EPCO-10. REPLICATION STRESS AND CHOLESTEROL BIOSYNTHESIS AS THERAPEUTIC VULNERABILITIES IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are universally fatal pediatric brain tumors driven by the oncogenic H3K27M mutation in histone variants H3.1 or H3.3. This mutation disrupts normal developmental programs by impeding cellular differentiation. Given the critical role of replication timing in regulating gene expression, cell fate, and therapeutic response, we applied an integrative multi-omics approach—including Repli-seq, cell cycle-resolved RNA-seq, and single-cell RNA-seq—to characterize DMG cells (H3.1K27M and H3.3K27M subtypes) and primary tumors in comparison to normal brain tissue.Our analysis revealed subgroup-specific alterations in replication timing, which were strongly associated with differential gene expression patterns, notably involving the cholesterol biosynthesis pathway. In line with these findings, DMG tumors exhibited pronounced signatures of replication stress and upregulated cholesterol biosynthesis. Functionally, DMG cells demonstrated heightened sensitivity to inhibitors targeting the cholesterol pathway as well as agents inducing replication stress, with enhanced efficacy observed in combination treatment. In summary, our integrative genomic profiling uncovered subtype-specific vulnerabilities in DMG, identifying potential therapeutic strategies for this currently untreatable disease.