Abstract 1800: Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells

Abstract

Abstract The genes that are required for the growth of mammalian cells can depend on an interplay of cell-intrinsic factors and environmental context. However, there is often little investigation into how nutrient availability impacts gene essentiality. Moreover, efforts such as DepMap have catalogued genes that contribute to cancer cell fitness using in vitro CRISPR screens in hundreds of cancer cell lines cultured in traditional media that poorly recapitulate the nutrient availability of human blood. Previously, we developed Human Plasma-Like Medium (HPLM), a physiologic medium designed to better model the nutrient availability of human blood. We recently tested the hypothesis that nutrient availability influences gene essentiality. By performing paired CRISPR-based screens in human blood cancer cells, we identified sets of genes that are differentially required to support cell growth in HPLM versus traditional media such as RPMI. Among the strongest scoring HPLM-essential genes was NAD kinase (NADK), which encodes an enzyme critical for the generation of cytosolic NADPH. NADK has been previously suggested as an anti-cancer therapeutic target, given the central importance of NADPH for enabling proliferative metabolism through macromolecule synthesis and ROS management. Interestingly, NADK has been annotated as an essential gene in only 1% of the cell lines included in DepMap, suggesting that the essentiality of NADK may have been largely masked by the nutrient conditions used to generate the DepMap. To investigate the conditional CRISPR phenotype for NADK, we engineered NADK-knockout cells and found that they showed a 50% stronger growth defect versus control cells in HPLM relative to RPMI. Next, we used localization and activity studies to confirm that cytosolic NAD+ kinase activity is required for the conditionally essential role of NADK. We then employed systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene-nutrient interaction and to gain insights into the conditionally essential role of NADK. Through these studies, we traced the cause of NADK essentiality to an inhibition of folate-dependent nucleotide synthesis linked to differential folic acid availability. Finally, we examined the pre-translational potential of NADK as an anti-cancer therapeutic target by demonstrating that the reported NADK inhibitor thionicotinamide largely phenocopies NADK-knockout in a folic-acid dependent manner in the K562 cell line. Collectively, our work improves understanding of how NADK contributes to human cell growth and reveals how the relative importance of cytosolic NAD+ kinase activity can depend on nutrient availability. Citation Format: Kyle M. Flickinger, Kimberly S. Huggler, Jason R. Cantor. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1800.