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 fu...