Abstract
Pediatric high-grade gliomas often contain mutations in the H3F3A gene encoding the histone variant H3.3 and more rarely in canonical histone H3 family genes, a feature distinguishing them from adult gliomas. To define specific functionally significant changes in epigenomic states driven by mutant H3.3, we have utilized CRISPR-Cas9 to introduce specific H3.3 mutations (K27M, G34R) into formerly H3.3 wildtype (WT) brain and glioma cells, while in parallel also precisely reverting the pre-existing K27M and G34R mutations in patient-derived glioma cells to WT. In each case, gene editing was conducted on endogenous H3F3A alleles. Analyses of this overall panel of H3.3 gene-edited cells indicate that CRISPR-introduced K27M or G34R mutations within formerly H3.3 WT cells leads to increased gliomatypic signatures: elevated expression of specific oncogenes as well as neurogenesis and Notch signaling pathway genes, and perturbation of specific histone post-translational marks. Conversely, gene editing-based reversion of histone mutations to WT in primary glioma cells partially reverses glioma-associated phenotypes. Gene editing of K27M also yields coherent phenotype changes in xenograft assays. K27M and G34R mutations appear to function via both shared and unique epigenomic mechanisms. Targeting the mutant H3.3 effector pathways identified by our analyses in our full panel of cells with specific inhibitory drugs plus or minus irradiation defines differential, largely opposite responses of the parental and gene-edited cells. These defined pathways may serve as entry points for development of novel therapies specific for H3.3-mutant pediatric glioma. Overall, this system of gene editing gain and loss of mutant H3.3 provides new insights into oncohistone mechanisms and therapeutic strategies.