The pseudo-caspase FLIP(L) regulates cell fate following p53 activation

Abstract

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Tumor suppressor p53: from engaging DNA to target gene regulation

“…However, recent data suggest that the overall net charge of the p53 C-terminus may play an important role in repressing its activity, with an un-acetylated ‘acidic’ C-terminus being read by SET, much like a histone, inhibiting its acetylation ( 118 ). Modulating the activities of regulators of p53 PTMs, such as inhibitors of HDACs and other co-factors, have shown potential to promote p53 induced cell death alone or in combination with DNA-damaging agents ( 130–133 ), but the underlying mechanisms remain poorly understood.…”

“…These differential effects of p53 activation are likely a combination of tissue specific accessibility of p53 target and differential ‘priming’ of cell types for cell death ( 212 ), as has recently been shown with HDAC inhibitors that synergistically induce p53-dependent cell death in combination with Nutlin-3a or chemotherapy. This is mediated through HDAC inhibitors blocking activation of a subset of p53 targets including the pro-survival caspase-8 inhibitor FLIP, which releases the brake on ‘priming’ of pro-apoptotic targets TRAILR2, BAX, and PUMA ( 130 ). Similarly, treatment of MCF7 cells with the DNA methylation inhibitor decitabine led to de novo chromatin accessibility and p53 binding, suggesting that changes in cell state and pharmaceutical treatments can alter the binding and activity of p53 ( 52 ).…”

Clinical positioning of the IAP antagonist tolinapant (ASTX660) in colorectal cancer

“…Mechanistically, the effects of FOLFOX on FLIP expression were not due to suppression of transcription; indeed, mRNA expression of FLIP(S) was upregulated at 24h by ~2-fold and FLIP(L) was upregulated by ~5-fold at 48h ( Figure 6A ). Inhibition of histone deacetylases (HDACs) has previously been reported by us and others to result in reduced expression of both splice forms of FLIP, at transcriptional and post-translational levels and augment cell death induced by activators of extrinsic cell death [30-32]. It was therefore notable that FOLFOX treatment downregulated expression of the Class-I HDACs (HDAC-1, −2 and −3) as did tolinapant/TNFα ( Figure 6B ).…”

“…Compared to a HCT116 caspase-8 CRISPR knockout model in which cell death induced by the tolinapant/TNFα-FOLFOX combination was almost completely abrogated ( Figure 5C/D ), significant cell death was still observed in response to the combination in the FLIP(S) overexpressing model ( Figure 5B ), suggesting that additional factors other than FLIP downregulation contribute to the effects of the combination. The role of caspase-8’s other paralog caspase-10/CASP10 as a regulator of apoptotic cell death is controversial, with both pro- and anti-apoptotic roles described [28-30]. We found that co-treatment of HCT116 cells with tolinapant/TNFα/FOLFOX dramatically reduced the levels of procaspase-10 expression ( Figure 5C ).…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 20, 2020. ; https://doi.org/10.1101/2020.11.20.391680 doi: bioRxiv preprint Figure 5B), suggesting that additional factors other than FLIP downregulation contribute to the effects of the combination. The role of caspase-8's other paralog caspase-10/CASP10 as a regulator of apoptotic cell death is controversial, with both pro-and anti-apoptotic roles described [28][29][30]. We found that co-treatment of HCT116 cells with tolinapant/TNF/FOLFOX dramatically reduced the levels of procaspase-10 expression ( Figure 5C).…”