Abstract 4709: Compartmental flexibility in mammalian folate metabolism

Abstract

Abstract Background: Folate metabolism occurs in parallel cytosolic and mitochondrial pathways. Increased mitochondrial, but not cytosolic, folate dependent serine catabolism is among the most consistent metabolic alterations in human tumors. This study employs genetics coupled with quantitative metabolic analysis to determine the respective requirements for compartmentalized folate metabolism in proliferating human cells. Methods: Using CRISPR-Cas9 technology, we created a library of deletion mutant cell lines for enzymes in folate metabolism. Cellular metabolites from these cells were quantified by liquid chromatography mass spectrometry (LC-MS). Compartmental specific folate metabolic fluxes were assayed by labeling experiments using isotopic tracers of serine, glycine and formate. Results: We show that in HEK 293T cells, as well as in many but not all human cancer cell lines, folate dependent serine catabolism occurs predominantly in the mitochondria. In mutant cell lines deficient for mitochondrial folate genes, the reversal of the direction of cytosolic one-carbon flux enables cell growth, albeit at a reduced rate and with metabolic hallmarks of 10-formyltetrahydrofolate deficiency. Addition of exogenous formate rescued the 10-formyltetrahydrofolate deficiency in all mutants, but the growth defect in only a subset. The growth defect persisted in cell lines mutant for enzymes required for the generation of mitochondrial one-carbon units and folate dependent generation of NAD(P)H but not for the production of formate. Conclusions: This study finds that folate metabolism is flexible with respect to compartmentalization. Cytosolic folate metabolism can provide sufficient one-carbon flux to fuel cell growth even in the complete absence of mitochondrial activity. High expression of mitochondrial folate genes in cancer may function to meet biosynthetic requirements under the stressful conditions of the tumor or to provide necessary compartmentalized one-carbon units and/or reducing equivalents. Citation Format: Gregory S. Ducker, Li Chen, Xin Teng, Joshua D. Rabinowitz. Compartmental flexibility in mammalian folate metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4709. doi:10.1158/1538-7445.AM2015-4709