Elucidating how a USP9X-COP1 axis regulates RIT1 protein abundance and reveals druggable targets in lung adenocarcinoma

阐明 USP9X-COP1 轴如何调节 RIT1 蛋白丰度并揭示肺腺癌中的药物靶标

基本信息

批准号：
10681250
负责人：
Amanda Riley
金额：
$ 4.26万
依托单位：
FRED HUTCHINSON CANCER CENTER
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 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10681250
关键词：
Address Adenocarcinoma Cell Affect Anchorage-Independent Growth Automobile Driving Binding Biochemistry Biological Assay Biology CRISPR screen Cancer Patient Cell Proliferation Cell Survival Cells Cessation of life Clinical Clinical Trials Co-Immunoprecipitations Cytotoxic Chemotherapy Data Deubiquitination Disease Drug resistance Epidermal Growth Factor Receptor Future Genes Genetic Genomics Goals Human In Vitro Individual Industry Knock-out Lung Adenocarcinoma Lysine Malignant neoplasm of lung Maps Mediating Medical Modeling Molecular Monomeric GTP-Binding Proteins Motivation Mus Mutation Oncogenic Pathogenicity Patients Phenotype Physiological Pre-Clinical Model Protein Family Proteins Reaction Recurrence Regulation Research Scientist Senior Scientist Signal Pathway Somatic Mutation Structure System Techniques Testing Therapeutic Tissues Training Translating Translational Research Tumor Burden Ubiquitination Validation Work cancer therapy career cell growth driver mutation druggable target effective therapy efficacy evaluation experimental study genome sequencing genome-wide improved in vivo inhibitor lung cancer cell metaplastic cell transformation mouse model mutant novel novel therapeutics personalized approach pharmacologic preclinical efficacy ras Proteins response skills small molecule standard care standard of care targeted treatment treatment strategy tumor tumor growth tumor xenograft ubiquitin isopeptidase ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY Targeted therapies have revolutionized cancer treatment and are becoming standard of care over cytotoxic chemotherapy. In lung cancer, where approximately 50% of tumors harbor druggable mutations in genes such as EGFR and ALK, targeted therapies are highly effective at reducing tumor burden; however, many mutations are not clinically actionable. Up to 13% of lung adenocarcinoma tumors are driven by mutation or amplification of the RAS-family protein RIT1, and RIT1 mutations do not co-occur with other canonical driver mutations. Because of this, there is a major unmet clinical need to identify effective targeted therapies for patients with RIT1-driven diseases. My career goal is to become a translational research scientist focused on identifying novel therapeutic options for cancer patients. The lack of therapeutic strategies for the treatment of RIT1-mutant lung cancer offers opportunities for me to build the skills, techniques, and expertise to address this problem and move towards my career objectives. This motivation led me to my thesis lab, where Dr. Berger developed a genome-wide CRISPR screening assay in human RIT1-mutant lung cancer cells. I helped analyze the CRISPR screen data and identified the deubiquitinase (DUB) USP9X and the E3 ligase COP1 as key regulators of RIT1 function. Validation experiments confirmed that individual loss of USP9X reverses RIT1-induced cell survival in lung cancer while loss of COP1 maintains RIT1-driven drug resistance. The results of the CRISPR screen provide rigorous, key support for this proposed project. Recent work suggests that the protein abundance of RIT1 is important for its oncogenic function; however, the exact DUBs and E3 ligases involved in regulating mutant RIT1 protein abundance have yet to be fully elucidated. Driven by this question, I initiated experiments to confirm that genetic knockout of USP9X decreases the abundance and stability of RIT1. I have also found that pharmacological inhibition of USP9X in vitro reduces RIT1 protein abundance, and preliminary in vivo experiments revealed that USP9X loss abrogates RIT1-driven xenograft tumor formation. Additionally, work from our collaborators demonstrates that RIT1 physically interacts with USP9X and COP1. Together, these data propose a regulatory axis of RIT1 protein abundance mediated by USP9X and COP1. I hypothesize that USP9X de-ubiquitinates and stabilizes RIT1 and that COP1 counteracts this regulation. Pharmacological inhibition of USP9X could therefore specifically target oncogenic RIT1. Ultimately, this work will reveal a novel mechanism of RIT1 protein regulation and could uncover the utility of USP9X inhibitors to address a major unmet clinical need for patients with RIT1-mutant or -amplified diseases. This project will build upon my skills in genomics and biochemistry while providing necessary training with in vivo murine systems. I will be able to translate my findings at the bench to mouse models, thereby expanding my skills in translational research and furthering my career objectives to become a senior scientist in an industry setting, working on developing new and better cancer treatments.

项目概要靶向治疗彻底改变了癌症治疗，并正在成为癌症治疗的标准细胞毒性化疗。在肺癌中，大约 50% 的肿瘤含有可药物突变 EGFR、ALK等基因，靶向治疗对于减轻肿瘤负荷非常有效；然而，许多突变在临床上不可行。高达 13% 的肺腺癌是由突变或 RAS 家族蛋白 RIT1 的扩增，并且 RIT1 突变不与其他典型驱动因素同时发生突变。因此，为患者找到有效的靶向治疗方案是一个未得到满足的重大临床需求患有 RIT1 驱动的疾病。我的职业目标是成为一名转化研究科学家，专注于识别新的治疗方法癌症患者的选择。缺乏治疗 RIT1 突变肺癌的治疗策略我有机会培养技能、技术和专业知识来解决这个问题并朝着我的目标迈进职业目标。这种动机引导我来到我的论文实验室，伯杰博士在那里开发了全基因组 CRISPR 人 RIT1 突变肺癌细胞的筛选试验。我帮助分析了 CRISPR 筛选数据并确定去泛素酶 (DUB) USP9X 和 E3 连接酶 COP1 是 RIT1 功能的关键调节因子。验证实验证实，USP9X 的个体缺失可逆转 RIT1 诱导的肺癌细胞存活，而 COP1 的丧失维持了 RIT1 驱动的耐药性。 CRISPR 筛选的结果提供了严格的、关键的支持该拟议项目。最近的研究表明 RIT1 的蛋白质丰度对于其发挥重要作用致癌功能；然而，参与调节突变 RIT1 蛋白的确切 DUB 和 E3 连接酶丰度尚未完全阐明。在这个问题的驱使下，我发起了实验来证实基因 USP9X 的敲除降低了 RIT1 的丰度和稳定性。我还发现药理作用体外抑制 USP9X 会降低 RIT1 蛋白丰度，初步体内实验表明 USP9X 缺失会消除 RIT1 驱动的异种移植肿瘤形成。此外，我们的合作者的工作表明 RIT1 与 USP9X 和 COP1 发生物理相互作用。这些数据共同提出了监管 USP9X 和 COP1 介导的 RIT1 蛋白丰度轴。我假设 USP9X 去泛素化并稳定 RIT1，而 COP1 抵消了这一调节。 USP9X 的药理学抑制作用因此专门针对致癌 RIT1。最终，这项工作将揭示RIT1蛋白的新机制监管并可能揭示 USP9X 抑制剂的效用，以解决患者未满足的主要临床需求患有 RIT1 突变或扩增疾病。该项目将建立在我在基因组学和生物化学方面的技能的基础上，同时提供体内小鼠系统的必要培训。我将能够将我在替补席上的发现转化为小鼠模型，从而扩展了我在转化研究方面的技能，并进一步实现了我的职业目标成为行业环境中的资深科学家，致力于开发新的、更好的癌症治疗方法。