Robinson, James

Abstract BACKGROUND Diffuse midline gliomas (DMGs) and posterior fossa group A (PFA) ependymomas are aggressive pediatric brain tumors. Most DMGs harbor histone H3.3K27M or G34R mutations, or exhibit EZHIP overexpression. With a median survival of just 9–15 months, DMGs are among the most lethal human cancers. PFAs, nearly universally overexpress EZHIP and have a 5-year survival rate of ~50%. Unlike K27M or EZHIP-overexpressing DMGs, G34R-mutant tumors retain H3K27me3. METHODS Using the RCAS/Nestin-TVA system, we introduced H3.3 K27M–, G34R–, or S31A–P2A–PDGFβ or H3.3WT–P2A–PDGFβ into neonatal mice. Controls included, histone mutants EZHIP alone and ΔEZHIP (PRC2-binding-deficient). Tumor bearing mice were assessed for incidence, grade, survival, and H3K27me3 and H3.3S31P status. RESULTS H3.3K27M, G34R, and S31A–P2A–PDGFβ induced high-grade gliomas with significantly reduced survival (p < 0.001). Mechanistically, G34R—like K27M—reduced mitotic Ser31 phosphorylation (S31P), causing chromosomal instability and impaired p53 checkpoint function. S31A directly modeled loss of S31P and similarly drove high-grade tumor formation. H3.3WT–P2A–PDGFβ induced only low-grade lesions. EZHIP–P2A–PDGFβ enhanced tumor grade but not survival; only EZHIP fully abolished H3K27me3. ΔEZHIP and all unlinked constructs were non-tumorigenic. CONCLUSION These models establish a robust platform to dissect glioma drivers and chromatin vulnerabilities. We previously showed that H3.3K27M reduces S31P, promoting chromosomal instability and impaired p53 function. We now demonstrate that H3.3G34R also reduces S31P and drives tumor formation in vivo. Direct loss of S31P via H3.3S31A is likewise sufficient for gliomagenesis. Although EZHIP does not alter S31P, it promotes genome instability through distinct mechanisms—disrupting PALB2–BRCA2-mediated DNA repair and mislocalizing TopoIIα, a process dependent on H3K27me3. H3.3K27M, G34R, and EZHIP thus converge on genome instability through distinct pathways. This platform supports mechanistic and therapeutic studies and is now being expanded using tetracycline-regulated expression to investigate tumor maintenance.