Defining and targeting the PAX3-FOXO1 interactome

定义和定位 PAX3-FOXO1 相互作用组

基本信息

批准号：
10680800
负责人：
Corinne Mary Linardic
金额：
$ 17.35万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10680800
关键词：
Achievement Alveolar Rhabdomyosarcoma Amino Acids Animal Model Binding Binding Proteins Biotin CHD4 gene CRISPR/Cas technology Catalogs Cell model Characteristics Chemicals Chimeric Proteins Chromatin Co-Immunoprecipitations DNA Binding Dependence Drug Targeting Emerging Technologies FOXO1A gene Face Fusion Oncogene Proteins Future Genes Goals Immunodeficient Mouse In Vitro Label Libraries Ligands Malignant Childhood Neoplasm Maps Mediating Molecular Mutagenesis Neighborhoods Oncogenic Oncoproteins PAX3 gene Patients Pharmaceutical Preparations Pharmacology Portraits Post-Translational Protein Processing Proteins Resolution Signal Transduction Techniques Technology Therapeutic Validation Work combinatorial drug development genetic approach genetic manipulation in vivo insight knockout gene lead candidate loss of function member mutant mutation screening neoplastic cell patient derived xenograft model protein function protein protein interaction screening therapeutic development therapeutic target tool tumorigenesis

项目摘要

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.

摘要 – 项目 2：定义和靶向 PAX3-FOXO1 相互作用组肺泡横纹肌肉瘤 (ARMS) 是一种由 PAX3-FOXO1 融合癌蛋白驱动的致命儿童恶性肿瘤，虽然遗传方法已严格验证这种癌蛋白作为治疗靶点，但缺乏抑制化合物。 PAX3-FOXO1功能为其赢得了“不可成药”的绰号。 “不可成药”的蛋白质可以被药理学靶向，这样做需要彻底了解蛋白质的功能，众所周知，PAX3-FOXO1 通过功能域和与其他细胞蛋白质（充当调节剂或蛋白质）的物理相互作用来发挥其转化活性。事实上，该团队的成员之前发现，CHD4 通过在染色质邻域中的共定位与癌蛋白间接关联来共同调节 PAX3-FOXO1 功能。总体目标 FusOnc2 中心的目标是通过全面鉴定 PAX3-FOXO1 的可药物辅助调节剂、调节剂和内在活性来提高 ARMS 中 PAX3-FOXO1 融合蛋白的治疗可处理性。该项目的目标是系统地鉴定 PAX3-FOXO1 的治疗上可利用的成分。致癌的 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至关重要。为了识别介导肿瘤发生的 PAX3-FOXO1 结构域和相互作用蛋白质，该项目提出了一种逐步的综合方法。具体目标如下： 1) 使用 BirA 邻近标记定义功能性 PAX3-FOXO1 的差异相互作用组；2) 在氨基处绘制 PAX3-FOXO1 功能域图使用饱和诱变进行酸解析；3) 定义功能和组合； PAX3-FOXO1 相互作用组内的依赖性；4) PAX3-FOXO1 ARMS 细胞和动物模型中的可信相互作用组依赖性。目标 1 中定义的蛋白质相互作用组将用于生成单靶向和双靶向 CRISPR/Cas9 功能丧失。 Aim 3 中的库。Aim 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，并完善介导 PAX3 的相互作用蛋白-FOXO1转化该项目的目标 3。目标 3 中出现的最佳候选者将被提名在目标 4 中使用已建立的细胞和动物模型进行深入分析，以确定针对关键相互作用蛋白本身的药物的近期和长期开发的经过验证的目标。或它们与 PAX3-FOXO1 的相互作用。