Abstract 3549: The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer

Abstract

Abstract Introduction. Oncolytic viruses preferentially replicate within and kill cancer cells and are a promising novel cancer treatment. However, the mechanisms by which oncolytic adenoviruses induce death in cancer remains unclear. It was long thought that DNA viruses trigger classical apoptosis but there is now evidence that cell death induced by both adenovirus and vaccinia displays features of necrosis-like programmed cell death. Methods and Results. In order to investigate the role of necrosis in cell death following oncolytic adenovirus infection, a panel of ovarian cancer cells with varying sensitivities was infected with the E1A CR2-deleted adenoviral mutant dl922-947. By electron microscopy, dl922-947 infection induces key morphological features of necrotic death, including membrane rupture, nuclear swelling and cytoplasmic vacuolation. Using specific necrosis inhibitors (necrostatin-1, necrosulfonamide, GSK’840B, GSK’872B and GSK’843A) as well as RNAi-mediated knockdown of receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), we show that adenovirus-infected ovarian cancer cells undergo RIPK3-dependent necrosis and that blockage of the downstream effector MLKL significantly attenuates cell death. Co-immunoprecipitation of caspase 8 shows formation of a complex containing RIPK1, FADD and caspase 8 during adenovirus infection. However, unlike Tumour Necrosis Factor-α (TNF-α)-induced programmed necrosis, which relies on the RHIM-dependent interaction of RIPK1 and RIPK3, RIPK1 appears redundant in adenovirus-induced death. Furthermore, the addition of a TNF-α blocking antibody to virus-infected cells has no effect on either cell death or overall cell survival. In a RIPK3 overexpression model, we show a direct correlation between adenovirus-induced cell death and the extent of RIPK3 expression. Moreover, RIPK3 expression has no effect on infectivity, viral protein expression or infectious virus production. Finally, expression of RIPK3 increases necrosis induction in vivo following direct intra-tumoural dl922-947 injection, and significantly improves anti-tumour efficacy. Conclusions. Our data suggest that cell death induced by oncolytic adenoviruses differs from TNF-induced programmed necrosis but still relies on the kinase RIPK3 and its downstream component MLKL, making these two proteins possible targets for future oncolytic virotherapies. Citation Format: Melanie Weigert, Alexander Binks, Stephen Tait, Iain McNeish. The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer. [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 3549.