The role of a histone point mutation in pediatric gliomagenesis

项目摘要 小儿高级别胶质瘤（pHGG）是儿童中最常见和最恶性的原发性脑肿瘤。 编码组蛋白变体H3.3的基因之一H3 F3 A中的突变是pHGG和H3.3的定义 突变状态与临床变量如存活率和肿瘤位置相关。pHGG与赖氨酸27至 甲硫氨酸取代（H3.3K27M）发生在脑干，与特别差的生存相关。H3.3K27M 不足以驱动小鼠模型中的肿瘤发生。然而，它与PDGFRA激活共同发生在人类中。 以及TP 53和ATRX缺失，表明H3.3K27M与这些致癌突变协同作用， 肿瘤的此外，H3.3K27M pHGG和其他pHGG之间的表型差异表明， 表观遗传改变和肿瘤发生机制。这项建议的重点是确定表观遗传 H3.3K27M及其共表达的H3.3K27M在小鼠神经干细胞（NSC）中诱导的转录组学变化 突变以及这些突变如何导致神经胶质瘤细胞表型。为准确建模H3.3K27M pHGG， 如果可能的话，我将使用基于RCAS-TVA的模型，其中NSC从Nestin tv-a; Trp 53 fl/fl或Nestin tv-a中收获。 用编码Cre重组酶、PDGFβ和H3.3K27 M的RCAS病毒感染a; Trp 53 fl/fl; Atrxfl/fl小鼠。的 结果NSC将用于表征诱导的全局和局部表观遗传和转录组学变化 通过H3.3K27M及其在NSC中的共发生突变，使用蛋白质印迹、ChIP-seq和RNA-seq。此外，我将 使用免疫细胞化学表征由这些改变引起的表型变化， 稀释、增殖测定和原位同种异体移植。最后，我将验证表观遗传学和转录组学 一组H3.3WT和H3.3K27M患者来源的异种移植（PDX）细胞系中H3.3K27M小鼠NSC的特征 通过ChIP-seq和RNA-seq，以及来自人类肿瘤样本的现有数据集。这些研究将 确定H3.3K27M pHGG发病机制和潜在的表观遗传治疗靶点， 在小鼠中易于处理并且与人类相关。通过完成这份研究计划，我将获得技术技能 适用于癌症生物学，神经科学，表观遗传学和生物化学，同时也发展了我的关键 思维，科学沟通和道德研究技能。

PROJECT SUMMARY Pediatric high-grade gliomas (pHGG) are the most common and malignant primary brain tumors in children. Mutations in H3F3A, one of the genes that encodes histone variant H3.3, are definitional for pHGG and H3.3 mutation status correlates with clinical variables such as survival and tumor location. pHGG with a lysine 27 to methionine substitution (H3.3K27M) occur in the brain stem and correlate with particularly poor survival. H3.3K27M is insufficient to drive tumorigenesis in mouse models. However, it co-occurs in humans with PDGFRA activation and TP53 and ATRX deletion, suggesting that H3.3K27M cooperates with these oncogenic mutations to induce tumors. Further, the phenotypic differences between H3.3K27M pHGG and other pHGG suggest differing epigenetic alterations and mechanisms of tumorigenesis. This proposal is focused on identifying the epigenetic and transcriptomic changes induced in murine neural stem cells (NSC) by H3.3K27M and its co-occurring mutations and how those mutations lead to glioma cell phenotypes. To model H3.3K27M pHGG as accurately as possible, I will use an RCAS-TVA-based model in which NSC harvested from Nestin tv-a; Trp53fl/fl or Nestin tv- a; Trp53fl/fl; Atrxfl/fl mice are infected with RCAS viruses encoding Cre recombinase, PDGFβ, and H3.3K27M. The resulting NSC will be used to characterize the global and local epigenetic and transcriptomic changes induced by H3.3K27M and its co-occurring mutations in NSC using western blots, ChIP-seq, and RNA-seq. Further, I will characterize the phenotypic changes caused by these alterations using immunocytochemistry, extreme limiting dilution, proliferation assays, and orthotopic allografts. Finally, I will validate the epigenetic and transcriptomic signatures of H3.3K27M mouse NSC in a panel of H3.3WT and H3.3K27M patient derived xenograft (PDX) cell lines via ChIP-seq and RNA-seq, as well as with existing datasets from human tumor samples. These studies will identify mechanisms of pathogenesis for H3.3K27M pHGG and potential epigenetic therapeutic targets that are both tractable in mice and relevant to humans. By completing this research proposal, I will gain technical skills applicable to cancer biology, neuroscience, epigenetics, and biochemistry while also developing my critical thinking, scientific communication, and ethical research skills.