Project 1: Decoding, Modeling and Targeting Oncohistones and an Oncohistone-like Protein

项目摘要 我们是两个小组之一，首先确定氨基酸取代组蛋白H3变体（癌组蛋白）， 人类疾病在过去的资助周期中，我们表明，除了在特定亚型中的初步鉴定外， 在高级别胶质瘤和骨肿瘤中，这些癌组蛋白也出现在肉瘤、头和 颈部鳞状细胞癌和急性髓性白血病。与该项目的成员合作， 我们展示了H3 K27 M和H3 K36 M（K到M的替换）是如何导致发育停滞和受阻的， 通过表观基因组重新布线的分化，因为它们促进关键染色质的异常扩散/重新分布 标志着他们的初始边界，这反过来又促进肿瘤发生。此外，我们与 刘易斯/Garcia实验室发现，睾丸特异性蛋白EZHIP在后颅窝组异常表达 A室管膜瘤，生物化学反映H3 K27 M癌组蛋白。我们的初步数据表明，EZHIP也是 在骨肉瘤的亚组中异常激活。与越来越多的关于K到M突变的信息相反， 然而，对于肿瘤的致瘤功能、分子机制和肿瘤特异性 癌组蛋白模拟EZHIP的脆弱性和突变影响H3.3G34。在很大程度上，这是由于 缺乏资源和模型，忠实地概括了这些变化介导的细胞效应。 因此，本项目的主要目标是开发和使用新的患者样本、细胞培养物和动物模型。 基于模型的系统，用于对癌组蛋白相关癌症进行建模并研究潜在的致病性 机制等几乎所有的G34突变都影响组蛋白变体H3.3，而H3.3G34 R/V经常被发现， 在小儿神经胶质瘤中，巨细胞瘤主要由H3.3G34W突变定义。这种二分法是 这反映在我们开发的H3 f3 a G34 W、R或V敲入小鼠模型的不同表型上。我们 目的是描述H3.3G34突变导致的密码子特异性表型背后的机制， 特异性突变伙伴关系对G34 R/V驱动的肿瘤发生的作用，以及H3.3变体在 介导G34依赖性表型（Aim 1）。我们将研究EZHIP在发展中的作用， 在骨肉瘤和室管膜瘤中实现异常表达并促进肿瘤发生-比较 并与H3 K27 M突变（Aim 2）作对照。最后，我们将建立在我们的发现，病毒模仿诱导 K-to-M突变，以评估这些肿瘤在基线时和治疗后免疫浸润的程度和性质。 治疗表观遗传药物（目标3）。值得注意的是，这些研究是由独特的同基因小鼠 我们已经为各种肿瘤细胞生成了CRISPR/Cas9编辑的等基因患者来源的肿瘤细胞系， 癌组蛋白和EZHIP。该项目将与本方案项目的其他努力协同作用， 机械的见解和相关的和可靠的临床前模型，以社区，建立一个知识和 可以设想和验证的治疗靶点的资源基础。

PROJECT SUMMARY We were one of two groups to first identify amino acid substitutions on histone H3 variants (oncohistones) in human disease. In the past funding cycle, we showed that, beyond their initial identification in specific subtypes of high-grade gliomas and bone tumors, these oncohistones also occur in subgroups of sarcomas, head and neck squamous cell carcinomas, and acute myeloid leukemias. Working with members of this Program Project, we showed how H3K27M and H3K36M (K-to-M substitutions) lead to stalled development and blocked differentiation through epigenome rewiring as they promote aberrant spreading/redistribution of key chromatin marks from their initial boundaries, which in turn facilitate oncogenesis. Furthermore, our collaboration with Lewis/Garcia labs uncovered that, EZHIP, a testis-specific protein aberrantly expressed in posterior fossa group A ependymomas, biochemically mirrors H3K27M oncohistone. Our preliminary data indicate that EZHIP is also aberrantly activated in subgroups of osteosarcomas. In contrast to the growing information on K-to-M mutations, however, there is limited knowledge on the oncogenic function, molecular mechanisms and tumor-specific vulnerabilities of the oncohistone-mimic EZHIP and mutations affect H3.3G34. In large part, this is due to a paucity of resources and models that faithfully recapitulate the cellular effects mediated by these alterations. Therefore, a major goal of this project is to develop and employ novel patient sample-, cell culture- and animal model-based systems to model oncohistone-associated cancers and investigate the underlying pathogenic mechanisms. Nearly all G34 mutations affect histone variant H3.3, and while H3.3G34R/V are frequently found in pediatric gliomas, giant cell tumors are predominantly defined by H3.3G34W mutations. This dichotomy is reflected by the distinct phenotypes of the H3f3a G34W, R or V knock-in mouse models we have developed. We aim to delineate the mechanisms behind the codon-specific phenotypes resulting from H3.3G34 mutations, the role of specific mutational partnership for G34R/V-driven tumorigenesis, and the necessity of H3.3 variant in mediating G34-dependent phenotypes (Aim 1). We will investigate the role of EZHIP in development, how its aberrant expression is achieved and promotes oncogenesis in osteosarcomas and ependymomas - comparing and contrasting to H3K27M mutation (Aim 2). Lastly, we will build on our findings of the viral mimicry induced by K-to-M mutations to assess the degree and nature of immune infiltration in these tumors at baseline and upon treatment of epigenetic drugs (Aim 3). Notably, these studies are enabled by the unique syngeneic mouse models and CRISPR/Cas9-edited isogenic patient-derived tumor cell lines we have generated for various oncohistones and EZHIP. This project will synergize with other efforts of this Program Project to provide mechanistic insights and relevant and reliable pre-clinical models to the community, building a knowledge and resource base upon which targets amenable to therapy that can be conceived and validated.