Nitrogen metabolism profiling reveals cell state-specific pyrimidine synthesis pathway choice

SUMMARY Conventional stable isotope tracing assays track one or several metabolites. However, cells use an array of nutrients to sustain nitrogen metabolic pathways. This incongruency hampers a system level understanding of cellular nitrogen metabolism. Therefore, we created a platform to simultaneously trace 30 nitrogen isotope-labeled metabolites. This platform revealed that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine despite expressing de novo pathway enzymes. This link between cell state and pyrimidine synthesis routes persisted in physiological contexts, including primary murine and human tissues and tumor xenografts. Mechanistically, we found that Ser1900 phosphorylation of CAD, the first enzyme of the de novo pathway, was enriched in primitive cells and that mimicking this modification in differentiated cells abrogated their preference for pyrimidine salvage. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice. Competing Interest Statement S.K.M. receives research funding from Servier Pharmaceuticals. S.K.M. and K.G.A. have intellectual property interests related to brain tumor metabolism and are co-founders of Gliomet. R.J.D. is a founder and advisor at Atavistik Bioscience and an advisor for Vida Ventures and Faeth Therapeutics. N.Y.R.A. is a consultant for Bruker, serves on the Scientific Advisory Board for National Brain Tumor Society, and receives research support from Thermo, EMD Serono and iTeos Therapeutics. T.E.R. has received consulting fees from Servier pharmaceuticals.