STEM-03. Mapping the epigenetic consequences and associated developmental programs in H3G34-mutant diffuse hemispheric glioma.

Abstract Diffuse hemispheric gliomas (DHGs) account for 30% of aggressive brain tumors in children with abysmal prognoses. These tumors are characterized by co-occurring H3.3G34R/V histone mutations and inactivating mutations in TP53 and ATRX, a chromatin remodeler that integrates H3.3 into chromatin, preserving epigenome integrity. Master transcription factors are known to engage core regulatory circuitries (CRCs) via super-enhancers, control cell fate, and have been implicated in oncogenesis across cancers. Epigenetic reprogramming is central to DHG tumorigenesis, with stalled progenitors suggested as cells of origin. However, the mechanisms by which these genetic alterations disrupt neurodevelopmental programs and drive DHG remain unclear. We propose that ATRX loss in G34-mutant glioma alters chromatin remodeling at enhancers, engaging CRCs that disrupt normal neurodevelopment and initiate oncogenesis. To investigate this, we profiled the transcriptomic and epigenetic landscape of a CRISPR-edited human-induced pluripotent stem cell (h-iPSC) isogenic system containing combinations of TP53 (P), ATRX (A), and H3.3G34R (G) alterations and mapped genetic contributions to epigenetic dysfunction. Differentiating h-iPSCs into neural stem cells (NSCs) reveals distinct transcriptional profiles for each model (P, GP, AP, GAP), with AP (ATRXKD/TP53mut) and GAP (H3.3G34R/ATRXKD/TP53mut) signatures aligning with G34-mutant tumor profiles, emphasizing disease relevance. Single-sample gene-set enrichment analysis identified enrichment of prenatal precursor genes in the AP and GAP models, with the GAP model displaying restriction of postnatal cell signatures. Using H3K27 acetylation CUT&RUN, we identified candidate CRCs in our models and patient samples. Key regulators involved in neuronal cell differentiation (SOX2, POU3F2, POU3F3) were shared between AP and GAP models and represented in ATRX-altered G34-mutant tumor samples. We discovered distinct transcriptional and epigenetic changes corresponding to alterations found in DHGs, implicating ATRX as a guardian of neural cell fate specification. Our findings show that loss of ATRX disrupts neurodevelopmental programs through dysregulated CRCs, while G34 mutations refine these oncogenic networks.