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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：PAX 3-FOXO 1相互作用组的定义和靶向腺泡状横纹肌肉瘤（ARMS）是一种由PAX 3-FOXO 1融合引起的致命的儿童恶性肿瘤癌蛋白虽然遗传学方法已经严格验证了这种癌蛋白作为治疗靶点，由于缺乏抑制PAX 3-FOXO 1功能的化合物，因此获得了“不可用药”的绰号。虽然 “不可药物化的”蛋白质可以被靶向，这样做需要彻底了解如何蛋白质的功能。已经确定PAX 3-FOXO 1通过以下途径发挥其转化活性：功能结构域和与其他细胞蛋白质的物理相互作用，这些蛋白质要么作为调节剂，要么作为共监管部门事实上，该团队的成员先前发现CHD 4通过以下方式共同调节PAX 3-FOXO 1功能：通过在染色质邻域中的共定位与癌蛋白间接相关。总体目标 FusOnc 2中心的目标是通过以下方式提高PAX 3-FOXO 1融合蛋白在ARMS中的治疗易处理性：全面鉴定PAX 3-FOXO 1的可药用辅助调节剂、调节剂和内在活性。该项目的目标是系统地鉴定致癌基因的治疗上可利用的成分， PAX 3-FOXO 1“相互作用组”-与PAX 3-FOXO 1相互作用的蛋白质目录，这些蛋白质也是其致癌活性。为了鉴定PAX 3-FOXO 1结构域和介导肿瘤发生的相互作用蛋白，本项目通过以下具体目标提出了一个逐步的、综合的方法：1）确定使用BirA邻近标记的功能性PAX 3-FOXO 1的差异相互作用组; 2）定位PAX 3-FOXO 1 使用饱和诱变进行氨基酸解析的功能结构域; 3）定义功能和组合结构域， PAX 3-F0 X 01相互作用组内的依赖性; 4）细胞内的凭证相互作用组依赖性， PAX 3-FOXO 1 ARMS动物模型。目的1中定义的蛋白质相互作用组将用于产生单- 和双靶向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的致癌活性所必需的结构域和氨基酸残基，这是一项成就，在整个中心进行利用，以告知项目1中涉及PAX 3-FOXO 1稳定性和优先级的区域项目3中的化学探针，并完善相互作用的蛋白质介导的PAX 3-FOXO 1转化，项目目标3。目标3中出现的最佳候选人将被提名在目标4中进行深入分析使用已建立的细胞和动物模型来确定近期和长期发展的有效目标，靶向关键相互作用蛋白本身或其与PAX 3-FOXO 1相互作用的药物。