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RIPK3 promotes adenovirus type 5 activity
Melanie Weigert1, Alex Binks1, Suzanne Dowson1
1Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, G61 1QH, UK.
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.
Insights
Oncolytic adenoviruses cause cancer cell death, but the mechanism is unclear. This study reveals adenovirus-induced cell death differs from classical necroptosis, involving RIPK3 but not MLKL, offering new therapeutic insights.
Area of Science:
- Oncolytic virotherapy
- Cell death mechanisms
- Immunology
Background:
- Oncolytic adenoviruses selectively infect and kill cancer cells, potentially activating immune responses.
- The precise mechanism of adenovirus-induced cell death, particularly its resemblance to necroptosis, remains incompletely understood.
Purpose of the Study:
- To investigate the roles of key necrosis proteins (RIPK1, RIPK3, MLKL) in adenovirus-mediated cytotoxicity.
- To elucidate the mechanism of cell death induced by the oncolytic adenovirus mutant dl922-947 and compare it to TNF-α-induced necroptosis (TSZ).
Main Methods:
- Electron microscopy to analyze cell morphology.
- CRISPR/Cas9 gene editing to assess the roles of RIPK1, RIPK3, and MLKL.
- In vitro and in vivo efficacy studies of adenovirus mutants.
Main Results:
- Adenovirus dl922-947 induced necrotic morphology similar to TSZ but was independent of TNF-α signaling and RIPK1.
- Adenovirus-induced cell death did not require MLKL, distinguishing it from classical necroptosis.
- Caspase-8 inhibition enhanced dl922-947 cytotoxicity in a RIPK3- and MLKL-dependent manner.
- RIPK3 expression promoted adenovirus efficacy independently of caspase inhibition.
Conclusions:
- Adenovirus-induced programmed necrosis is mechanistically distinct from classical necroptosis.
- RIPK3 plays a crucial role in enhancing adenovirus oncolytic efficacy, even without caspase inhibition.
- Understanding these distinct cell death pathways can inform the development of more effective oncolytic virotherapies.
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