Abstract

Diffuse midline glioma (DMG) is an aggressive malignancy driven by the H3K27M oncohistone, leading to global epigenetic dysregulation and limited efficacy of single-agent epigenetic therapies. We previously observed clinical benefit from an HDAC/EZH2 inhibitor-based therapeutic regimen, but the mechanistic basis was unclear. A panel of patient-derived DMG glioma stem cell (GSC) cultures from brainstem and spinal cord tumors was used for drug-response and functional assays, with representative models further analyzed for transcriptomic alterations, cell-cycle distribution, global histone-modification changes, self-renewal, and in vivo efficacy. Antitumor efficacy was validated in orthotopic xenografts. Panobinostat and tazemetostat showed robust synergy across patient-derived DMG GSC cultures, suppressing GSC growth, proliferation, and self-renewal while inducing apoptosis and G0/G1 cell-cycle arrest. Transcriptomic analyses revealed coordinated repression of cell-cycle programs and downregulation of OPC-like and stemness-associated programs, accompanied by the induction of differentiation-associated neuronal genes. Functionally, dual treatment reduced stem-cell frequency by >90% to >99%. In orthotopic xenografts, combination therapy significantly decreased tumor burden, reduced spinal dissemination, and prolonged survival. Together, these findings indicate that dual HDAC/EZH2 inhibition disrupts epigenetically maintained proliferative and stem-like programs through coordinated cell-cycle repression, apoptosis induction, and impaired self-renewal. This work supports HDAC/EZH2 co-targeting as a rational therapeutic backbone for H3K27-altered DMG and its incorporation into future rational combination strategies.