The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models [RNA-seqII]. The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models [RNA-seqII]

Background: Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. Methods: The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq and multiplexed immunofluorescence staining. Results: The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. Conclusions: The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone. Overall design: XFM-bearing mice were treated with vehicle, Delta-24-RGD, ONC201 and Delta-24-RGD+ONC201 at 10^7 pfu for the viruses group and 125 mg/kg for ONC201 groups. Then, mice were sacrificed and tumor tissue was recovered. RNA extraction for RNAseq was performed.

背景：儿童高级别胶质瘤（pediatric high-grade gliomas, pHGGs）包括弥漫中线胶质瘤（diffuse midline gliomas, DMGs），是一类极具侵袭性的儿童肿瘤，预后极差。溶瘤病毒Delta-24-RGD与小分子化合物ONC201单药治疗这类肿瘤已展现出良好的应用前景，但二者联合用药的疗效尚未被评估。
方法：通过免疫印迹（immunoblotting）与病毒复制实验评估功能性病毒的制备效果；在一组人源及鼠源pHGG、DMG细胞系中评估联合治疗的抗肿瘤活性；采用RNA测序（RNAseq）、海马代谢压力检测（Seahorse stress test）、线粒体DNA含量检测及γH2A.X免疫荧光染色（γH2A.X immunofluorescence）开展机制研究；利用两种疾病的小鼠模型在体内评估联合治疗的疗效；通过流式细胞术（flow cytometry）、RNA测序（RNAseq）及多重免疫荧光染色（multiplexed immunofluorescence staining）分析肿瘤免疫微环境的变化。
结果：Delta-24-RGD与ONC201联合用药未影响人源pHGG、DMG模型体外培养中的病毒复制能力；细胞毒性分析显示，联合治疗可产生协同或相加的抗肿瘤效应。机制研究表明，联合治疗可增强核DNA损伤，并维持单药分别引发的代谢紊乱与线粒体损伤。Delta-24-RGD/ONC201联合治疗可延长植入人源及鼠源pHGG、DMG细胞的小鼠的总生存期，且该效应不受H3突变状态及肿瘤位置的影响。此外，鼠源DMG模型的联合治疗研究显示，肿瘤微环境被重塑为促炎表型。
结论：相较于单药治疗，Delta-24-RGD与ONC201联合用药可通过增强核DNA损伤，进而改善机体对单药的抗肿瘤（免疫）应答，在体外及体内模型中提升抗肿瘤疗效。
整体实验设计：将携带XFM的小鼠分为四组，分别给予赋形剂、Delta-24-RGD、ONC201及Delta-24-RGD+ONC201治疗；其中病毒给药组的病毒剂量为10^7噬斑形成单位（plaque forming unit, pfu），ONC201给药组剂量为125 mg/kg。给药后处死小鼠，获取肿瘤组织，提取总RNA用于RNA测序分析。