The cell death regulator, FLIP

: evaluating novel inhibitors in colorectal cancer

Abstract

Colorectal cancer (CRC) is the second most common cause of cancer related mortality. For patients with metastatic disease, 5-year survival is One mechanism of drug resistance involves the cell death regulator, FLIP (Fas-associated death domain (FADD)-like interleukin-1β-converting enzyme (FLICE) inhibitory protein). Frequently overexpressed in CRC, FLIP is a well described negative prognostic factor with multiple pro-tumour functions, most notably its role in regulating caspase-8 activation. Through recruitment to FADD during the formation of the death-inducing signalling complex (DISC) by the TRAIL (DR4/DR5) and FAS/CD95 death receptors, FLIP is responsible for regulating death-receptor-mediated apoptosis by preventing the homodimerisation of procaspase-8. FLIP is therefore capable of abrogating apoptotic signalling and promoting cell survival. Identification of a unique FLIP/FADD protein:protein interaction site led to the development of specific small molecule inhibitors of FLIP (FLIPi) capable of binding to FLIP and disrupting its interaction with FADD at the DISC. This thesis focuses on evaluating the efficacy and mechanism of action of three lead FLIPi compounds in CRC.

FLIP inhibitors were shown to reduce FLIP recruitment to the DISC and induce cell death in several CRC cell models, consistent with their sensitivity to RNAi-mediated FLIP depletion. The pro-apoptotic effect of FLIP inhibition was enhanced in the presence of TRAIL receptor agonist, izTRAIL. This suggests a potential application for FLIP inhibitors in tumours with high immune cell infiltration or for improving the clinical activity of TRAIL receptor agonists. Likewise, FLIPi enhanced the sensitivity of CRC cells to 5-fluorouracil (5-FU) in a manner dependent on the expression of caspase-8 and FADD and also p53. This highlighted the potential application of FLIP inhibitors for improving the response of CRC to standard of care 5-FU therapy. Moreover, Apc-null, Kras-mutant, Trp53-null (AKP) murine organoids models were shown to be highly sensitive to FLIP inhibition which abrogated organoid growth and enhanced sensitivity to 5-FU. These AKP organoids map to the Cancer Cell Intrinsic Subtype-E (CRIS-E), a subgroup of tumours enriched for KRAS and TP53 mutations in which adjuvant 5-FU-based chemotherapy does not confer clinical benefit in the adjuvant disease setting.

The on-target activity of FLIP inhibitors was confirmed through DISC immunoprecipitation assays and phenotypic studies in CRISPR knockout cell line models. These confirmed that FLIP inhibitors sensitise CRC cells to caspase-8-dependent apoptosis which requires the adaptor protein FADD and downstream expression of the mitochondrial proteins, BAX and BAK. When cell death is prevented in CASP8/10 double knockout or BAX/BAK knockout cells, FLIP inhibition induces a growth inhibitory effect in CRC cells and induces changes in the cell cycle, particularly increasing the G2M phase. This was replicated following RNAi-mediated FLIP depletion, implying that this effect is FLIP-dependent. FLIP inhibitors were also shown to downregulate FLIP protein expression through proteasomal degradation.

Overall, these studies show that these novel FLIP inhibitors recapitulate the phenotypes induced in CRC by FLIP-targeted siRNAs and highlight the potential for their application in CRC in combination with the standard-of-care chemotherapeutic 5-FU.

Thesis is embargoed until 31 December 2028.

Date of Award	Dec 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Medical Research Council & Northern Ireland Department for the Economy
Supervisor	Daniel Longley (Supervisor), Tim Harrison (Supervisor) & Simon McDade (Supervisor)