Abstract LB-016: Nutlin and oxaliplatin induce p53-dependent addiction to FLIP

Abstract

Abstract Background: The tumour suppressive functions of the p53 transcription factor are inactivated through mutations or suppressed through non-mutational mechanisms in almost all cancer cells. This work focusses on identifying mechanisms through which, tumours retaining wild-type p53 evade apoptosis in response to chemotherapies and p53 agonists such as Nutlin-3A. Methods: A panel of matched p53 wild-type and deficient colorectal cancer cell line models were studied, using Nutlin-3A and oxaliplatin as direct and indirect p53 activating agents. A number of molecular (Western blots, RT-PCR), phenotypic (cell death) and genomic analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results: We demonstrate the importance of p53 in priming cancer cell sensitivity to apoptotic cell death following treatment with Nutlin-3A or oxaliplatin through activation of pro-apoptotic p53 targets, such as TRAIL-R2, Fas/CD95, PUMA and NOXA. Surprisingly, our analysis further reveals that this priming is accompanied by a parallel early p53-dependent pro-survival response that imposes a critical dependence on the caspase-8 inhibitor FLIP. Preventing FLIP up-regulation with siRNA resulted in synergistic increases in apoptosis in response to Nutlin-3A and oxaliplatin in p53 wild-type, but not p53 null cells. p53-mediated FLIP up-regulation was blocked by pharmacological inhibition of HDACs1-3 using Entinostat, which caused hyper-acetylation of the p53 C-terminus and altered transactivation of a number of other p53 apoptotic target genes. Notably, the synergy between Nutlin-3A or oxaliplatin and Entinostat was abrogated in cells over-expressing FLIP trans-genes or in cells in which caspase-8 was depleted using RNAi. Conclusion: These results uncover a p53-regulated apoptosis priming event, in which a number of key pro-apoptotic genes are upregulated in response to direct p53 agonism (Nutlin-3A) or indirect activation downstream of DNA damage (oxaliplatin). However, immediate commitment to apoptosis is prevented by the concomitant p53-mediated up-regulation of FLIP. This may be a mechanism that has evolved to allow DNA damage repair time to take place, while priming the cell to undergo apoptosis if the damage is irreparable. From a cancer therapeutics point-of-view, this work identifies FLIP upregulation in p53 wild-type cancers as a key target for overcoming resistance to direct and indirect p53 activating therapies. Citation Format: Alexander McIntyre, Fiammetta Falcone, Patrick G. Johnston, Daniel B. Longley, Simon S. McDade. Nutlin and oxaliplatin induce p53-dependent addiction to FLIP. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-016.