Introduction

Oncolytic viruses are a promising new cancer therapy, since they can infect cancer cells, selectively multiply within them and induce direct cytotoxicity, leading to the release of mature viral particles that can infect other neighboring cancer cells. However, the mechanisms by which oncolytic adenoviruses induce cell death remains uncertain. It was long thought that DNA viruses induce apoptosis, but there is now evidence that cell death induced by adenovirus, vaccinia and HSV-1 displays features strongly resembling a form of programmed necrosis.

Methods and Results

In order to investigate the role of necrosis in cell death as a result of oncolytic adenovirus infection, cancer cells were infected with the E1A CR2-deleted adenoviral mutant dl922-947, which specifically replicates in cells that have abnormalities in the pRB-pathway. We specifically sought to investigate the role of the core necrotic proteins RIPK1, RIPK3 and MLKL in adenovirus cytotoxicity.

Electron microscopy indicated that dl922-947 infection induces key morphological changes similar to necrotic death induced by TSZ (TNF-α, Smac-mimetic, Z.Vad.fmk) treatment. Using specific inhibitors of programmed necrosis (necrostatin-1, necrosulfonamide, GSK'840B and GSK'843A) as well as RNAi-mediated knockdown of RIPK1, RIPK3 and MLKL, we showed that adenovirus-infected cancer cells undergo RIPK3-dependent necrosis. We further found that, while TNF-α-induced programmed necrosis relies on the (RHIM)-dependent interaction of RIPK1 and RIPK3, dl922-947-induced cell death is independent of TNF-α signalling, does not involve RIPK1 and does not rely on the presence of MLKL. Caspase-8 inhibition, however, induces RIPK3-dependent necrosis that significantly enhances dl922-947 cytotoxicity. Using CRISPR/Cas9 gene editing, we have demonstrated that this increase in cytotoxicity during caspase inhibition was MLKL dependent.

Using a RIPK3 overexpression model, we observed that the extent of adenovirus-induced cell death correlated with RIPK3 expression even in the absence of caspase inhibition. Using RIPK3 co-immunoprecipitation, we identified an interaction between RIPK3 and MLKL as well as an interaction between RIPK3 and adenoviral proteins. In vivo experiments using human xenografts showed that expression of RIPK3 significantly improved anti-tumor activity following intra-tumoral injection of dl922-947.

Conclusions

Our data suggest that adenovirus infection induces a novel form of programmed necrosis that differs from classical TSZ-induced necroptosis, but still relies on the kinase RIPK3. Unfortunately, many cancer cells do not express RIPK3 and can therefore not undergo programmed necrosis. The integration of human RIPK3 into an adenoviral vector offers a therapeutic window to eradicate cancer cells that are resistant to apoptosis.

Citation Format: Melanie Weigert, Alex Binks, Suzanne Dowson, Elaine Leung, Dimitris Athineos, Xinzi Yu, Margaret Mullin, Josephine Walton, Clare Orange, Darren Ennis, Karen Blyth, Stephen Tait, Iain McNeish. Oncolytic adenovirus type 5 induces a novel form of programmed necrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4379.