TMET-51. EGFR metabolically suppresses differentiation in H3K27M-mutant diffuse midline glioma

EGFR-altered H3K27M-mutant diffuse midline glioma (H3K27M-DMG) represent a recently recognized yet poorly understood subpopulation of H3K27M-DMG characterized by increased malignancy. To better understand the role of EGFR signaling in this patient population, we generated isogenic H3K27M-DMG cell lines expressing wild-type and commonly observed activating mutant isoforms of EGFR (p.A289T, p.A767delinsASVG). EGFR alterations increased H3K27M-DMG cell proliferation in vitro and in in vivo mouse flank tumor xenograft models. Surprisingly, overexpression of EGFR alterations further decreased H3K27M-associated H3K27me3 and significantly downregulated “late” stemness genes (e.g., PDGFRA, SOX10, OLIG2). Furthermore, gene expression programs of EGFR-altered H3K27M-DMG mapped to “early” OPC-like-3 cells that are enriched in brainstem H3K27M-DMG tumors in single-cell RNA-sequencing datasets. Mechanistically, EGFR-altered H3K27M-DMG cells decrease chromatin accessibility and expression of lactate dehydrogenase A (LDHA), leading to decreased L-2-hydroxyglutarate (L-2HG) metabolite levels, a known inhibitor of the JmjC domain family of histone demethylases. Consistent with this, EGFR alterations in H3K27M-DMG cells conferred resistance to the ClpP agonist ONC201 in vitro, whose efficacy in H3K27M-DMG depends on LDHA-mediated production of L-2HG. Indeed, through secondary analysis of clinical outcomes and tumor sequencing from the Phase I ONC014 trial (NCT03416530), we identified high expression of wild-type EGFR and EGFR mutations as key biomarkers of ONC201 sensitivity. EGFRHIGH (“OPC-like-3”) expression was associated with negative radiographic responses while PDGFRAHIGH (“OPC-like-1”) expression correlated with positive radiographic responses. Pharmacologic inhibition of EGFR (EGFRi) variably restored H3K27me3, supporting the idea that EGFR signaling reinforces the hypomethylated state. Treatment of an in vivo orthotopic model of H3K27M-DMG resulted in combinatorial survival benefit with ONC201 and EGFRi, with multiple additional in vivo studies ongoing. Our findings demonstrate a previously undiscovered role of EGFR in the metabolic suppression of epigenetic differentiation, providing a novel pathway to therapeutically target EGFR-altered H3K27M-DMG and OPC-like-3 cell sub-populations of all H3K27M-DMG.