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

项目总结 我们是首次确定组蛋白H3变异体(原癌组蛋白)上氨基酸替换的两个小组之一。 人类疾病。在过去的筹资周期中，我们表明，除了在特定亚型中最初确定外， 在高级别胶质瘤和骨肿瘤中，这些癌组织蛋白也出现在肉瘤、头部和 颈部鳞状细胞癌和急性髓系白血病。与此计划项目的成员合作， 我们展示了H3K27M和H3K36M(K-to-M替换)如何导致开发停滞和受阻 通过表观基因组重新连接促进关键染色质异常扩散/重新分布的分化 从它们最初的边界留下的痕迹，这反过来又促进了肿瘤的发生。此外，我们与 Lewis/Garcia实验室发现，EZHIP是一种睾丸特异蛋白，在后颅窝中异常表达 室管膜瘤，生化镜下见H3K27M癌基因。我们的初步数据显示，EZHIP也 在骨肉瘤亚群中异常激活。与不断增长的关于K-M突变的信息相反， 然而，对其致癌功能、分子机制和肿瘤特异性的认识有限。 模仿癌组蛋白的EZHIP和突变的漏洞会影响H3.3G34。在很大程度上，这是由于 缺乏资源和模型来忠实地概括这些变化所介导的细胞效应。 因此，该项目的一个主要目标是开发和使用新的患者样本、细胞培养和动物 基于模型的系统来模拟癌组蛋白相关的癌症并调查潜在的致病因素 机械装置。几乎所有G34突变都会影响组蛋白变异体H3.3，而H3.3G34R/V是常见的 在儿童胶质瘤中，巨细胞瘤主要由H3.3G34W突变来定义。这种二分法是 我们开发的H3F3A G34W、R或V敲入小鼠模型的独特表型反映了这一点。我们 目的探讨H3.3G34基因突变导致密码子特异性表型的机制。 特异性突变伙伴关系在G34R/V驱动的肿瘤发生中的作用以及H3.3变异在肿瘤发生中的必要性 调节依赖G34的表型(目标1)。我们将调查EZHIP在开发中的作用，它是如何 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H3K27M突变对照(目标2)。最后，我们将建立在我们的发现基础上，诱导病毒模仿 用K-to-M突变来评估这些肿瘤中免疫渗透的程度和性质 表观遗传药物的治疗(目标3)。值得注意的是，这些研究是由独特的同基因小鼠实现的 我们建立的模型和CRISPR/Cas9编辑的等基因患者来源的肿瘤细胞系 肿瘤组蛋白和EZHIP。该项目将与本计划项目的其他工作协同，以提供 对社区的机械性见解和相关可靠的临床前模型，构建知识和 可以构思和验证可接受治疗的目标的资源基础。

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.