Genome-wide synthetic lethal screening for vulnerabilities in a cell model of succinate dehydrogenase-loss paraganglioma

全基因组合成致死筛查琥珀酸脱氢酶缺失副神经节瘤细胞模型中的漏洞

• 批准号: 10572019
• 负责人: LOUIS JAMES MAHER
• 金额: $ 22.3万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-08 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10572019
• 关键词: Address; Allografting; Alzheimer' s Disease; Automobile Driving; Bar Codes; Breast Cancer Cell; CRISPR interference; CRISPR screen; Candidate Disease Gene; Cell Line; Cell model; Cells; Chromaffin Cells; Citric Acid Cycle; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Data Analyses; Defect; Diabetes Mellitus; Dioxygenases; Disease; Electron Transport; Enzymes; Estrogen Receptor alpha; Estrogen Receptor beta; Family; Gastrointestinal Stromal Tumors; Gene Expression; Gene Targeting; Genes; Genomic DNA; Growth; Guide RNA; Heart Diseases; Hereditary Paraganglioma; Histones; Hypoxia; Inherited; Lethal Genes; Link; Malignant Neoplasms; Measures; Metabolic; Methods; Mitochondria; Modeling; Molecular; Monitor; Mus; Mutation; Paraganglioma; Pathologic; Pathway interactions; Patients; Pheochromocytoma; Positioning Attribute; Pre-Clinical Model; RNA library; Recipe; Renal carcinoma; Resistance; Running; Signal Transduction; Succinate Dehydrogenase; Succinates; Synthetic Genes; Testing; Therapeutic; Time; Tissues; Transcription Repressor; Tumor Suppressor Genes; Validation; Work; candidate identification; cell growth; cell type; clinical application; deep sequencing; demethylation; experimental study; gene repression; genome-wide; hormone therapy; insight; knock-down; molecular pathology; neoplastic cell; neuroendocrine cancer; novel; novel therapeutics; promoter; rare cancer; screening; targeted treatment; tumor; tumorigenesis

Abstract: Genome-wide synthetic lethal screening for vulnerabilities in a cell model of succinate dehydrogenase-loss paraganglioma This proposal focuses on the molecular pathology of familial paraganglioma (PGL). Paradoxically, this remarkable neuroendocrine cancer is caused by inherited mutations that inactivate succinate dehydrogenase (SDH), an enzyme of the mitochondrial tricarboxylic acid (TCA) cycle. A fundamental scientific understanding of the molecular basis of this tumor is lacking, limiting powerful approaches that might be identified to exploit unique vulnerabilities due to the fundamental metabolic defect in this cancer. The links between SDH loss and tissue-specific tumorigenesis remain unknown, and conventional preclinical models have been unavailable. The current hypothesis for PGL tumorigenesis invokes loss or inactivation of both parental copies of any of the four SDH subunit genes (A-D). The subsequent accumulation of succinate competitively inhibits the activities of several dioxygenase enzymes that normally suppress hypoxic signaling and demethylate histones and DNA. Despite this general mechanistic model, other mechanisms are possible and it is unclear what unique vulnerabilities may be present in SDH-loss cells that could permit targeted therapies. We hypothesize that an unbiased genome-wide lentiviral CRISPR screen will identify genes whose loss displays synthetic lethality with SDH loss. This hypothesis is supported by previous successful work in our labs identifying genes whose loss confers resistance to multiple forms of endocrine therapy in ERα+ and ERβ+ breast cancer cells. Important new PGL cell models have become available in the form of Sdhblox/lox and Sdhb-/- immortalized mouse chromaffin cells (imCC). This positions us ideally to conduct and analyze an unbiased genome-wide synthetic lethal screen using an available CRISPR single guide RNA (sgRNA) library. Aim 1 will undertake a paired unbiased lentiviral CRISPR screen in matched normal and SDH-loss imCC lines. Aim 2 will complete data analysis to identify candidate synthetic lethal genes. Aim 3 will complete independent validation of representative synthetic lethal genes and pathways. Finally, Aim 4 will implement a mouse allograft tumor model to monitor selective inhibitory effects of nominated target gene knockdown in Sdhb-/- vs. Sdhblox/lox imCC. This project is unique in being the first unbiased synthetic lethal screen addressing the unmet cancer needs of SDH-loss familial PGL patients. Because SDH genes are tumor suppressors in other cancers, including gastrointestinal stromal tumor (GIST) and some kidney cancers, identifying potential vulnerabilities in SDH-loss cells may be broadly applicable clinically.

摘要：全基因组的合成致死性筛查对琥珀酸酯细胞模型中的脆弱性 脱氢酶 - 降低paraganglioma 该提议侧重于家族性旁帕氏瘤（PGL）的分子病理学。矛盾的是，这 显着的神经内分泌癌是由遗传突变引起的 脱氢酶（SDH），线粒体三核酸（TCA）周期的酶。基本科学 缺乏对该肿瘤的分子基础的理解，限制了可能是强大的方法 由于该癌症的基本代谢缺陷，因此被确定可利用独特的漏洞。链接 SDH损失与组织特异性肿瘤发生之间的发生尚不清楚，并且常规临床前 模型不可用。 PGL肿瘤发生的当前假设引起了 四个SDH亚基基因中的任何一个父母的副本（A-D）。随后的琥珀酸酯积累 竞争性抑制通常抑制低氧信号传导的几种二氧酶的活性 和脱甲基元素组蛋白和DNA。尽管采用了这种通用的机械模型，但其他机制还是可能的 目前尚不清楚SDH损坏单元中可能存在哪些独特漏洞 疗法。我们假设公正的全基因组慢病毒CRISPR筛选将识别基因 其损失表现出与SDH损失的合成致死性。该假设得到以前的成功的支持 在我们的实验室中工作，识别其对多种形式内分泌治疗的损失贡献的基因 ERα+和ERβ+乳腺癌细胞。重要的新PGL细胞模型已以 SDHBLOX/LOX和SDHB - / - 永生的小鼠铬蛋白细胞（IMCC）。这是我们理想情况下的定位和 使用可用的CRISPR单引导RNA分析无偏基因组的合成致死筛查 （SGRNA）库。 AIM 1将在匹配的正常和 SDH-loss IMCC线路。 AIM 2将完成数据分析以识别候选合成致死基因。目标3意志 完全验证代表性的合成致死基因和途径。最后，AIM 4将 实施小鼠同类肿瘤模型，以监测指定靶基因的选择性抑制作用 SDHB中的敲低 - / - 与SDHBLOX/LOX IMCC。这个项目是第一个公正的合成的独特之处 致命的筛查解决了SDH-LOSS家族PGL患者未获得的癌症需求。因为SDH 基因是其他癌症中的肿瘤补充剂，包括胃肠道肿瘤（GIST）和一些 肾脏癌症，鉴定SDH损伤细胞中潜在的脆弱性可能在临床上广泛适用。