Nominating vulnerabilities in fusion oncoprotein-driven rhabdomyosarcoma

项目摘要 这项提案的重点是400名儿童和年轻人分别患上横纹肌肉瘤(RMS)这一事实 到今年，绝大多数高危患者将不会是长期无病的幸存者。其中之一 PAX3-FOXO1融合蛋白的存在是高危疾病的主要驱动因素。在通常的情况下 称为融合阳性(FP)RMS、新一代DNA和RNA测序工具以及分子和细胞 生物学方法尚未发现有针对性的癌症驱动因素。因此，对这些儿童的治疗 而年轻的成年人几十年来没有根本的改变！ 解开FP-RMS和PAX3-FOXO1生物学的主要挑战至少有两个：首先，我们知道 关于PAX3-FOXO1融合蛋白的生物学，包括合作遗传或 它需要表观遗传变化来驱动RMS的形成和进展。但我们的知识并不是 足够复杂，足以专注于可以利用的协同遗传/表观遗传变化的子集 作为治疗的弱点。其次，尽管FP-RMS存在一些优雅的实验模型，但它们是纯粹的 PAX3-FOXO1表达可以快速而完全地打开和关闭的同基因系统不是 可用。我们相信，解决这两个挑战将提供实现以下目标的基础步骤 识别FP-RMS中由致癌融合蛋白驱动的可操作靶点。 在接下来的两年里，我们可以通过完成两个相互补充但独立的目标来实现这一目标。第一, 我们应用一种创新的计算管道来提名致癌驱动因素和肿瘤抑制因素，基于 FP-RMS中的遗传和表观遗传学变化，并在基于CRISPR/Cas9的“迷你池”中进行功能验证 同时使用FP和融合阴性RMS模型进行检测。第二，我们将开发和验证一个新的降级- 一种可在本地以动态和可逆方式控制人PAX3-FOXO1的基于系统 RMS单元和PDX模型。除其他外，在AIM 2中创建的这个系统将被用来识别 AIM 1的关键驱动因素和抑制因素由PAX3-FOXO1控制。我们的成功将为 未来，以假设为导向的FP-RMS研究，为科学界产生可共享的数据和工具， 并说明了应对其他移位驱动的癌症的一般方法，这些癌症构成了类似于 FP-RMS。

Project Summary This proposal focuses on the fact that 400 children and young adults develop rhabdomyosarcoma (RMS) each year, and the vast majority of those with high-risk disease will not be long term, disease-free survivors. One of the major drivers of high-risk disease is the presence of PAX3-FOXO1 fusion protein. In what is commonly referred to as fusion-positive (FP) RMS, next generation DNA and RNA sequencing tools and molecular and cell biological approaches have yet to uncover targetable cancer drivers. As such, the treatment for these children and young adults has not fundamentally changed for several decades! Major challenges to unraveling FP-RMS and the biology of PAX3-FOXO1 are at least two-fold: First, we know much about the biology of the PAX3-FOXO1 fusion protein, including the fact that cooperating genetic or epigenetic changes are needed for it to drive RMS formation and progression. But our knowledge is not sophisticated enough to focus on the subset of cooperating genetic/epigenetic changes that can be leveraged as therapeutic vulnerabilities. Second, though some elegant, experimental models exist for FP-RMS, pure isogenic systems in which PAX3-FOXO1 expression can be quickly and completely turned “on” and “off” are not available. We are convinced that solving both of these challenges will provide a foundational step toward identifying actionable targets that are driven by the oncogenic fusion protein in FP-RMS. Over the next two years, we can accomplish this by completing two complementary but independent aims. First, we apply an innovative computational pipeline to nominate oncogenic drivers and tumor suppressors based on genetic and epigenetic changes in FP-RMS, and functionally validate them in a CRISPR/Cas9-based “mini-pool” assay using both FP and fusion-negative RMS models. Second, we will develop and validate a new degron- based system in which human PAX3-FOXO1 can be controlled in a dynamic and reversible fashion in native RMS cells and PDX models. Among other things, this system created in Aim 2 will be utilized to identify how the key drivers and suppressors from Aim 1 are controlled by PAX3-FOXO1. Our success will lay the foundation for future, hypothesis-directed studies of FP-RMS, generate sharable data and tools for the scientific community, and illustrate a general approach to tackling other translocation-driven cancers that pose challenges similar to FP-RMS.