Abstract

Isocitrate dehydrogenase-mutant (IDH-MUT) gliomas exhibit distinct metabolic profile marked by 2-hydroxyglutarate (2-HG) accumulation at the expense of α-ketoglutarate. How these tumors maintain the tricarboxylic acid (TCA) cycle, however, remained unclear. We conducted comprehensive metabolomic profiling using clinical cohorts, cell lines, and patient-derived organoids (PDOs). The metabolic dynamics of TCA and urea cycles were interrogated using stable isotope tracing with 13 C-aspartate, U5- 13 C- 15 N-aspartate, 15 NH 4 Cl and 15 N-glutamate. The functional role of carbamoyl-phosphate synthase 1 (CPS1), the key enzyme of the urea cycle, was validated through inhibition experiments in vitro , in vivo and in PDO model, followed by seahorse respirometry and electron microscopy. Metabolomic profiling of two glioma cohorts consistently showed elevated urea cycle metabolites in IDH-MUT tumors. We identified CPS1 as a metabolic checkpoint sustaining TCA cycle through half urea cycle (from ammonia to arginine). Of note, CPS1 upregulation drove fumarate anaplerosis to sustain TCA flux in IDH-MUT gliomas. Thus, CPS1 inhibition not only reduced fumarate levels but also decreased oncometabolite 2-HG both in vitro and in vivo . Consistently, CPS1 inhibition impaired mitochondrial respiration and suppressed tumor growth in vitro , in vivo and in PDOs. Taken together, metabolic checkpoint CPS1 orchestrates half urea cycle to replenish the TCA cycle in IDH-MUT gliomas. Targeting CPS1 represents a promising metabolic therapeutic target for this glioma subtype.