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

• 批准号: 10536485
• 负责人: Amanda Riley
• 金额: $ 4.16万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 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/10536485
• 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; Lead; Lung Adenocarcinoma; Lysine; Malignant neoplasm of lung; Maps; Mediating; Medical; Modeling; Molecular; Monomeric GTP-Binding Proteins; Motivation; Mus; Mutation; Oncogenic; Pathogenicity; Patients; Pharmacology; 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; base; cancer therapy; career; cell growth; driver mutation; druggable target; effective therapy; experimental study; genome sequencing; genome-wide; improved; in vivo; inhibitor; lung cancer cell; metaplastic cell transformation; mouse model; mutant; novel; novel therapeutics; personalized approach; preclinical efficacy; response; skills; small molecule; standard care; standard of care; targeted treatment; treatment strategy; tumor; tumor growth; tumor xenograft; ubiquitin-protein ligase

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.

项目总结 靶向疗法使癌症治疗发生了革命性变化，并正在成为治疗癌症的标准疗法。 细胞毒性化疗。在肺癌中，大约50%的肿瘤含有可药物突变的 如EGFR和ALK等基因，靶向治疗在减轻肿瘤负担方面非常有效；然而，许多 突变在临床上是不可操作的。高达13%的肺腺癌肿瘤是由突变或 Ras家族蛋白RIT1的扩增和RIT1突变不与其他典型驱动因素共存 突变。正因为如此，为患者确定有效的靶向治疗是一个尚未得到满足的主要临床需求。 患有由RIT1驱动的疾病。 我的职业目标是成为一名专注于识别新疗法的转化研究科学家 癌症患者的选择。缺乏治疗RIT1突变肺癌的治疗策略 我有机会培养解决这个问题的技能、技术和专业知识，并朝着我的 职业目标。这种动机将我带到了我的论文实验室，在那里，伯杰博士开发了一个全基因组的CRISPR 人RIT1突变肺癌细胞的筛选试验。我帮助分析了CRISPR的屏幕数据 DUB基因USP9X和E3连接酶COP1是RIT1功能的关键调节基因。验证 实验证实，USP9X的个体缺失逆转了RIT1诱导的肺癌细胞存活，而 COP1的缺失维持了RIT1驱动的耐药性。CRISPR筛查的结果提供了严格、关键的 支持这一拟议的项目。最近的研究表明，RIT1的蛋白丰度对其 致癌功能；然而，确切的DUBS和E3连接酶参与调节突变的RIT1蛋白 丰度还没有完全阐明。在这个问题的驱使下，我开始了实验，以证实基因 USP9X基因敲除降低了RIT1的丰度和稳定性。我还发现药理上的 体外抑制USP9X降低RIT1蛋白丰度，体内初步实验显示 USP9X缺失消除了RIT1驱动的异种移植瘤的形成。此外，我们的合作者的工作 演示了RIT1与USP9X和COP1的物理相互作用。总而言之，这些数据提出了一种监管 USP9X和COP1介导的RIT1蛋白丰度轴。我假设USP9X去泛素化 稳定RIT1，而COP1抵消了这一调控。USP9X的药理抑制作用可能 因此，特异性靶向致癌基因RIT1。最终，这项工作将揭示RIT1蛋白的一种新机制 并可能发现USP9X抑制剂的用途，以解决患者未得到满足的主要临床需求 患有RIT1突变或扩增的疾病。这个项目将建立在我在基因组学和生物化学方面的技能上，同时 利用体内小鼠系统提供必要的训练。我将能够将我在替补席上的发现转化为 鼠标模型，从而扩展了我在翻译研究方面的技能，并进一步推动了我的职业目标 成为行业中的资深科学家，致力于开发新的更好的癌症治疗方法。