Identification of enzymes inhibited by oncometabolites in Succinate Dehydrogenase mutant tumors

项目摘要 三羧酸循环（TCA循环，也称为Krebs循环）是真核生物中的基本过程。 细胞，作为ATP产生的来源，并在细胞代谢下产生还原当量。 有氧条件近年来，已经清楚的是，编码TCA循环中的酶的基因可以 可能是人类癌症的致病遗传病变，包括与肾脏疾病相关的遗传性肿瘤综合征。 癌症、副神经节瘤/嗜铬细胞瘤（PPGL）和胃肠道间质瘤（GIST）， 其他包括上皮性甲状腺癌。它们也在来自这些相同组织的散发性肿瘤中得到了很好的描述。 组织，以及其他癌症，包括急性髓性白血病（AML）和胶质母细胞瘤。 克雷布斯循环酶及其相关辅因子的突变被认为是通过肿瘤形成的原因。 过量代谢物积累的致癌作用（致癌代谢物）。这些中间代谢物通过以下方式起作用： 干扰需要代谢辅因子α-酮戊二酸（aKG，也称为 作为2-酮戊二酸酯）。人体内大约有70种aKG依赖性酶，它们在人体内发挥着多种作用。 基本的细胞功能，包括介导DNA，RNA和组蛋白的修饰。酶 这类蛋白还负责蛋白质（包括结构蛋白胶原）的氧化羟基化。 我们先前已经证明，使用甲状腺肿瘤的体外和体内模型， 琥珀酸脱氢酶D亚基（SDHD）引起指示癌症早期阶段的表型变化。 此外，这种遗传变化导致细胞获得干细胞样表型，如通过在细胞中表达干细胞样表型所证明的。 干细胞相关转录因子Nanog和Oct 4以及干细胞标记物乙醛的产生 脱氢酶（ALDH）。尽管已经提出了干扰aKG依赖性加氧酶的事实， 作为一种肿瘤机制，以前没有努力确定负责的家庭成员， 肿瘤的变化。为了填补这一知识空白，我们建议使用基于高通量CRISPR的 转录抑制筛选以鉴定其抑制导致重演的aKG依赖性酶 干细胞样表型。将使用细胞模型和计算机模拟相结合的方法对识别出的命中进行验证。 基于肿瘤的组学数据分析。本R 03试点提案的目标如下： 1)使用CRISPRi筛选来鉴定α-酮戊二酸（aKG）依赖性酶，其抑制 导致获得茎样表型 2)验证细胞系模型和人类肿瘤数据集中识别的酶 鉴定其功能受癌代谢物琥珀酸盐积累影响的酶， 为Sdhx和其他TCA循环相关疾病的分子机制提供了重要的新线索 突变，并将提供一个垫脚石，为未来的研究赠款，将允许详细划定 基因和/或蛋白质靶标，其驱动甲状腺和其他组织中的肿瘤形成（例如神经胶质瘤、白血病）。

PROJECT SUMMARY The tricarboxylic acid cycle (TCA cycle, also known as the Krebs cycle) is a fundamental process in eukaryotic cells, serving as the source for ATP generation and producing reducing equivalents for cell metabolism under aerobic conditions. In recent years, it has become clear that genes encoding the enzymes in the TCA cycle can be causative genetic lesions in human cancers, including in inherited tumor syndromes associated with renal cancer, paragangliomas/pheochromocytomas (PPGL), and Gastrointestinal Stromal Tumors (GISTs) among others, including epithelial thyroid cancer. They are also well described in sporadic tumors from these same tissues, as well as in a spectrum of other cancers, including acute myeloid leukemia (AML) and glioblastoma. Mutations in Krebs cycle enzymes and their related cofactors are thought to cause tumor formation through the oncogenic effects of excess metabolite accumulation (oncometabolites). These intermediate metabolites act by interference with the function of enzymes requiring the metabolic cofactor alpha-ketoglutarate (aKG, also known as 2- oxoglutarate). There are approximately 70 aKG-dependent enzymes in humans, and they perform a variety of essential cellular functions, including mediating modification of DNA, RNA, and histone proteins. Enzymes of this class are also responsible for oxidative hydroxylation of proteins, including the structural protein collagen. We have previously demonstrated using both in vitro and in vivo models for thyroid neoplasia that loss of the Succinate Dehydrogenase D subunit (SDHD) causes phenotypic changes indicative of early stages of cancer. Further, this genetic changes causes cells to gain a stem-like phenotype, as evidenced by expression of the stem cell associated transcription factors Nanog and Oct4 and production of the stem cell marker Aldehyde Dehydrogenase (ALDH). Despite the fact that interference with aKG-dependent oxygenases has been proposed as a neoplastic mechanism, no prior efforts have been made to identify family members which are responsible for the neoplastic change. To fill this knowledge gap, we propose to use a high throughput CRISPR-based transcriptional repression screen to identify aKG-dependent enzymes whose inhibition leads to a recapitulation of the stem-like phenotype. Identified hits will be validated using a combination of cellular models and in silico analysis of tumor-based omics data. The Aims for this R03 pilot proposal are as follows: 1) To use a CRISPRi screen to identify alpha-ketoglutarate (aKG) dependent enzyme(s) whose inhibition leads to the acquisition of a stem-like phenotype 2) To validate identified enzymes in cell line models and in human tumor datasets Identification of the enzymes whose functions are affected by accumulation of the oncometabolite succinate will shed important new light on the molecular mechanism of disease associated with Sdhx and other TCA cycle mutations, and will provide a stepping stone for future research grants that will allow detailed delineation of genetic and/or protein targets that drive tumor formation in the thyroid and other tissues (e.g. glioma, leukemia).