Mutant BRAF Upregulates MCL-1 to Confer Apoptosis Resistance that Is Reversed by MCL-1 Antagonism and Cobimetinib in Colorectal Cancer

Oncogenic BRAF(V600E) mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAF(V600E)-mutant colorectal cancers, treatment failure may be related to BRAF(V600E)-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAF(V600E) can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF. BRAF-BRAF(V600E) induced MCL-1 upregulation was confirmed by ectopic BRAF(V600E) expression that activated MEK/ERK signaling to phosphorylate (MCL-1(Thr163)) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAF(V600E) cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction ofMCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAF(V600E)-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAF(V600E)-mutant CRCs. (C) 2016 AACR.