Investigation of Histone H3 Post-Translational Modifications in Pediatric Brainstem Glioma

项目概要/摘要 弥漫性内在脑桥胶质瘤（DIPG）是儿童最致命的实体瘤， 疾病进展、对治疗反应差、诊断后平均生存期不到一年。后生 基因表达的调节与包括癌症在内的多种人类疾病有关。最近， 在高达80%的DIPG中发现了组蛋白H3的点突变，导致染色质功能改变 和广泛的转录组重编程。在这里，我们描述了一个假设驱动的研究计划，以阐明 组蛋白H3翻译后修饰在儿童脑干胶质瘤中的作用机制 （DIPG）的形成和进展。还介绍了职业发展计划，以促进成功 从一个有指导的调查员转变为一个独立的调查员。为了支持组蛋白 H3 K27 M突变型儿童脑干胶质瘤的修饰有助于疾病的发病机制，我们将 表征与特异性组蛋白H3转录相关的DIPG中基因表达的改变 监管标志。此外，我们将研究负责这些组蛋白的酶机制， 修改，以确定这些是否代表合理的治疗目标。为了研究 DIPG中组蛋白H3对基因转录的修饰，我们将在DIPG中进行染色质免疫沉淀。 与DNA测序（ChIP-Seq）相结合，以表征特定H3标记的基因组位置，以及 将这些发现与基因表达（RNA-Seq）相关联。然后，我们将描述改变的影响， 特异性癌基因（PDGFRA）和肿瘤抑制基因（p21）的表达，以及负责 鉴定了H3修饰对体外和体内DIPG肿瘤生物学的影响。人H3 K27 M突变体和野生型 DIPG肿瘤细胞将用于体外研究，而DIPG的小鼠异种移植模型将用于体内研究。 体内研究。本提案的目的是：1）确定DIPG转录组相对于组蛋白 2）阐明DIPG中组蛋白H3修饰的酶促基础，并确定DIPG中组蛋白H3修饰的酶促基础。 特异性酶活性是否可用于治疗DIPG;和3）验证特异性癌基因 与DIPG恶性表型相关的肿瘤抑制基因激活和失活 这项研究的结果将证明表观遗传失调对基因表达的影响， 儿童脑干胶质瘤，提供了新的见解DIPG肿瘤发生的机制和潜在的 为患有这种疾病的儿童开发合理有效的治疗方法。

PROJECT SUMMARY / ABSTRACT Diffuse intrinsic pontine glioma (DIPG) is the most deadly solid tumor in children, with rapid disease progression, poor response to therapy, and average survival less than one year from diagnosis. Epigenetic regulation of gene expression has been implicated in a variety of human diseases, including cancer. Recently, point mutations in histone H3 have been identified in up to 80% of DIPG, causing altered chromatin function and extensive transcriptome reprogramming. Here, we describe a hypothesis-driven research plan to elucidate the mechanism by which histone H3 post-translational modifications contribute to pediatric brainstem glioma (DIPG) formation and progression. A career development plan is also described to facilitate successful transition from a mentored investigator to an independent investigator. In support of the hypothesis that histone modifications in H3K27M mutant pediatric brainstem glioma contribute to disease pathogenesis, we will characterize alterations in gene expression in DIPG associated with specific histone H3 transcriptional regulatory marks. Further, we will investigate the enzymatic mechanisms responsible for these histone modifications, to determine if these represent rational therapeutic targets. In order to investigate the effect of Histone H3 modifications on gene transcription in DIPG, we will perform chromatin immunoprecipitation in association with DNA sequencing (ChIP-Seq) to characterize genomic locations of specific H3 marks, and correlate these findings with gene expression (RNA-Seq). We will then characterize the effects of altered expression of specific oncogenes (PDGFRA) and tumor suppressor genes (p21), and enzymes responsible for identified H3 modifications, on DIPG tumor biology in vitro and in vivo. Human H3K27M mutant and wild type DIPG tumor cells will be used for in vitro studies, while a mouse xenograft model of DIPG will be used for in vivo investigations. The aims of this proposal are: 1) To determine DIPG transciptomes with respect to histone H3 modifications; 2) To elucidate the enzymatic basis of histone H3 modifications in DIPG, and determine whether specific enzymatic activities are actionable for treating DIPG; and 3) To validate specific oncogene activations and tumor suppressor gene inactivations as causally associated with DIPG malignant phenotypes The findings of this proposal will demonstrate the effect of epigenetic dysregulation on gene expression in pediatric brainstem glioma, providing novel insight on the mechanism of DIPG tumorigenesis and a potential for developing rational, effective treatment for children with this disease.