Defining and targeting the PAX3-FOXO1 interactome

摘要-项目2：定义和靶向PAX3-FOXO1相互作用组泡状横纹肌肉瘤(ARMS)是由PAX3-FOXO1融合癌蛋白驱动的一种致命的儿童恶性肿瘤。虽然遗传方法已经严格验证了这种癌蛋白作为治疗靶点的有效性，但由于缺乏抑制PAX3-FOXO1功能的化合物，它获得了“无法下药”的绰号。虽然 “无法下药的”蛋白质可以是药理学靶向的，要做到这一点，需要彻底了解蛋白质的功能。已经证实，PAX3-FOXO1通过功能结构域和与其他细胞蛋白的物理相互作用来发挥其转化活性，这些蛋白既可以作为调节剂，也可以作为辅助调节因子。事实上，该团队的成员之前发现，CHD4通过在染色质附近的共同定位间接与癌蛋白相关联来共同调节PAX3-FOXO1的功能。首要目标是 该FusOnc2中心的目的是通过全面鉴定PAX3-FOXO1的可药物共调节因子、调节剂和内在活性，提高PAX3-FOXO1融合蛋白在ARM中的治疗可操作性。该项目的目标是系统地识别致癌物质中可用于治疗的成分 Pax3-FOXO1“相互作用组”--与PAX3-FOXO1相互作用的蛋白质目录，这些蛋白质对其致癌活性也是必不可少的。为了确定介导肿瘤发生的PAX3-FOXO1结构域和相互作用的蛋白质，本项目提出了一种循序渐进的综合方法，通过以下具体目标：1)使用BIRA邻近标记定义功能PAX3-FOXO1的差异交互作用组；2)使用饱和突变在氨基酸分辨率上映射PAX3-FOXO1功能结构域；3)定义功能和组合 PAX3-FOXO1相互作用组内的依赖关系；4)PAX3-FOXO1臂的细胞和动物模型中凭证相互作用组的依赖关系。目标1中定义的蛋白质相互作用组将用于在目标3中生成单靶向和双靶向CRISPR/Cas9功能丧失文库。在目标2中的工作将揭示关键功能 融合癌蛋白致癌活性所需的结构域和氨基酸残基，整个中心利用这一成果来告知项目1中涉及PAX3-FOXO1稳定性的区域和项目3中化学探针的优先顺序，并提炼介导PAX3-FOXO1转化的相互作用蛋白。 本项目的目标3。然后，从AIM 3中脱颖而出的最佳候选药物将被提名用于AIM 4的深入分析，使用已建立的细胞和动物模型来定义针对关键相互作用蛋白本身或其与PAX3-FOXO1相互作用的药物的近期和长期开发的有效目标。

ABSTRACT – Project 2: Defining and targeting the PAX3-FOXO1 interactome Alveolar rhabdomyosarcoma (ARMS) is a deadly childhood malignancy driven by the PAX3-FOXO1 fusion oncoprotein. While genetic approaches have rigorously validated this oncoprotein as a therapeutic target, the lack of compounds inhibiting PAX3-FOXO1 function has earned it the moniker of being “undruggable”. Although “undruggable” proteins can be pharmacologically targeted, doing so requires a thorough understanding of how the protein functions. It is well established that PAX3-FOXO1 exerts its transforming activities both through functional domains and physical interactions with other cellular proteins that either serve as modulators or co-regulators. Indeed, members of this team previously found that CHD4 co-regulates PAX3-FOXO1 function by indirectly associating with the oncoprotein via co-localization in chromatin neighborhoods. The overarching goal of this FusOnc2 Center is to advance the therapeutic tractability of the PAX3-FOXO1 fusion protein in ARMS by comprehensively identifying the druggable co-regulators, modulators, and intrinsic activities of PAX3-FOXO1. This Project’s objective is to systematically identify therapeutically exploitable components of the oncogenic PAX3-FOXO1 “interactome” – the catalog of proteins that interact with PAX3-FOXO1 that are also essential for its oncogenic activity. To identify the PAX3-FOXO1 domains and interacting proteins that mediate oncogenesis, this Project proposes a stepwise, comprehensive approach through the following Specific Aims: 1) Define the differential interactome of functional PAX3-FOXO1 using BirA proximity labeling; 2) Map PAX3-FOXO1 functional domains at amino acid resolution using saturation mutagenesis; 3) Define functional and combinatorial dependencies within the PAX3-FOXO1 interactome; 4) Credential interactome dependencies in cellular and animal models of PAX3-FOXO1 ARMS. The protein interactome defined in Aim 1 will be used to generate single-and dual-targeting CRISPR/Cas9 loss-of-function libraries in Aim 3. Work in Aim 2 will reveal the key functional domains and amino acid residues necessary for the oncogenic activity of the fusion oncoprotein, an achievement exploited throughout the Center to inform regions involved in PAX3-FOXO1 stability in Project 1 and prioritization of chemical probes in Project 3, and to refine the interacting proteins mediating PAX3-FOXO1 transformation in Aim 3 of this Project. Top candidates emerging from Aim 3 will then be nominated for in-depth analysis in Aim 4 using established cellular and animal models to define validated targets for near- and long-term development of drugs targeting either key interacting proteins themselves or their interactions with PAX3-FOXO1.