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的儿童软组织恶性肿瘤， 迫切需要的。在过去的三年里，RMS患者的临床管理基本上没有变化。 几十年来，并且目前仅限于手术切除、放疗和联合化疗。在这 背景下，基于机制的靶向治疗可能对RMS患者产生变革性影响 成果。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，可解除染色质的调节， 转录以促进转化。我们专注于领域的CRISPR筛选验证了RMS肿瘤保留了 对PAX融合的强烈成瘾，但直接或间接靶向这种“不可药物化”蛋白的策略 还没有成功。这一奋进的一个障碍是我们对上游的不完全理解， 支持PAX融合功能的下游因素，我们寻求通过研究解决这一问题。 在这里提出。通过对PAX融合缺失的RMS细胞系进行深度分子分析，我们最近 开发了与基于流式细胞术的测量和细胞分选兼容的报告基因。这个现在 允许我们进行饱和的遗传筛选，以描绘PAX融合途径的所有组成部分， 疾病在本研究的第一个目的中，我们将对内源性PAX融合基因进行CRISPR外显子扫描。 基因座，这是我们以前开发的一种检测方法，用于暴露癌症的重要功能域 维持基因这些实验将确定融合癌蛋白的关键亚区， 去调节转录以维持肌分化的阻断。该提案的第二个目标将利用 我们最近开发的paradox结构域共靶向方法，以暴露所有关键基因， 冗余的旁系同源基因对，其对于PAX融合体执行其功能至关重要。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领域建立了重要的资源;遗传基础 用于PAX融合癌蛋白的机制研究，以实现其药理学调节 有治疗的意图。

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.