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Abstract
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.
The role of NONO in innate immune activation. (A) Heatmap comparing the transcriptional signatures of human fibroblasts infected with adenovirus serotype 5 versus mock-infected. The log2-normalized expression levels of genes with significant adjusted P-values. (B) Gene ontology (GO) biological process enrichment analysis for human fibroblasts treated with adenovirus versus mock-infection. The ten most significant GO biological processes are shown. (C) Ingenuity pathway analysis (IPA) NONO network genes with significantly altered expression levels in Ad infected human fibroblasts (vs. mock). Green intensity indicates the log2 fold-change levels of each gene. (D) The most significantly altered upstream regulators in WI-38 human fibroblasts treated with adenovirus vs. mock. Activation z-scores are plotted in the graph.
NONO is activated in response to adenoviral infection. Detection of NONO and cGAS protein levels in human fibroblasts, as assessed by Western blot. Quantified levels (relative to GAPDH) as mean from 2 independent experiments.
NONO complexes with cGAS and adenoviral proteins. Human fibroblast cells were infected with adenovirus serotype 5, and 48hrs. (A) Potential binders of NONO and protein coverage percentage determined by tandem mass spectrometry.
NONO complexes with cGAS and adenoviral proteins. (A-B) Human fibroblast cells were infected with adenovirus serotype 5, and 48hrs. Whole cell lysates were processed for metallo-bead immunoprecipitation (IP) of NONO or IgG. Western blot was performed to detect cGAS (A) and adenoviral fiber (B). GAPDH levels are shown as control. Data show a representative experiment of n=3. *, unbound NONO; **, unbound IgG flowthrough.
NONO is required for innate immune responses against adenovirus. (A-D) WI38 human fibroblasts were stably transduced with shRNA targeting NONO, cGAS or a non-targeting scramble. Transduced cells were then infected with 100MOI adenovirus or mock infection. The levels of induction of IFN-α and IFN-β mRNAs were measured by RT-qPCR. The graph shows mean ± SD (n=3) or the fold induction relative to the control condition (wildtype WI38 0hr mock). (E) Whole-cell extracts from the experiment performed in the upper panel were analyzed by Western blotting using the indicated antibodies.
NONO is required for efficient adenovirus replication. WI38 fibroblasts were infected with adenovirus (100MOI) before viral DNA was collected and used to compare adenovirus replication at each time point. qPCR was used to analyze total viral fiber DNA relative to known amount of plasmid DNA containing adenovirus fiber DNA.
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