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，目前正在开发新的治疗方法 迫切需要。过去三年来，RMS 患者的临床管理基本没有变化 几十年来，目前仅限于手术切除、放疗和联合化疗。在这个 背景下，基于机制的靶向治疗将有可能对 RMS 患者产生变革性影响 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，可以解除染色质和 转录以促进转化。我们以领域为中心的 CRISPR 筛选验证了 RMS 肿瘤保留 对 PAX 融合的强烈成瘾，但直接或间接靶向这种“不可成药”蛋白质的策略 尚未成功。这一努力的一个障碍是我们对上游和下游的不完全了解。 支持 PAX-fusion 功能的下游因素，我们寻求通过研究解决这些问题 在这里提出。通过对 PAX 融合耗尽的 RMS 细胞系进行深入的分子分析，我们最近发现 开发了与基于流式细胞术的测量和细胞分选兼容的报告器。现在这个 使我们能够进行饱和遗传筛选，以描绘出 PAX 融合途径的所有组成部分 疾病。在本研究的第一个目标中，我们将对内源性 PAX 融合进行 CRISPR 外显子扫描 位点，这是我们之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。这些实验将定义融合癌蛋白的关键亚区域 解除转录调控以维持肌分化的阻断。该提案的第二个目标将利用 我们最近开发了旁系同源域共同靶向方法来暴露所有关键基因，以及 冗余的旁系同源基因对，对于 PAX 融合发挥其功能至关重要。此次活动的最终目的 该项目将鉴定关键的 E3 连接酶，该酶可抑制 RMS 细胞中的 PAX 融合表达，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 基于 CRISPR 的基因筛选，为 RMS 领域建立重要资源；遗传基础 用于 PAX 融合癌蛋白的基于机制的研究，这将使其药理调节成为可能 具有治疗目的。

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.