Abstract 4659: Depicting ONC201/Delta-24-RGD combination for the treatment of pHGGs and DMGs reveals a therapeutic benefit and a proinflammatory tumor microenvironment remodelation
Authors: de la Nava Daniel , Laspidea Virginia , Ausejo-Mauleón Iker , García-Moure Marc , Mueller Sabine , Nazarian Javad , Allen Joshua E. , Becher Oren , Fueyo Juan , Gomez-Manzano Candelaria , Patiño-Garcia Ana , Koschmann Carl , Alonso Marta M. 
Published: April 2023

Abstract
Purpose of the work: Pediatric High Grade Gliomas (pHGGs) and Diffuse Midline Gliomas (DMGs) are two of the most aggressive tumors developed during childhood. Its poor overall survival emphasizes the need of alternative treatments. ONC201, an imipiridone small molecule, and the oncolytic virus Delta-24-RGD have shown safety and effectiveness in preclinical and clinical settings for these diseases. In this work, we set to characterize the antitumor effect of these two agents combined for the treatment of pHGGs and DMGs.
Experimental procedures: A panel of human pHGG (CHLA-03-AA, SF188, SJ-GBM2), DMG (SU-DIPG IV, SF8628, TP-54, HSJD-DIPG-007, UMPED83), and murine DMG cell lines (XFM, NP-53) were used. We assessed viral replication by hexon titration, protein expression by immunoblotting/immunohistochemistry, and oxygen consumption by Seahorse analyses. Viability was measured by MTS. Mitochondrial DNA (mtDNA) were measured by qPCR. CHLA-03-AA/XFM were orthotopically engrafted in athymic nude/Balb/c mice. For in vivo experiments, animals were treated with PBS/Delta-24-RGD (107 pfu/mice) intratumorally, followed by ONC201 oral gavage administration (125 mg/kg/twice/weekly). The tumor microenvironment immune infiltration was evaluated by flow cytometry.
Results: We checked the potential negative interaction between both agents in vitro. Adenovirus replication was not affected by ONC201 cotreatment, indicating the suitability of this combination. In vitro citotoxycity analyses showed the cotreatment is either synergistic or additive in 80% of the cells evaluated. Then, we set to characterize the mechanistic effects of the combination. Analysis of mitochondria status showed the decrease in oxygen consumption previously reported as ONC201 effect is maintained with Delta-24-RGD presence. Confirmation of higher mitochondria damage was assessed by a reduction of mtDNA in the combination. Independently, endoplasmic reticulum stress (ERS) previously described in both agents was found potentiated in cotreatment via ATF4. Altogether, these data suggest an improved response in the combination by mitochondria and ERS perturbation. In vivo, cotreatment led to a significant increase in the median OS (Control: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)) in CHLA-03-AA bearing mice, leading to 20% long-term survivors, free of disease. Of importance, a reshaping of the tumor microenvironment towards a proinflammatory phenotype were described (increases of lymphoid (T and NK cells) and myeloid (macrophages, granulocytes, monocytes, denditric cells and microglia) linage.
Conclusions: Our data support the combination ONC201/Delta-24-RGD leads to a superior therapeutic effect due to the generation of a proinflammatory microenvironment in pHGG and DMGs preclinical models.
Citation Format: Daniel de la Nava, Virginia Laspidea, Iker Ausejo-Mauleón, Marc García-Moure, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Carl Koschmann, Marta M. Alonso. Depicting ONC201/Delta-24-RGD combination for the treatment of pHGGs and DMGs reveals a therapeutic benefit and a proinflammatory tumor microenvironment remodelation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4659.