A Novel Mouse Model of Diffuse Midline Glioma Initiated in Neonatal Oligodendrocyte Progenitor Cells Highlights Cell- of-origin Dependent Effects of H3K27M I

Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in Olig2-expressing progenitors and Nestin-expressing progenitors in the neonatal mouse brainstem. PDGF-A or PDGF-B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is increased when tumorigenesis is initiated in Nestin+ cells. Therefore, we need to find how the tumorigenic effects of H3.3K27M are more oncogenic in Nestin+ cells than Olig2+ cells. Overall design: Five independent tumors with H3.3K27M (PaKC) and three independent tumors without H3.3K27M (PaHC) were snap frozen, RNA extracted, and RNAseq analysis was performed (PDGF-A; H3.3K27M; p53 loss vs. PDGF-A; H3.3WT; p53 loss)

弥漫内生型桥脑胶质瘤（Diffuse midline glioma, DMG）是一类主要发生于儿童的致命性脑肿瘤。大多数DMG存在组蛋白H3的K27M突变。脑干内的少突胶质细胞祖细胞（Oligodendrocyte progenitor cells, OPCs）是DMG的候选起源细胞，但目前尚无在OPCs中起始构建的基因工程DMG小鼠模型。本研究利用RCAS/Tv-a禽逆转录病毒系统，在新生小鼠脑干内的表达Olig2的祖细胞与表达Nestin的祖细胞中构建DMG模型。PDGF-A或PDGF-B过表达联合p53缺失，可在两种模型中诱导胶质瘤生成。与Nestin+细胞中的H3.3WT组相比，Nestin+细胞中外源性过表达H3.3K27M可显著影响肿瘤潜伏期与肿瘤细胞增殖能力，而该效应在Olig2+细胞中未被观测到。此外，在PDGF-A和PDGF-B驱动的模型中，Olig2+细胞起源的DMG内H3.3K27M阳性细胞比例均显著低于Nestin+细胞起源的DMG，提示当肿瘤发生起始于Nestin+细胞时，对H3.3K27M的需求更高。因此，本研究旨在阐明H3.3K27M的致瘤效应为何在Nestin+细胞中强于Olig2+细胞。整体实验设计：将5株独立的H3.3K27M阳性（PaKC）肿瘤与3株独立的H3.3K27M阴性（PaHC）肿瘤快速冷冻后提取RNA，并进行RNA测序分析（PDGF-A; H3.3K27M; p53缺失 vs. PDGF-A; H3.3WT; p53缺失）