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

摘要：在琥珀酸细胞模型中进行全基因组合成致死性筛选 脱氢酶缺失型副神经节瘤 本研究的重点是家族性副神经节瘤的分子病理学。矛盾的是，这 显著的神经内分泌癌是由使琥珀酸失活的遗传突变引起的 脱氢酶(SDH)，线粒体三羧酸(TCA)循环的一种酶。一项基础科学 对这种肿瘤的分子基础缺乏了解，限制了可能的有效方法 由于这种癌症的基本代谢缺陷，被确定为利用独特的脆弱性。这些链接 SDH缺失和组织特异性肿瘤发生之间的关系尚不清楚，而传统的临床前研究 型号一直无法获得。目前关于PGL肿瘤发生的假说涉及到基因缺失或失活。 四个SDH亚单位基因(A-D)中任何一个的双亲拷贝。随之而来的琥珀酸积累 竞争性地抑制几种通常抑制低氧信号的双加氧酶的活性 去甲基化组蛋白和DNA。尽管有这种一般的机械论模型，其他机制也是可能的。 目前还不清楚SDH丢失信元中可能存在哪些独特的漏洞，从而允许有针对性的 治疗。我们假设，一个没有偏见的全基因组慢病毒CRISPR筛查将识别基因 其损失表现为SDH损失的综合致命性。这一假设得到了以前成功的支持 我们实验室的工作是识别基因的丢失会导致对多种形式的内分泌治疗产生抵抗 ERα+和ERβ+乳腺癌细胞。重要的新的PGL细胞模型已经以 Sdhblox/lox和SDHB-/-永生化小鼠嗜铬细胞(IMCC)。这使我们在理想的情况下进行和 使用可用的CRISPR单引导RNA分析无偏见的全基因组合成致死筛查 (SgRNA)文库。目标1将进行配对的无偏慢病毒CRISPR筛查，在匹配的正常和 SDH-损耗IMCC线路。Aim 2将完成数据分析，以确定候选合成致死基因。目标3将 完成代表性合成致死基因和致死途径的独立验证。最后，目标4将 建立小鼠同种异体移植瘤模型监测指定靶基因的选择性抑制作用 击倒在SDHB-/-与Sdhblox/lox IMCC。这个项目的独特之处在于它是第一个无偏见的合成 致命筛查解决SDH缺失家族性PGL患者未得到满足的癌症需求。因为SDH 基因在其他癌症中是肿瘤抑制基因，包括胃肠道间质瘤(GIST)和一些 肾癌，识别SDH丢失细胞的潜在脆弱性，可能在临床上得到广泛应用。

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.