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：
10902753
负责人：
Corinne Mary Linardic
金额：
$ 15.77万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10902753
关键词：
Achievement Alveolar Rhabdomyosarcoma Amino Acids Animal Model Base Sequence Binding Binding Proteins Biotin CHD4 gene CRISPR/Cas technology Catalogs Cell model Characteristics Chemicals Chimeric Proteins Chromatin Co-Immunoprecipitations Credentialing DNA Binding Dependence Drug Targeting Emerging Technologies FOXO1A gene Face Fusion Oncogene Proteins Future Genes Goals Immunodeficient Mouse In Vitro Label Libraries Malignant Childhood Neoplasm Maps Mediating Modeling Molecular Mutagenesis Neighborhoods Oncogenes Oncogenic Oncoproteins PAX3 gene Patients Pharmaceutical Preparations Portraits Post-Translational Protein Processing Proteins Resolution Signal Transduction Techniques Technology Therapeutic Validation Work combinatorial design 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 pharmacologic protein function protein protein interaction screening therapeutic development therapeutic target tool transmission process 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：定义和瞄准 PAX3-FOXO1 相互作用组腺泡状横纹肌肉瘤 (ARMS) 是一种由 PAX3-FOXO1 融合驱动的致命儿童恶性肿瘤虽然基因方法已经严格验证了这种癌蛋白作为治疗靶点，缺乏抑制 PAX3-FOXO1 功能的化合物使其赢得了“不可成药”的绰号。 “不可成药”的蛋白质可以成为药理学目标，这样做需要彻底了解如何众所周知，PAX3-FOXO1 通过以下方式发挥其转化活性。功能域和与其他细胞蛋白质的物理相互作用，这些蛋白质要么充当调节剂，要么充当协同作用事实上，该团队的成员之前发现 CHD4 通过以下方式共同调节 PAX3-FOXO1 功能。通过染色质邻域的共定位与癌蛋白间接关联总体目标。该 FusOnc2 中心的目标是通过以下方式提高 PAX3-FOXO1 融合蛋白在 ARMS 中的治疗可处理性：全面鉴定 PAX3-FOXO1 的可药物协同调节剂、调节剂和内在活性。该项目的目标是系统地识别致癌物质的治疗上可利用的成分 PAX3-FOXO1“相互作用组”——与 PAX3-FOXO1 相互作用的蛋白质目录，这些蛋白质对于鉴定介导肿瘤发生的 PAX3-FOXO1 结构域和相互作用蛋白，该项目通过以下具体目标提出了一种逐步、全面的方法： 1) 定义使用 BirA 邻近标记的功能性 PAX3-FOXO1 的差异相互作用组；2) Map PAX3-FOXO1 使用饱和诱变在氨基酸分辨率上确定功能域；3) 定义功能和组合 4) 细胞和细胞中的凭证相互作用组依赖性 PAX3-FOXO1 ARMS 的动物模型。目标 1 中定义的蛋白质相互作用组将用于生成单- 目标 3 中的双靶向 CRISPR/Cas9 功能丧失文库。目标 2 中的工作将揭示关键功能融合癌蛋白致癌活性所必需的结构域和氨基酸残基，一项成就在整个中心内用于通知涉及项目 1 中 PAX3-FOXO1 稳定性和优先级的区域项目 3 中相互作用的化学探针的研究，并精炼介导 PAX3-FOXO1 转化的蛋白质该项目的目标 3 然后将提名目标 3 中出现的最佳候选人，以便在目标 4 中进行深入分析。使用已建立的细胞和动物模型来定义近期和长期开发的经过验证的目标靶向关键相互作用蛋白本身药物或其与 PAX3-FOXO1 的相互作用。