Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered H3K27me3 activity including within super-enhancers, which exhibited perturbed transcriptional function. This was largely independent of H3.3 DNA binding. The K27M and G34R mutations induced several of the same pathways suggesting key shared oncogenic mechanisms including activation of neurogenesis and NOTCH pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH pathway gene knockdown and drug inhibition, reducing their viability in culture. Reciprocal editing of cells generally produced reciprocal effects on tumorgenicity in xenograft assays. Overall, our findings define common and distinct K27M and G34R oncogenic mechanisms, including potentially targetable pathways.

组蛋白H3.3突变是小儿神经胶质瘤的标志，但其核心致癌机制尚不明确。为了识别主要效应子，我们使用了CRISPR-Cas9将H3.3K27M和G34R突变引入以前的H3.3野生型脑细胞中，同时将神经胶质瘤细胞中的突变恢复为野生型。ChIP-seq分析广泛地将K27M与改变的H3K27me3活性联系起来，包括在超级增强子中，后者表现出受干扰的转录功能。这在很大程度上与H3.3 DNA结合无关。K27M和G34R突变诱导了几种相同的途径，提示了关键的共同致癌机制，包括激活神经发生和NOTCH途径基因。H3.3突变型神经胶质瘤对NOTCH途径基因敲低和药物抑制也特别敏感，从而降低了它们在培养物中的生存能力。在异种移植测定中，细胞的相互编辑通常对致瘤性产生相互影响。总体而言，我们的发现定义了常见且独特的K27M和G34R致癌机制，包括潜在的可靶向途径。