1097 New immunomodulatory roles of NONO pathway during glioma virotherapy

Background

Despite aggressive therapeutic interventions, glioblastoma (GBM) patients face a median survival rate of just seven months after tumor progression. To address these obstacles, oncolytic-virotherapy has emerged as a propitious approach, offering the potential to reprogram the immunosuppressed microenvironment of GBMs and enhance anti-tumor immune responses. Among the oncolytic adenoviruses, Delta-24-RGD has showcased favorable outcomes in phase I/II clinical trials (NCT00805376, NCT03178032, and NCT02798406). Nevertheless, the immune response functionally eliminated the virus, limiting its efficacy to approximately 20% of patients. In the context of gliomas, Non-POU Domain Containing Octamer Binding (NONO) has been identified as a gene that is overexpressed and associated with diminished patient survival.

Methods

We conducted bulk RNA sequencing to identify upstream regulators triggered by adenovirus infection. To explore protein interactions, liquid chromatography mass spectrometry was employed, and the identified interactions were confirmed through immunoprecipitation. The activation of innate immune responses was assessed using qPCR and further validated through western blot analysis. The replication of the virus was evaluated by qPCR and virus titration assays.

Results

Our study employed bulk RNA sequencing to uncover a notable seven-fold upregulation of the NONO pathway in response to adenoviral infection (figure 1). Western-blot analyses confirmed a significant increase in NONO expression following adenovirus infection (figure 2). Through mass spectrometry analyses, we identified both cellular and viral components that interact with NONO (figure 3). Immunoprecipitation assays confirmed these findings and revealed the binding of NONO to adenoviral proteins during infection, as well as its association with the cyclic GMP AMP synthase (cGAS), a sensor of foreign DNA (figure 4). To investigate the functional significance of NONO, we employed shRNA knockdown and discovered that NONO is crucial for initiating innate immune responses in the context of adenovirus infection. Notably, we observed a significant increase in type I interferon levels upon virus infection, which was reversed in NONO-deficient cells (figure 5). Intriguingly, adenovirus replication was found to be enhanced in NONO knockdown cells (figure 6). Collectively, our findings establish NONO as an unrecognized innate immune sensor of adenoviruses. Furthermore, the interaction between NONO and adenoviral proteins suggests a novel double-sensor mechanism involving the recognition of both foreign DNA and viral proteins.

Conclusions

The implications of our findings underscore the significance of incorporating the interactions between NONO and adenoviral proteins into the design of future oncolytic adenoviruses for glioma therapy. By leveraging the knowledge obtained from this study, our objective is to augment the effectiveness of oncolytic-virotherapy and improve outcomes for individuals with glioblastoma.