Elucidating the function of PAX3-FOXO1 in rhabdomyosarcoma with molecular reporters and next-generation genome editing

利用分子报告基因和下一代基因组编辑阐明 PAX3-FOXO1 在横纹肌肉瘤中的功能

• 批准号: 10064134
• 负责人: CHRISTOPHER VAKOC
• 金额: $ 22.44万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-02 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064134
• 关键词: Address; Biological Assay; CRISPR screen; Categories; Cell Line; Cell Separation; Cells; Chemicals; Chromatin; Chromosomal Rearrangement; Clinical Management; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Combination Drug Therapy; Complement; Complementary DNA; Complex; DNA Binding Domain; Degradation Pathway; Dependence; Development; Disease; Dropout; Enzymes; Epigenetic Process; Event; Excision; Exons; Exposure to; Expression Library; FOXO1A gene; Flow Cytometry; Fluorescence; Follow-Up Studies; Fostering; Foundations; Funding; Fusion Oncogene Proteins; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Growth; Hydrophobicity; Intervention; Knock-in; Knock-out; Laboratories; Libraries; Maintenance; Malignant Childhood Neoplasm; Malignant Neoplasms; Maps; Measurement; Mediating; Methodology; Modernization; Molecular; Molecular Profiling; Muscle; Oncogenes; Oncology; Oncoproteins; Operative Surgical Procedures; PAX3 gene; Pathogenesis; Pathway interactions; Patient-Focused Outcomes; Patients; Pediatric Neoplasm; Pharmacology; Phenocopy; Phenotype; Point Mutation; Positioning Attribute; Proteins; Radiation therapy; Reporter; Reporting; Research; Research Personnel; Research Project Grants; Resolution; Resources; Rhabdomyosarcoma; Scanning; Structure; Surface; Techniques; Technology; Testing; Therapeutic; Time; Undifferentiated; Withdrawal; addiction; base; cofactor; drug discovery; effective therapy; experience; experimental study; gene complementation; gene function; gene induction; genetic approach; genetic resource; genome editing; genome-wide; innovation; logarithm; multicatalytic endopeptidase complex; new therapeutic target; next generation; novel therapeutics; paralogous gene; prevent; response; screening; soft tissue; targeted treatment; therapeutic target; therapy development; transcription factor; tumor; ubiquitin-protein ligase

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.

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