Description:
Abstract Introduction Inhibitor of Apoptosis Proteins (IAPs) are anti-apoptotic proteins which suppress the activation of caspases. IAPs have been shown to be overexpressed in several cancers, including colorectal cancer (CRC) and correlate with poor prognosis. IAP antagonists, including SMAC mimetics, can convert TNFα from a pro-inflammatory and pro-survival signal into a potent pro-death signalling molecule. In the presence of IAP antagonists, TNFα signalling is driven to cell death induction through the formation of a complex containing FADD/procaspase 8 and RIPK1, termed complex-II. However, high expression of FLIP, an endogenous regulator of procaspase-8 processing and activation, can inhibit cell death signalling from this complex. We have previously shown that FLIP is frequently overexpressed in CRC, and we and others have demonstrated that histone deacetylase (HDAC) inhibition results in down-regulation of FLIP protein expression. The aims of this study were to assess the efficacy of a novel, non-peptidomimetic, cIAP and XIAP antagonist, ASTX660 (Astex Pharmaceuticals), as single agent and in combination with the class I HDAC inhibitor Entinostat in CRC cell lines. Results ASTX660 was found to be a potent on-target IAP antagonist in a panel of human and murine CRC cell lines, resulting in reduced cIAP1 expression and formation of the apoptosis-inducing complex II, containing RIPK1 and caspase-8, in the presence of TNFα. However, viability of CRC cell lines was unaffected by the ASTX660 treatment alone or in the presence of TNFα, added to mimic the pro-inflammatory microenvironment frequently associated with CRC. Downregulation of FLIP expression using siRNA in a panel of CRC cell lines significantly enhanced cell death induced by ASTX660 in the presence of TNFα. Entinostat down-regulated FLIP protein expression at clinically achievable concentrations and potentiated ASTX660-mediated cell death in the panel of CRC cell lines. This cell death was confirmed to be apoptotic in nature and, more specifically, caspase-8-dependent using CRISPR-caspase-8 knockout models. Moreover, the mode of combination effects was demonstrated to be FLIP-dependent using CRC models engineered to overexpress wild-type FLIP. Conclusion High levels of FLIP expression in pre-clinical models of CRC may contribute to resistance to single agent IAP antagonist treatment. Pre-treatment with the class I HDAC inhibitor Entinostat can sensitize CRC cell lines toASTX660 by down-regulating FLIP expression. HDAC inhibitors could be used in novel combinations with IAP antagonists to promote apoptosis in pro-inflammatory CRC. Citation Format: Nyree T. Crawford, Katie Stott, Tomoko Smyth, John Lyons, Roberta Ferraldeschi, George Ward, Daniel B. Longley. Entinostat sensitizes colorectal cancer cell lines to the IAP antagonist, ASTX660 by down-regulating FLIP expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4399.