HGG-22. Modulation of GLIS1-driven metabolome epigenome signaling axis in pediatric high-grade gliomas (pHGGs)

Abstract Introduction Diffuse Midline Glioma (DMG) and Diffuse Hemispheric Glioma (DHG) are aggressive pediatric high-grade gliomas (pHGGs), with median OS of 11 and 17 months, respectively. The global reduction in H3K27 tri-methylation (H3K27me3) in K27M-altered DMG and the associated increase in H3K27ac drive tumorigenic transcriptional reprogramming. In contrast, although the nucleosomes harboring the glycine-to-arginine or -valine (H3G34R/V) mutation in DHG exhibit reduced H3K36me2/me3 levels on the cis histone tail, the complete spectrum of epigenetic alterations remains uncharacterized. Both K27M-expressing neuronal progenitor cells and DMG xenografts, and G34V mutant cells exhibit elevated central carbon metabolism, particularly glycolysis and tricarboxylic acid (TCA) cycle. The metabolites of these pathways are critical epigenetic regulators, but the specific regulatory elements involved in the aberrant oncometabolite- driven chromatin remodeling in pHGGs have not been elucidated. Methods We established single-cell knockout (KO) clones of GLI-Similar1 (GLIS1), a transcription factor that induces multi-level metabolic and epigenetic remodeling, by binding to and opening the chromatin at glycolytic genes (ChIP-qPCR), targeting its zinc-finger DNA binding domain (DBD) and transactivation domain (TAD). This study investigates whether modulating K27ac and K lactylation (Kla) enrichment and K27me3 depletion patterns at GLIS1-dependent gene promoters and (super) enhancers can reverse the altered transcriptional landscape. Results GLIS1 expression was significantly higher in pHGG cell lines relative to low-grade glioma, by immunoblot and qPCR. G34V cells show increased global H3K18la and 27la, novel active histone marks. Nuclear translocation of GLIS1 (confocal microscopy) and in-vitro tumor cell proliferation was significantly reduced in KO lines targeting its DBD. Ongoing work Includes isotope tracing and metabolomic analysis (liquid chromatography-mass spectrometry) to quantitate the direct contribution of metabolic activity on chromatin modifications, and RNA sequencing to assess downstream effects on gene expression. CUT&RUN profiling of H3-K27ac, -K18la and-K27me3 patterns to identify specific GLIS1-dependent regulatory elements that enhance glycolytic and TCA cycle activity is underway.