Limited evidence for chemo- and radiosensitization by HDAC inhibitors in gliomasphere models of pediatric high-grade glioma

Abstract Purpose Pediatric high-grade glioma (pedHGG), including diffuse intrinsic pontine glioma (DIPG) and non-pontine diffuse midline glioma, remains uniformly lethal despite radiotherapy and, in some protocols, temozolomide (TMZ). Recurrent histone H3 mutations reducing H3K27 trimethylation provide a rationale for histone deacetylase inhibitors (HDACi). We therefore asked whether HDAC inhibition can overcome intrinsic TMZ resistance. Methods Six HDACi (vorinostat, panobinostat, valproic acid, domatinostat, mocetinostat, resminostat) were tested as single agents in three pedHGG models (HSJD-DIPG-007 [H3K27M], SF188 and HSJD-GBM-001 [H3 wildtype]) under gliomasphere and adherent conditions; TMZ and 8 Gy combinations were tested in gliomaspheres, valproic acid also in monolayer. Viability, morphology and histone acetylation were assessed; drug interactions were scored with HSA, ZIP, Loewe and Bliss, and radiation/TMZ response ratios. Results All HDACi reduced viability concentration-dependently, panobinostat most and valproic acid least potently, and gliomaspheres were generally less sensitive than adherent cells. At concentrations referenced to reported plasma exposures, HDACi altered sphere morphology and showed a heterogeneous trend towards increased H3K27 acetylation, without statistically significant individual comparisons. Combination analyses revealed no reproducible synergy or radiosensitization in this screening-level assay; HSA scores above 10 were confined to SF188, and none exceeded this threshold across all four reference models. Only mocetinostat increased the TMZ-associated reduction in MTT signal in DIPG-007 at the supra-clinical TMZ concentration. Conclusions Despite a trend consistent with target engagement, HDACi did not consistently enhance TMZ or irradiation efficacy in pedHGG and DIPG models under the conditions tested, underscoring the need for alternative, mechanism-informed combination strategies.