Succinate dehydrogenase loss suppresses pyrimidine biosynthesis via succinate-mediated inhibition of aspartate transcarbamylase.

Decreased availability of the amino acid aspartate constrains cell function across diverse biological contexts, but the temporal interplay between aspartate abundance, downstream metabolic changes and functional effects remains poorly understood. Here we show that succinate dehydrogenase (SDH) inhibition suppresses pyrimidine synthesis via dual effects of cellular aspartate depletion and succinate accumulation. Using an aspartate biosensor and live-cell imaging, we monitor aspartate levels and cell proliferation across several models of aspartate limitation. While complex I inhibition or knockout of aspartate biosynthetic enzymes lead to a strict decrease in aspartate levels and impair proliferation, SDH inhibition produces a unique aspartate rebound, yet fails to restore proliferation. Mechanistically, we find that SDH loss impairs pyrimidine biosynthesis via succinate accumulation, which competitively inhibits aspartate utilization by mammalian aspartate transcarbamylase (ATCase), a key step in pyrimidine biosynthesis. This metabolic interaction occurs in multiple models of SDH deficiency, causing pyrimidine insufficiency, replication stress and sensitivity to ATR kinase inhibition. Taken together, these findings define an unexpected role for succinate in modulating cellular nucleotide homeostasis and demonstrate how cascading metabolic interactions can unfold to impact cell function.