EZH2 is a potential therapeutic target for H3K27M mutant paediatric gliomas [ChIP-Seq human cell lines]. Homo sapiens

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive brain tumour that is located in the pons and primarily affects children. Whole-exome sequencing studies have identified recurrent driver mutations in H3F3A (H3.3) and HIST1H3B (H3.1), leading to the expression of histone H3 in which lysine 27 is substituted with methionine (H3K27M) in nearly 80% of DIPGs1-5. H3K27M has been shown to inhibit Polycomb Repressive Complex 2 (PRC2) activity by binding to its catalytic subunit EZH2, and although DIPGs with H3K27M mutation show global loss of H3 with trimethylated lysine 27 (H3K27me3), several genes retain H3K27me3 (Refs. 1-8). Here, we describe a mouse DIPG model in which H3K27M potentiates tumourigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M expressing tumours require PRC2 for proliferation. Furthermore, we demonstrate that small molecule EZH2 inhibitors abolish tumour cell growth through a mechanism that is dependent on the induction of the tumour suppressor protein p16INK4A. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets and are highly enriched for H3K27me3/PRC2/PRC1 in cells prior to H3K27M expression. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the mouse DIPG model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M positive DIPGs, and inhibition of EZH2 is as a potential therapeutic strategy for the treatment of these tumours. Overall design: H3K27me3 ChIP-seq experiments in four human DIPG cell lines expressing K27M mutant histone H3.

弥漫内生型桥脑胶质瘤（Diffuse intrinsic pontine glioma, DIPG）是一种恶性程度极高的脑肿瘤，病变位于桥脑区域，主要发病人群为儿童。全外显子组测序研究已在H3F3A（H3.3）与HIST1H3B（H3.1）中发现复发性驱动突变，导致近80%的DIPG患者表达组蛋白H3赖氨酸27被甲硫氨酸取代的突变体（H3K27M）[1-5]。已有研究证实，H3K27M可通过结合多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）的催化亚基EZH2，抑制该复合体的活性；尽管携带H3K27M突变的DIPG会出现整体三甲基化赖氨酸27的组蛋白H3（H3K27me3）水平下降，但仍有部分基因保留H3K27me3修饰[参考文献1-8]。
本研究构建了一种可使H3K27M促进肿瘤发生的小鼠DIPG模型。利用该模型及患者来源的原代DIPG细胞系，我们证实表达H3K27M的肿瘤细胞增殖依赖于PRC2。进一步研究表明，小分子EZH2抑制剂可通过依赖抑癌蛋白p16INK4A诱导的机制，完全阻断肿瘤细胞的生长。
全基因组富集分析结果显示，在H3K27M突变细胞中保留H3K27me3修饰的基因均为典型的多梳靶基因，且在H3K27M突变表达前的细胞中，这些基因即高度富集H3K27me3/PRC2/PRC1修饰。此外，在小鼠DIPG模型与表达H3K27M的人类原代DIPG中，保留H3K27me3修饰的基因存在高度显著的重叠。
综合以上结果，本研究表明残留的PRC2活性是H3K27M阳性DIPG细胞增殖所必需的，而EZH2抑制有望成为治疗此类肿瘤的潜在治疗策略。
整体实验设计：对4株携带K27突变型组蛋白H3的人类DIPG细胞系开展H3K27me3染色质免疫共沉淀测序（ChIP-seq）实验。