Abstract 4667: Simultaneous Chk1 and MEK1/2 inhibition circumvents Mcl-1 anti-apoptotic function and bortezomib resistance in human multiple myeloma cells
Authors: Pei Xin-Yan , Dai Yun , Felthousen Jessica , Chen Shuang , Wang Li , Takabatake Yukie , Youssefian Leena E. , Bodie Wesley W. , Kramer Lora , Grant Steven 
Published: August 2012

Mcl-1, a critical survival factor for human multiple myeloma (MM) cells, confers resistance to various conventional and novel anti-MM agents, including bortezomib. We previously reported that a therapeutic strategy combining Chk1 with MEK1/2 inhibitors is highly active in both MM cell lines as well as primary MM cells, and that up-regulation of the BH3-only protein Bim plays a critical role in lethality. However, whether this strategy also targets Mcl-1 and/or circumvents bortezomib-resistance is unknown. To address these questions, the specific Chk1 inhibitor CEP3891 was used in combination with the MEK1/2 inhibitor PD184352. Exposure to CEP3891 triggered ERK1/2 activation and Bim phosphorylation/degradation in multiple MM cell lines (e.g., U266, MM.1S, RPMI8226, and H929). Blockade of ERK1/2 activation by PD184352 led to Bim upregulation, accompanied by striking induction of apoptosis. Significantly, the CEP3891/PD184352 regimen was highly active against primary CD138+ samples (7 of 9), while sparing all CD138- samples (9 of 9) as well as normal cord blood CD34+ cells. Notably, PD184352 +/− CEP3891 induced marked Mcl-1 downregulation in MM cell lines and primary CD138+ MM samples. This regimen also blocked Mcl-1 up-regulation induced by IL-6 or IGF-1, a mechanism responsible for the pro-survival function of these growth factors in MM cells. Of note, ectopic expression of Mcl-1 conferred marked resistance to bortezomib. However, in Mcl-1-overexpressing cells, PD184352 retained its ability to block CEP3891-mediated ERK1/2 activation as well as Bim degradation, and combined treatment resulted in marked apoptosis equivalent to that observed in empty vector-transfected controls. Co-immunoprecipitation showed a marked increase in the association between Mcl-1 with Bim in MM cells exposed to PD184352 +/− CEP3891, while co-administration of these agents resulted in the release of Bak from Mcl-1. Similar events occurred in Mcl-1-overexpressing MM cells. Finally, PS-R cells, a bortezomib-resistant subline generated from U266 cells continuously cultured in stepwise increases in bortezomib concentrations exhibited increased Mcl-1 levels but markedly diminished Bim expression. Co-treatment with PD184352/CEP3891 clearly down-regulated Mcl-1 and increased Bim levels in these cells, leading to Bax translocation to mitochondria, release of mitochondrial cytochrome c and Smac/DIABLO, activation of caspase-3, and PARP cleavage. Collectively, these findings demonstrate a mechanism wherein, in MM cells, a strategy combining Chk1/MEK1/2 inhibitors targets Mcl-1 by a) directly down-regulating Mcl-1 protein expression, and b) inactivating Mcl-1 by increasing its binding to up-regulated Bim. They also raise the possibility that the Chk1/MEK1/2 inhibitor regimen circumvents bortezomib-resistance by disabling the cytoprotective actions of Mcl-1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4667. doi:1538-7445.AM2012-4667