Functionizing the epigenome in Ewing sarcoma

描述(由申请人提供)： 人们越来越多地认识到，表观遗传变化有助于癌症的发展。由于表观遗传变化的定义是可逆的，而且许多表观遗传修饰酶高度受制于药物发现，因此人们对开发表观遗传靶向疗法的兴趣激增。尽管表观遗传疗法具有高度的“可药性”，但其临床翻译的一个主要障碍是缺乏对 这些药物应该应用于的临床疾病。因此，我们正处于一个十字路口，学术界和工业界的多个实体正在积极开发针对表观遗传修饰蛋白的药物，但这些药物应该应用于哪些疾病还没有明确的定义。我们假设，全面和系统地评估哪些表观遗传调节蛋白对体内肿瘤生长至关重要，将有助于确定表观遗传靶点，适合于特定癌症亚型的治疗应用。在这项提案中，我们 将使用汇集的shRNA分析来系统地确定哪些表观遗传修饰蛋白对尤文肉瘤细胞的体外和体内生长至关重要。在第一个特定目标中，我们使用基因注释和蛋白质结构域分析来鉴定449个已知或推测具有表观遗传调控功能的基因产物(表观基因组)。我们将包装一个由3040个针对表观基因组和对照的shRNA组成的shRNA文库。在第二个特定目的中，我们将使用这个文库转导4个尤文肉瘤细胞系，这些细胞系已经被反复选择为高致瘤性。下一代测序将用于确定输入样本中所有shRNA的频率，并将其与在细胞培养和体内生长后选择的样本作为异种移植瘤进行比较。一条分析管道将识别对体外和体内肿瘤细胞生长至关重要的基因产品。在第三个特定目标中，我们将全面评估与上述目标中确定的特定高优先级表观遗传调节蛋白的功能丧失相关的细胞效应， 并将评估已建立的尤文肉瘤异种移植的治疗靶向。总而言之，这些研究的总体目标是系统地确定尤文肉瘤生长所需的表观遗传调节蛋白，并实现通过表观遗传疗法进行尤文肉瘤靶向治疗的体内概念验证。这些研究的成功将建立一个范例，可以很容易地应用于几乎所有其他癌症亚型。

DESCRIPTION (provided by applicant): There is an increasing understanding that epigenetic changes contribute to cancer development. Because epigenetic changes are by definition reversible, and because many epigenetic modifying enzymes are highly amenable to drug discovery, there is an explosion in interest in developing epigenetic-targeted therapies. Despite being highly "druggable", a major impediment to clinical translation of epigenetic therapies is the lack of understanding as to the clinical diseases to which these drugs should be applied. We are therefore at a crossroad in which multiple entities within academia and industry are actively developing drugs targeting epigenetic modifying proteins, but the diseases to which they should be applied are as yet poorly defined. We hypothesize that a comprehensive and systematic assessment of which epigenetic regulating proteins are critical for in vivo tumor growth will help define the epigeneti targets appropriate for therapeutic application in specific cancer sub-types. In this proposal, we will use pooled shRNA analysis to systematically determine, which epigenetic-modifying proteins are critical for the in vitro and in vivo growth of Ewing sarcoma cells. In the first Specific Aim, we have used gene annotation and protein domain analysis to identify 449 gene products that have known or putative epigenetic regulatory function (the epigenome). We will package a shRNA library composed of 3040 shRNAs targeting the epigenome and controls. In the second Specific Aim, we will use this pooled library to transduce 4 Ewing sarcoma cell lines that have been iteratively selected for high tumorigenicity. Next-generation sequencing will be used to determine the frequency of all shRNAs in an input sample compared to samples selected after in vitro growth in cell culture and in vivo growth as xenograft tumors. An analysis pipeline will identify genes products essential for in vitro and in vivo tumor cell growth. In the third Specific Aim we will fully assess the cellular effects associated with loss o function of specific high priority epigenetic regulatory proteins identified in the preceding Aims, and will assess therapeutic targeting in established Ewing sarcoma xenografts. Together, the overall goal of these studies is to systematically determine which epigenetic regulatory proteins are required for Ewing sarcoma growth and to achieve in vivo proof-of-concept for therapeutic targeting of Ewing sarcoma with epigenetic therapies. Success in these studies will establish a paradigm that can be readily applied to almost all other cancer sub-types.