Reciprocal gene editing defines targetable mutant H3.3 oncohistone effectors in pediatric glioma

High-grade pediatric gliomas often contain histone H3.3 mutations, but open questions remain about oncogenic mechanisms. To address this gap, we performed 'reciprocal gene editing' using CRISPR-Cas9 to introduce H3.3 mutations (K27M, G34R) into H3.3-wildtype brain and glioma cells, while in parallel reverting pre-existing K27M mutations in glioma cells back to wildtype. Analyses of our reciprocally-edited cells indicate that H3.3 mutation leads to specific transcriptomic and epigenetic events, and associated cell biological changes including in xenograft assays. We used these data and the reciprocally-edited cells to screen selected drugs and identify specific putative treatments that are mutant H3.3-specific. Overall, reciprocal gene editing provides new insights into mutant H3.3 oncogenic mechanisms and more broadly may prove useful for studying other cancer-associated mutations. Overall design: We used CRISPR-Cas9 gene editing of H3F3A to precisely revert preexisting K27M mutations to WT in patient-derived glioma cells to identify consistently functionally important effectors. Two replicates of H3K27me3 and two replicates of H3.3 ChIP-Seq were performed in each of the following cell lines: Line XIII, Line XIII WT, Line XVII, Line XVII WT.

高级别儿童胶质瘤（high-grade pediatric gliomas）常携带组蛋白H3.3（histone H3.3）突变，但其致癌机制仍存在诸多未解问题。为填补该研究空白，我们采用CRISPR-Cas9开展双向基因编辑（reciprocal gene editing），将H3.3突变（K27M、G34R）引入至H3.3野生型（wildtype）脑及胶质瘤细胞中，同时同步将胶质瘤细胞内预先存在的K27M突变恢复为野生型。对上述双向编辑细胞的分析表明，H3.3突变可引发特异性转录组与表观基因组变化，以及相关细胞生物学改变，涵盖异种移植实验中的表型变化。我们依托本研究产生的数据及双向编辑细胞系筛选了多款候选药物，鉴定出针对突变型H3.3的特异性潜在治疗方案。总体而言，双向基因编辑为突变型H3.3的致癌机制提供了全新见解，且该方法可推广应用于其他癌症相关突变的研究。整体实验设计：我们通过CRISPR-Cas9对H3F3A进行基因编辑，将患者来源的胶质瘤细胞中预先存在的K27M突变精准恢复为野生型，以鉴定始终具有功能重要性的效应因子。在以下细胞系中分别完成两组H3K27me3的ChIP-Seq实验，以及两组H3.3的ChIP-Seq实验：XIII细胞系、XIII野生型细胞系、XVII细胞系、XVII野生型细胞系。