p53 activation induces targetable dependence on FLIPL.


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Andrea Lees1,Alex McIntyre1,Fiammetta Falcone1,Tamas Sessler1,Nyree Crawford1,Gemma Gregg1,Kirsty McLaughlin1,Wendy Allen1,Caitriona Holohan1,Larry Egan2,Aideen Ryan2,Phillip Dunne1,Mark Wappet1,Patrick Johnston1,Daniel Longley1,Simon McDade1
1CCRCB Queen's University Belfast,2NUI Galway

Abstract

Background

The mechanisms through which the tumour suppressor p53 differentiates between activating cell cycle arrest or apoptosis and the underlying transcriptional programs responsible for this decision-making process remain poorly understood. This work focuses on identifying mechanisms through which tumours retaining wild-type p53 evade apoptosis.

Method

A panel of matched p53 wild-type and deficient colorectal cancer cell lines were studied using Nutlin-3A as a direct p53 activating agent. The HDAC dependency of the p53 response was assessed using the clinically relevant Class-I selective HDAC inhibitor, Entinostat. Molecular (Western blot, RT-PCR), phenotypic (Flow cytometry, High Content Microscopy) and genomic analyses were used to investigate the role of p53 and its downstream transcriptional programs.

Results

Here, we report that activation of pro-apoptotic p53 transcriptional targets in colorectal cancer cells imposes a critical, targetable dependence on the long splice form of the caspase-8 regulator FLIP (FLIPL) for survival. Upon Nutlin-3A-induced stabilisation, p53 directly upregulates FLIPL expression in a manner dependent on Class-I HDAC activity. Preventing FLIPL upregulation with Entinostat promotes apoptosis in response to Nutlin-3A, which otherwise predominantly induces growth arrest despite upregulation of pro-apoptotic target genes. Cell death in response to Nutlin-3A in FLIPL-depleted cells is mediated through two of p53’s canonical transcriptional targets, TRAIL-R2 and BAX, and is caspase-8-dependent.

Conclusion

These results uncover a p53-regulated apoptosis priming event in which key pro-apoptotic genes are up-regulated in response to p53 stabilisation. However, immediate commitment to apoptosis is prevented by the concomitant p53-mediated up-regulation of FLIPL.From a cancer therapeutics point of view, this work uncovers a novel mechanism for the synergy previously reported between DNA damage and HDACi, while identifying FLIPLas a key target for overcoming resistance to p53-stabilising agents in cancers harbouring the WTp53 allele.