Elucidating the function of PAX3-FOXO1 in rhabdomyosarcoma with molecular reporters and next-generation genome editing

项目摘要： 横纹肌肉瘤是一种高度转移性的儿童软组织恶性肿瘤，新的治疗方法是 迫切需要。在过去的三年中，RMS患者的临床管理基本上没有变化 几十年，目前仅限于手术切除、放射治疗和联合化疗。在这 背景：基于机制的靶向治疗将对RMS患者产生潜在的变革性影响 结果。RMS发病机制中最常见的遗传事件是染色体重排 产生PAX融合癌蛋白，这是一种嵌合转录因子，解除对染色质和 转录促进转化。我们专注于领域的CRISPR筛查验证RMS肿瘤保留 对PAX融合的强烈上瘾，但仍有直接或间接靶向这种“无法下药的”蛋白质的策略 都还没有成功。这一努力的一个障碍是我们对上游和 支持PAX-Fusion功能的下游因素，我们试图通过研究解决这一问题 在这里求婚。通过对去除PAX融合的RMS细胞系进行深度分子图谱分析，我们最近 开发了与基于流式细胞仪的测量和细胞分选兼容的报告。这就是现在 允许我们进行饱和的基因筛选来描绘PAX-融合途径的所有组件 疾病。在本研究的第一个目的中，我们将对内源性PAX融合进行CRISPR外显子扫描 这是我们之前开发的一种用于揭示癌症重要功能区域的分析方法 维持基因。这些实验将定义融合癌蛋白的关键亚区， 放松转录调控以维持肌肉分化的阻断。这项提案的第二个目标将利用 我们最近开发的Paralog结构域共靶向方法可以揭示所有关键基因，以及 多余的平行基因对，这对PAX融合执行其功能是关键的。这样做的最终目的是 该项目将确定在RMS细胞中抑制PAX融合表达的关键E3连接酶，其功能 可以被刺激来降解这种癌蛋白。这个为期两年的研究项目将采用最新的创新技术。 在基于CRISPR的遗传筛选中为RMS领域建立重要的资源；遗传基础 对PAX融合癌蛋白进行基于机制的研究，使其能够进行药理调节 带着治疗意图。

Project Abstract: Rhabdomyosarcoma is a highly metastatic soft tissue malignancy of childhood for which new therapies are desperately needed. The clinical management of RMS patients has been largely unchanged over the past three decades, and is currently limited to surgical resection, radiotherapy, and combination chemotherapy. In this context, a mechanism-based targeted therapy would have potential for a transformative impact on RMS patient outcomes. The most common genetic event in RMS pathogenesis is a chromosomal rearrangement that produces the PAX-fusion oncoprotein, which is a chimeric transcription factor that deregulates chromatin and transcription to promote transformation. Our domain-focused CRISPR screens validate that RMS tumors retain a powerful addiction to the PAX-fusion, yet strategies for direct or indirect targeting of this ‘undruggable’ protein have yet to be successful. One obstacle in this endeavor is our incomplete understanding of the upstream and downstream factors that support the function of the PAX-fusion, which we seek to address with the research proposed here. Through deep molecular profiling of RMS cell lines depleted of the PAX-fusion, we have recently developed reporters which are compatible with flow cytometry-based measurements and cell sorting. This now allows us to perform saturating genetic screens to delineate all components of the PAX-fusion pathway in this disease. In the first aim of this study, we will perform CRISPR exon-scanning of the endogenous PAX-fusion locus, which is an assay we previously developed for exposing functionally important domains of cancer maintenance genes. These experiments will define the critical subregions of the fusion oncoprotein that deregulate transcription to sustain the block in myo-differentiation. The second aim of this proposal will leverage our recently developed paralog domain co-targeting methodology to expose all of the critical genes, and redundant paralogous gene pairs, that are critical for the PAX-fusion to carry out its function. The final aim of this project will identify the critical E3 ligase that acts to restrain PAX-fusion expression in RMS cells, whose function could be stimulated to degrade this oncoprotein. This two-year research project will employ the latest innovations in CRISPR-based genetic screening to establish an important resource for the RMS field; a genetic foundation for mechanism-based research of the PAX-fusion oncoprotein that will enable its pharmacological modulation with therapeutic intent.