Hepatocellular carcinoma (HCC) accounts for >80% of patients with liver cancer and is one of the leading causes of cancer-associated mortalities, both worldwide and in the United States. Recent evidence highlights the involvement of YRDC (YrdC-threonylcarbamoyltransferase domain-containing protein), a tRNA modifying enzyme, in tumor growth via N6-threonylcarbamoylation (t6A) of tRNA in various cancer types, including HCC. Additionally, YRDC has been shown to be overexpressed in different types of cancer and associated with chemotherapeutic drug resistance as well as poor prognosis in HCC patients. However, the precise molecular mechanisms underlying YRDC overexpression in cancers and its involvement in the regulation of tumor metabolism remain unknown. The objective of our study is to define the mechanisms by which YRDC drives cellular growth and investigate the potential of targeting YRDC in cancers, with a specific focus on HCC. Our preliminary data, employing metabolomic and isotope tracing approaches, have shown that YRDC can act as a novel regulator of one-carbon metabolism and purine synthesis in several cancer cells, including HCC cells. We demonstrate that YRDC depletion reduces flux through one-carbon metabolism, thereby restricting the availability of substrates for the synthesis of de novo purine nucleotides. In light of these findings, this proposal focuses on studying YRDC and its impact on tumor metabolism using HCC as a model. We hypothesize that YRDC promotes tumor growth by activating one-carbon metabolism, thereby stimulating de novo purine synthesis. Consequently, targeting YRDC represents a potentially effective strategy against HCC. We will define the mechanisms underlying YRDC upregulation in HCC, we will use isogenic cell settings deleted for the tumor suppressor P53 and expressing mutant KRAS, and examine the role of the MAPK/ERK downstream of KRAS in the regulation of YRDC mRNA and protein levels (Aim1). Then, we will determine the molecular mechanisms by which YRDC stimulates one-carbon metabolism and purine synthesis in HCC cells (Aim2). Finally, we will examine the therapeutic potential of suppressing YRDC to improve HCC, and/or sensitize HCC to chemotherapy (Aim3). Overall, the proposal is significant because it will identify novel regulators of onecarbon metabolism and the de novo purine synthesis pathways, clarify the tumor pro-growth function of YRDC, and identify a novel drug target in HCC treatment. The project is highly innovative because, to date, YRDC has not been linked to the regulation of one-carbon metabolic pathway. The proposal will provide a stepping-stone to aid the applicant to establish an independent research career and the work performed under this award will be used to generate NIH R01 grant proposals.