Abstract 3800: Physiological nutrient levels reveal nucleotide salvage as a dependency in B-cell acute lymphoblastic leukemia

Abstract Acquiring nucleotides is essential for all dividing cells, and failure to maintain sufficient levels and balance of nucleotides impairs DNA replication. Eukaryotic cells meet nucleotide demands through either de novo synthesis or salvage of nucleotide precursors from the extracellular environment. Although it is known that levels of salvageable nucleotide precursors vary across tissues, the contribution of nucleotide salvage to total nucleotide acquisition in malignant cells remains underexplored. Using a mouse model of B-cell acute lymphoblastic leukemia (B-ALL), we formulated a mouse-plasma-like medium (MPM) to investigate how cells balance nucleotide acquisition strategies in nutrient environments that mimic mouse plasma during leukemogenesis. We used stable-isotope tracing and LC-MS to measure the contribution of de novo synthesis and salvage pathways for nucleotide acquisition in B-ALL cells. We found that cells cultured in MPM preferentially acquire some nucleotide species through salvage pathways, and genetic perturbation of nucleotide salvage pathways leads to deoxynucleotide triphosphate (dNTP) depletion, induction of replication stress, and reduced proliferation. We found that the dependence on nucleotide salvage arises because physiological levels of the vitamin folate, a molecule used in de novo nucleotide synthesis, are low enough to constrain de novo nucleotide biosynthesis in physiological conditions, driving increased reliance on salvage pathways. Accordingly, culturing cancer cells in conventional cell culture media with folate levels found in physiological conditions attenuates cell proliferation due to decreased de novo nucleotide synthesis. In vivo, genetically perturbing pyrimidine nucleotide salvage slows B-ALL proliferation, and this is further exacerbated when mice were fed folate-depleted diets. On the other hand, dietary folate supplementation increased the proliferation of salvage deficient cells in vivo. Together, these findings demonstrate that physiological folate availability limits de novo nucleotide synthesis in B-ALL, creating dependence on nucleotide salvage pathways. This work informs potential combination therapy strategies targeting folate metabolism and nucleotide salvage. Citation Format: Ryan Elbashir, Keene L. Abbott, Ahmed Ali, Diya L. Ramesh, Michelle Wu, Brian T. Do, Anya Shevzov-Zebrun, Tenzin Kunchok, Millenia Waite, Wontaek Chung, Chelsea Zhang, Sharanya Sivanand, Azucena Ramos, Jacob A. Hansen, Raphael Ferreira, Alexander Muir, Michael H. Hemann, Matthew G. Vander Heiden. Physiological nutrient levels reveal nucleotide salvage as a dependency in B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3800.