The oncolytic adenovirus Delta-24-RGD in combination with ONC201 displays a potent antitumor effect in pediatric high-grade glioma models [RNA-seqI]

Background: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs) are aggressive pediatric tumors harboring one of the poorest prognoses. Previous preclinical and clinical studies have demonstrated that oncolytic adenovirus Delta-24-RGD and the imipridone ONC201 are two promising therapies for these type of tumors as single agents. However, the combination of both agents has not been previously evaluated. Methods: Potential negative effects of ONC201 on the production of functional viruses were assessed by immunoblotting and replication assays. Antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, mTORC1 pathway analysis, Seahorse Stress Test, mitochondrial DNA content, and pH2A.X immunofluorescence were used to perform mechanistical studies. Models of both diseases were used to assess the efficacy of the combination in vivo. Tumor immune microenvironment was evaluated using flow cytometry. Results: ONC201 cotreatment with Delta-24-RGD did not affect the virus replication capability in human and murine pHGG and DMG models in vitro. Cytotoxicity analysis showed that cotreatment was either synergistic or additive. Mechanistically, combination treatment produces an increase of nuclear DNA damage and maintains the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extends the overall survival of mice implanted with human and murine pHGG and DMG cells, independently of H3 mutational status. Finally, combination treatment in murine DMG models revealed a proinflammatory phenotype reshaping of the tumor microenvironment. Conclusions: Delta-24-RGD/ONC201 combination improves the efficacy of each agent alone in vitro and in vivo models, maintaining transcriptional and phenotypical changes, producing exacerbated nuclear DNA damage, and leading to an augmented antitumor immune response.