Abstract 3153: High-throughput identification of radiosensitizers and synergistic drug combinations with potent therapeutic efficacy against diffuse midline glioma

Abstract Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine glioma (DIPG), are the most aggressive pediatric brain tumors with a median survival of less than one year. Over 85% of DMGs harbor the H3K27M mutation in histone genes H3F3A (H3.3) and H3C2 (H3.1), which inhibits EZH2, the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), leading to global loss of H3K27 trimethylation (H3K27me3). This epigenetic disruption drives oncogenesis by reprogramming gene expression and activating oncogenic pathways. Current treatments, including chemotherapy and radiotherapy (IR), offer marginal benefits, with 95% of patients dying within 1.5 years of diagnosis. ONC201 has shown clinical benefits in H3K27M-mutant DMGs, extending median survival to 21.7 months by disrupting metabolic and epigenetic pathways and reversing H3K27me3 loss. Novel therapeutic strategies targeting H3K27M oncohistone are urgently needed. We performed high-throughput screening (HTS) of 1, 520 FDA-approved compounds to identify drugs that selectively target H3K27M-mutant primary DMG cell lines, but not CRISPR-corrected H3K27M-KO lines, derived from distinct neuroanatomical location: the thalamus (BT-245) and the pons (DIPGXIIIP). From this screening, we identified 86 promising drugs, further narrowed down to five lead compounds: floxuridine, gemcitabine, cytarabine, pyrimethamine, and cladribine (HTS-identified lead candidates, HTS-ILCs). These HTS-ILCs exhibited potent cytotoxicity against other patient-derived H3K27M-mutant DMG lines (DIPG007, SF8628) and murine PPK cells (DNp53/PDGFRA/H3.3K27M) from genetically engineered mouse models (GEMMs). HTS-ILCs were further tested for radiosensitizing potential, with floxuridine, gemcitabine, cytarabine, and pyrimethamine showing enhanced efficacy when combined with IR. Western blot analysis confirmed H3K27me3 restoration, indicating epigenetic modulation and reduced DMG growth. Combining IR, HTS-ILCs, and ONCs (201/206/212) showed synergistic effects and reversed epigenetic dysregulation. Gemcitabine exhibited strong anti-invasive properties, followed by floxuridine against H3K27M mutant DIPGXIIIP and SF8628 cells. Conditioned medium from HTS-ILCs-treated PPK cells showed enhanced HMGB1 secretion, suggesting activation of an immune-stimulatory response. Ongoing in vivo testing includes orthotopic implantation of PPK cells in immune-competent murine models and IUE-generated GEMMs of H3K27M DMGs. Spatial transcriptomics and phosphoproteomic microarray analysis are being used to elucidate the molecular mechanisms. Our findings suggest that combining HTS-ILCs with IR, alone or with ONC therapies, offers a promising strategy for treating H3K27M-mutant gliomas, potentially leading to more effective, personalized therapies for children with DMGs. Citation Format: Syed M. Faisal, Monika Yadav, Garrett R. Gibson, Adora T. Klinestiver, Ryan Sorenson, Anuradha Roy, Carl Koschmann, Scott J. Weir, Stefan H. Bossmann, Viveka N. Yadav. High-throughput identification of radiosensitizers and synergistic drug combinations with potent therapeutic efficacy against diffuse midline glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3153.