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: 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
• 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

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.

项目摘要 靶向治疗已经彻底改变了癌症治疗，并正在成为标准的护理， 细胞毒化疗在肺癌中，大约50%的肿瘤在基因组中含有可药用突变。 尽管靶向治疗对EGFR和ALK等基因的治疗非常有效，但许多靶向治疗在降低肿瘤负荷方面非常有效， 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RIT 1的扩增，并且RIT 1突变不与其他典型驱动因子共同发生 突变。正因为如此，有一个主要的未满足的临床需要，以确定有效的靶向治疗的患者 RIT 1驱动的疾病。 我的职业目标是成为一名翻译研究科学家，专注于发现新的治疗方法。 癌症患者的选择。缺乏治疗RIT 1突变型肺癌的治疗策略， 我有机会建立技能，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。这个动机把我带到了我的论文实验室，在那里，伯杰博士开发了一种全基因组CRISPR， 在人RIT 1突变型肺癌细胞中的筛选测定。我帮助分析了CRISPR筛选数据， 鉴定了去泛素化酶（DUB）USP 9 X和E3连接酶COP 1作为RIT 1功能的关键调节因子。验证 实验证实，USP 9 X的个体缺失逆转了RIT 1诱导的肺癌细胞存活， COP 1的缺失维持了RIT 1驱动的耐药性。CRISPR筛选的结果提供了严格的、关键的 支持这个项目。最近的研究表明，RIT 1的蛋白质丰度对于其生物学功能的实现是重要的。 致癌功能;然而，确切的DUBs和E3连接酶参与调节突变RIT 1蛋白 丰度尚未完全阐明。在这个问题的驱使下，我开始进行实验，以证实基因 USP 9 X的敲除降低了RIT 1的丰度和稳定性。我还发现药理学上 体外抑制USP 9 X会降低RIT 1蛋白丰度，初步体内实验显示， USP 9 X缺失消除了RIT 1驱动的异种移植肿瘤形成。此外，我们合作者的工作 证明RIT 1与USP 9 X和COP 1发生物理相互作用。总之，这些数据提出了一个监管 由USP 9 X和COP 1介导的RIT 1蛋白丰度轴。我假设USP 9 X去泛素化 并稳定RIT 1，COP 1抵消了这种调节。USP 9 X的药理学抑制可以 因此特异性靶向致癌RIT 1。最终，这项工作将揭示RIT 1蛋白的新机制 监管，并可能揭示USP 9 X抑制剂的效用，以解决患者的主要未满足的临床需求 患有RIT 1突变或RIT 1扩增的疾病。这个项目将建立在我的基因组学和生物化学的技能， 用体内鼠系统提供必要的训练。我将能够在法官席上把我的发现翻译成 小鼠模型，从而扩大了我在转化研究方面的技能，并促进了我的职业目标， 成为一名资深科学家在一个行业设置，致力于开发新的和更好的癌症治疗。