HDAC inhibitors overcome resistance to IAP inhibitors in colorectal cancer by downregulating FLIP


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Katie Stott1,Nyree Crawford1,Daniel Longley1
1CCRCB Queens University Belfast

Abstract

Background

Inhibitors of apoptosis proteins (IAPs) are E3 ligases that promote cell survival in response to TNFα signalling. Recently, IAP inhibitors have been developed which mimic SMAC an endogenous IAP antagonist. In the presence of IAP inhibitors, TNFα signalling is driven to cell death induction through the formation of a complex containing FADD/procaspase 8 and RIPK1, termed complex-II. However, FLIP, an endogenous regulator of procaspase-8, can inhibit cell death through complex-II. It has been previously demonstrated that FLIP is frequently overexpressed in CRC and that histone deacetylase (HDAC) inhibitors down-regulate FLIP; therefore, we hypothesised that HDAC inhibitors could sensitise CRC cells to IAP inhibitors.

Results

To assess the role of FLIP as a resistance mechanism to IAP inhibitors, siRNA transfections were used to down-regulate FLIP in a panel of CRC cell lines. Down-regulation of FLIP synergistically enhanced cell death induced by the IAP inhibitor ASTX660 (Astex Pharmaceuticals) in cells co-cultured with TNFα to mimic the pro-inflammatory microenvironment associated with CRC. Subsequently, the recently FDA approved class I-specific HDAC inhibitor Entinostat was found to down-regulate FLIP at clinically achievable concentrations and synergistically enhance ASTX660-mediated cell death. This cell death was demonstrated to be apoptotic in nature and caspase-8-dependent, using CRISPR-caspase-8 knockout models. Moreover, the mode of synergy was demonstrated to be FLIP-dependent using CRC models engineered to overexpress wild-type FLIP. The mode of cell death was further demonstrated to be dependent on FLIP’s interaction with FADD as overexpression of a mutant form of FLIP (F114A) that is unable to bind FADD was unable to prevent cell death induced by the combination of Entinostat and ASTX660.

Conclusion

This work indicates that IAP antagonists are relatively ineffective in pre-clinical models of CRC due to their relatively high expression of FLIP. However, FLIP-mediated resistance to IAP inhibitors can be overcome by treatment with the HDAC inhibitor Entinostat.