DDDR-35. THERAPEUTIC STRATEGIES FOR DIFFUSE MIDLINE GLIOMA: INSIGHTS FROM HIGH-THROUGHPUT COMBINATION DRUG AND RADIATION SENSITIZATION SCREENING

Abstract Diffuse midline gliomas (DMGs) are the most aggressive pediatric high-grade gliomas. Over 85% DMGs harboring the H3K27M mutation in histone genes H3F3A (H3.3) and H3C2 (H3.1). H3K27M mutation disrupts epigenetic regulation by inhibiting PRC2, causing loss of H3K27 trimethylation and oncogenic gene expression. Presently, there are no effective therapies for DMG, and 95% of the patients die within 1.5 years of diagnosis. Thus, there is an urgent need to identify novel therapeutic strategies targeting H3K27M oncohistone. To assess the H3K27M mutation’s vulnerability, we used paired isogenic H3K27M-mutated and CRISPR-corrected H3M27K (H3-WT) primary DMG cell lines derived from thalamus (BT-245) and pons (DIPGXIIIP). We performed comprehensive single-agent high-throughput screens (HTS) with 1,520 FDA-approved drugs and further assessed drug combinations of ONC201 with the 86 most promising drugs from the HTS. A third combination experiment evaluated radio-sensitizing potential, where most effective top-six drugs (A-F) were tested in combination with radiation therapy across an expanded panel of patient-derived DMG cell cultures. To test drug efficacy in vivo, we developed a genetically engineered isogenic mouse model (GEMM) of DMGs using intrauterine electroporation (IUE) with DNp53/PDGFRA/H3.3K27M (PPK) and DNp53/PDGFRA/H3.3WT (PPW) genetic alterations. Our initial single-agent HTS screening identified 86 drugs with significantly higher cytotoxicity, specifically against H3K27M-mutated DMG cell lines. Combination HTS of the top six drugs revealed that drug E displayed significantly synergistic cytotoxicity with ONC201. We also evaluated drugs A-E in combination with radiation in both human and mouse H3K27M-mutated DMG cell lines. Drugs D and E showed selective radiosensitivity in both human and mouse H3K27M-mutated DMG cell lines. Preliminary findings also suggested drug D acts as an anti-invasive agent. Our findings identify novel drugs via HTS that enhance ONC201 efficacy and act as radiation sensitizers in DMGs. Future studies will test these compounds with ONC201 and radiation in our in vivo IUE-mediated H3K27M mouse model.