Identification of enzymes inhibited by oncometabolites in Succinate Dehydrogenase mutant tumors

琥珀酸脱氢酶突变肿瘤中癌代谢物抑制的酶的鉴定

• 批准号: 10579575
• 负责人: Lawrence S Kirschner
• 金额: $ 7.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10579575
• 关键词: Acute Myelocytic Leukemia; Aerobic; Affect; Biological Models; CRISPR interference; CRISPR/Cas technology; Cancer Etiology; Cell Line; Cell model; Cell physiology; Cells; Cellular Metabolic Process; Characteristics; Citric Acid Cycle; Clear Cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Correlative Study; DNA; DNA Modification Process; Data; Data Set; Disease; Elderly; Endocrine; Enzyme Inhibition; Enzymes; Epithelium; Eukaryotic Cell; Familiarity; Family; Family member; Fumarate Hydratase; Future; Gastrointestinal Stromal Tumors; Generations; Genes; Genetic; Glioblastoma; Glioma; Hereditary Paraganglioma; Histones; Human; Hydroxylation; In Vitro; Inherited; Investigation; Isocitrate Dehydrogenase; Knowledge; Lead; Lesion; Light; Localized Disease; Malate Dehydrogenase; Malignant Neoplasms; Malignant neoplasm of thyroid; Mediating; Metabolic; Missense Mutation; Mitochondrial Proteins; Mixed Function Oxygenases; Modeling; Molecular; Mutation; Neoplasms; Nuclear; Oncogenic; Operative Surgical Procedures; Oxygenases; Paraganglioma; Patients; Phenotype; Pheochromocytoma; Process; Production; Property; Protein Isoforms; Proteins; Quality of life; RNA; Reactive Oxygen Species; Renal Cell Carcinoma; Renal carcinoma; Reporting; Research Project Grants; Role; Somatic Mutation; Source; Structural Protein; Study models; Succinate Dehydrogenase; Succinates; Survival Rate; Syndrome; System; Thyroid Gland; Time; Tissues; Tumor Promotion; United States; United States National Institutes of Health; Validation; Variant; Work; aldehyde dehydrogenases; alpha ketoglutarate; cofactor; gene repression; high risk; histone demethylase; in silico; in vivo; in vivo Model; knock-down; leukemia; member; mouse model; mutant; neoplastic; small hairpin RNA; stem; stem cell biomarkers; stem cells; therapy development; transcription factor; tumor; tumorigenesis

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).

项目概要 三羧酸循环（TCA循环，也称为克雷布斯循环）是真核生物的一个基本过程。 细胞，作为 ATP 生成的来源，并在条件下产生细胞代谢的还原当量 有氧条件。近年来，人们已经清楚，编码 TCA 循环酶的基因可以 是人类癌症的致病基因病变，包括与肾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。它们在来自这些相同区域的散发性肿瘤中也得到了很好的描述。 组织以及一系列其他癌症，包括急性髓系白血病 (AML) 和胶质母细胞瘤。 人们认为三羧酸循环酶及其相关辅因子的突变可通过以下途径导致肿瘤形成： 过量代谢物积累（致癌代谢物）的致癌作用。这些中间代谢物的作用是 干扰需要代谢辅因子α-酮戊二酸（aKG，也称为 如2-酮戊二酸）。人类体内大约有 70 种 aKG 依赖性酶，它们执行多种功能 重要的细胞功能，包括介导 DNA、RNA 和组蛋白的修饰。酶的 此类还负责蛋白质的氧化羟基化，包括结构蛋白胶原。 我们之前已经使用甲状腺肿瘤的体外和体内模型证明了甲状腺肿瘤的丧失 琥珀酸脱氢酶 D 亚基 (SDHD) 会引起表明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类似干的表型，这一点可以通过 干细胞相关转录因子 Nanog 和 Oct4 以及干细胞标记物醛的产生 脱氢酶（ALDH）。尽管事实上已经提出干扰 aKG 依赖性加氧酶 作为一种肿瘤机制，事先没有做出努力来查明对此负责的家庭成员 对于肿瘤性改变。为了填补这一知识空白，我们建议使用基于 CRISPR 的高通量 转录抑制筛选，以鉴定 aKG 依赖性酶，其抑制会导致重演 的茎样表型。将使用细胞模型和计算机模型的组合来验证已识别的命中 基于肿瘤的组学数据分析。 R03 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，其抑制作用 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已识别的酶 鉴定其功能受致癌代谢物琥珀酸积累影响的酶将 为与 Sdhx 和其他 TCA 循环相关的疾病分子机制提供重要的新线索 突变，并将为未来的研究资助提供垫脚石，从而详细描述 驱动甲状腺和其他组织（例如神经胶质瘤、白血病）中肿瘤形成的遗传和/或蛋白质靶点。