mcl-1 binding to Bim, which was robustly up-regulated, accompanied by release of Bak from Mcl-1 and Bak/Bax activation. IL-6 or IGF-1 exposure up-regulates Mcl-1 via Stat3 activation, and this phenomenon was blocked by PD/CEP, suggesting that this strategy may circumvent microenvironment-mediated drug resistance. Indeed, co-culture of MM cells with stromal HS-5 cells conferred resistance to dexamethasone and melphalan, but not to PD/CEP. Significantly, equivalent results were obtained in btz-resistant PS-R cells as well as other OPM2/btz-resistant MM cells co-cultured with HS-5 cells. Significantly, the PD/CEP regimen was also highly active against primary CD138+ MM samples (n = 8 of 10), while sparing their normal CD138− counterparts as well as normal cord blood CD34+ cells (n = 3). Analogous to mechanisms observed in MM cell lines, PD/CEP inhibited Stat3 activation, induced Mcl-1 downregulation, and triggered Bim up-regulation in primary CD138+ MM but not in normal CD138−, cells. Collectively, these findings identify novel mechanisms by which combined Chk1 and MEK1/2 inhibition targets Mcl-1 in MM cells: a) down-regulation of Mcl-1 through inhibition of Stat3 activation and b) disabling Mcl-1 by increasing its binding to up-regulated Bim, thereby circumventing both Mcl-1-related bortezomib- and microenvironment-mediated resistance.
Citation Format: Xin-Yan Pei, Yun Dai, Jessica Felthousen, Shuang Chen, Yukie Takabatake, Leena E. Youssefian, Wesley W. Bodie, Lora B. Kramer, Robert Z. Orlowski, Steven Grant. Simultaneous Chk1 and MEK1/2 inhibition circumvents Mcl-1-mediated anti-apoptotic functions and bortezomib resistance in human multiple myeloma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3390. doi:10.1158/1538-7445.AM2013-3390