Figure S1. HDACi promote GSK3β-mediated Mcl-1 phosphorylation, but not Mcl-1 degradation, in colon cancer cells. SAHA or MS-275 treatment did not affect Mcl-1 protein half-life in HCT116 cells, or induce Mcl-1 degradation in DLD1 and RKO cells. The 4A (S121A/E125A/S159A/T163A) mutant was more effective than WT Mcl-1 in suppressing SAHA- and MS-275-induced apoptosis in DLD1 and RKO cells. Figure S2. Knock-in of the 4A (S121A/E125A/S159A/T163A) mutant Mcl-1 suppressed apoptosis induced by HDACi and other anticancer agents. Figure S3. FBW7 was induced by the kinase inhibitors regorafenib and sorafenib, but not by the HDACi SAHA and MS-275 in colon cancer cells. Transfection of WT but not mutant FBW7 was sufficient to promote Mcl-1 degradation in cells treated with SAHA or MS-275. Figure S4. PUMA, Bim and Noxa mRNA was induced in HCT116 cells in response to SAHA and MS-275 treatment. Bim and Noxa were knocked out by homologous recombination in HCT116 cells. Figure S5. SAHA- and MS-275-induced apoptosis in HCT116 cells was suppressed by knockout of PUMA, Bim, Noxa or BAX. Figure S6. Immunoprecipitation analysis showed that the mutant Mcl-1 in the Mcl-1-KI cells had enhanced binding to Bim and Noxa in response to SAHA or MS-275 treatment. Figure S7. Mcl-1 inhibitors restored SAHA- and MS-275-induced apoptosis analyzed by annexin V/PI staining in Mcl-1-KI cells.
ARTICLE ABSTRACT
Mcl-1, a prosurvival Bcl-2 family protein, is frequently overexpressed in cancer cells and plays a critical role in therapeutic resistance. It is well known that anticancer agents induce phosphorylation of Mcl-1, which promotes its binding to E3 ubiquitin ligases and subsequent proteasomal degradation and apoptosis. However, other functions of Mcl-1 phosphorylation in cancer cell death have not been well characterized. In this study, we show in colon cancer cells that histone deacetylase inhibitors (HDACi) induce GSK3β-dependent Mcl-1 phosphorylation, but not degradation or downregulation. The in vitro and in vivo anticancer effects of HDACi were dependent on Mcl-1 phosphorylation and were blocked by genetic knock-in of a Mcl-1 phosphorylation site mutant. Phosphorylation-dead Mcl-1 maintained cell survival by binding and sequestering BH3-only Bcl-2 family proteins PUMA, Bim, and Noxa, which were upregulated and necessary for apoptosis induction by HDACi. Resistance to HDACi mediated by phosphorylation-dead Mcl-1 was reversed by small-molecule Mcl-1 inhibitors that liberated BH3-only proteins. These results demonstrate a critical role of Mcl-1 phosphorylation in mediating HDACi sensitivity through a novel and degradation-independent mechanism. These results provide new mechanistic insights on how Mcl-1 maintains cancer cell survival and suggest that Mcl-1–targeting agents are broadly useful for overcoming therapeutic resistance in cancer cells.Significance: These findings present a novel degradation–independent function of Mcl-1 phosphorylation in anticancer therapy that could be useful for developing new Mcl-1–targeting agents to overcome therapeutic resistance. Cancer Res; 78(16); 4704–15. ©2018 AACR.
