Abstract
Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes. By electron microscopy, we show that dl922-947 induces similar necrotic morphology as TSZ treatment (TNF-α, Smac mimetic, zVAD.fmk). However, dl922-947-mediated death is independent of TNF-α signalling, does not require RIPK1 and does not rely upon the presence of MLKL. However, inhibition of caspases, specifically caspase-8, induces necroptosis that is RIPK3 dependent and significantly enhances dl922-947 cytotoxicity. Moreover, using CRISPR/Cas9 gene editing, we demonstrate that the increase in cytotoxicity seen upon caspase inhibition is also MLKL dependent. Even in the absence of caspase inhibition, RIPK3 expression promotes dl922-947 and wild-type adenovirus type 5 efficacy both in vitro and in vivo. Together, these results suggest that adenovirus induces a form of programmed necrosis that differs from classical TSZ necroptosis.
Highlights
Oncolytic viruses are a promising new therapy for cancer
Induction of necroptosis in tumour cells We first investigated the expression of RIPK1, RIPK3, caspase-8 and Mixed lineage kinase domain-like (MLKL) in a panel of ovarian cancer cell lines as well as HeLa cells (Fig. 1a)
TOV21G cells expressed all three proteins in equal quantities and were sensitive to necroptosis induced by TSZ (TNF-α, Smac mimetic, zVAD.fmk) that was reversible by treatment with necrostatin-1 and necrosulphonamide (NSA) (Fig. 1b)
Summary
Oncolytic viruses are a promising new therapy for cancer. They can infect cancer cells, multiply selectively within them and cause cell death, with release of mature viral particles that infect neighbouring cells. We have previously shown that the E1A CR2-deleted adenovirus type 5 mutant dl[922-947] has considerable activity in ovarian cancer and is more potent than both E1A wildtype adenoviruses and the E1B-55K mutant dl1520 (Onyx-015)[1,2]. The exact mechanisms by which adenoviruses cause cell death remain uncertain. We previously showed that classical apoptosis is not the primary mode of cell death following E1A CR2-deleted adenovirus infection in ovarian cancer and that autophagy was likely to be a survival mechanism[5]. Our overall conclusion was that adenovirus cytotoxicity had the features of a programmed necrotic process
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