Multi-omic analysis of cell-of-origin and epigenomic state in pediatric H3K27M gliomas

It is not known whether midline pediatric gliomas driven by Histone 3 K27M mutations (oncohistones) or EZHIP expression (oncohistone-mimics) share a common cell-of-origin, or arise from distinct cell types with unique vulnerabilities to PRC2 inhibition and partner alterations. Here, we define the etiological and oncogenic relationship between pediatric midline gliomas characterized by inhibition of K27M and K27M-like oncohistones. We assemble an extensive reference for gliogenesis from the developing mouse and human fetal brain. With bulk and single-cell transcriptomics and epigenomics, we profiled a large cohort of primary tumors comprising H3.1K27M and H3.3K27M pontine gliomas, H3.3K27M thalamic gliomas, and EZHIP+ posterior fossa ependymomas. We focus on differences across axes of location (thalamus vs. pons vs. posterior fossa), tumor type (diffuse midline gliomas vs. ependymomas), and oncohistone (H3.1/2K27M vs. H3.3K27M vs. EZHIP). We use tumor molecular features to delineate transcriptional states and cell-of-origin in each entity. Finally, we use culture models in isogenic contexts to interrogate the effect of each oncohistone or mimic. 8 single-cell RNA-seq samples from healthy mouse forebrains, hindbrain and pons were sequenced at E10.5, E13.5, E16.5 and E18.5. H3K27ac, H3K27me2 and H3K27me3 ChIP-seq experiments on multiple human cell lines were also performed (102 samples)目前尚不明确，由组蛋白H3 K27M突变（Histone 3 K27M mutation，癌组蛋白（oncohistone））或EZHIP表达（癌组蛋白模拟物（oncohistone-mimic））驱动的儿童中线胶质瘤，是否共享共同的起源细胞（cell-of-origin），亦或是起源于不同细胞类型，且对多梳抑制复合体2（PRC2）抑制及伴发改变具有独特易感性。本研究明确了以K27M及K27M样癌组蛋白介导的PRC2抑制为特征的儿童中线胶质瘤之间的病因学与致癌学关联。我们构建了源自发育中小鼠及人类胎儿大脑的神经胶质发生广泛参考数据集。通过批量转录组学（bulk transcriptomics）、单细胞转录组学（single-cell transcriptomics）及表观基因组学（epigenomics）技术，我们对大型原发性肿瘤队列进行了分型，该队列包含H3.1K27M与H3.3K27M型桥脑胶质瘤、H3.3K27M型丘脑胶质瘤，以及EZHIP阳性后颅窝室管膜瘤。本研究重点关注不同维度的差异：包括解剖部位（丘脑 vs. 桥脑 vs. 后颅窝）、肿瘤类型（弥漫性中线胶质瘤 vs. 室管膜瘤），以及癌组蛋白类型（H3.1/2K27M vs. H3.3K27M vs. EZHIP）。我们利用肿瘤分子特征，解析了各肿瘤实体的转录状态（transcriptional states）与起源细胞。最后，我们采用同基因背景（isogenic contexts）下的培养模型，探究每种癌组蛋白或模拟物的作用效果。本研究对8个取自健康小鼠前脑、后脑及桥脑的单细胞RNA测序（single-cell RNA-seq）样本进行了测序，采样时间覆盖胚胎发育第10.5天（E10.5）、E13.5、E16.5及E18.5。此外，我们还针对多种人类细胞系开展了H3K27ac、H3K27me2及H3K27me3的染色质免疫共沉淀测序（ChIP-seq）实验，共计102个样本。