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.

翻译后摘要：全基因组的合成致死筛选的漏洞，在细胞模型的琥珀酸 副神经节瘤 该建议的重点是家族性副神经节瘤（PGL）的分子病理学。巧合的是， 一种引人注目的神经内分泌癌是由遗传突变引起的， 脱氢酶（SDH）是线粒体三羧酸（TCA）循环的酶。的一个基本的科学 缺乏对这种肿瘤的分子基础的理解，限制了可能是有效的方法。 由于这种癌症的基本代谢缺陷，确定利用独特的弱点。的联系 SDH损失和组织特异性肿瘤发生之间的关系仍然未知， 模型已经不可用。目前PGL肿瘤发生的假设是， 四种SDH亚基基因（A-D）中任一种的双亲拷贝。随后琥珀酸的积累 竞争性抑制通常抑制缺氧信号传导的几种双加氧酶的活性 去甲基化组蛋白和DNA尽管有这种一般的机制模型，但其他机制也是可能的 目前还不清楚SDH丢失细胞中可能存在哪些独特的漏洞， 治疗我们假设，无偏倚的全基因组慢病毒CRISPR筛选将识别基因 其损失表现为SDH损失的综合致死性。这一假设得到了先前成功的支持。 在我们的实验室工作，确定基因的损失赋予抵抗多种形式的内分泌治疗， ERα+和ERβ+乳腺癌细胞。重要的新型PGL电池模型以以下形式可用： Sdhblox/lox和Sdhb-/-永生化小鼠嗜铬细胞（imCC）。这使我们能够理想地进行， 使用可用的CRISPR单向导RNA分析无偏倚的全基因组合成致死筛选 （sgRNA）文库。目标1将在匹配的正常和非正常人群中进行配对的无偏慢病毒CRISPR筛选。 SDH-损失IMCC线。目标2将完成数据分析，以确定候选的合成致死基因。目标3将 代表性合成致死基因和途径的完整独立验证。最后，Aim 4将 建立小鼠同种异体移植肿瘤模型以监测指定靶基因选择性抑制效应 在Sdhb-/- vs. Sdhblox/lox imCC中的敲低。这个项目的独特之处在于它是第一个 致死筛选解决了SDH缺失家族性PGL患者未满足的癌症需求。因为SDH 基因是其他癌症的肿瘤抑制因子，包括胃肠道间质瘤（GIST）和一些癌症。 肾癌，鉴定SDH损失细胞中的潜在脆弱性可能在临床上广泛适用。