ASS1-Mediated Reductive Carboxylation of Cytosolic Glutamine Confers Ferroptosis Resistance in Cancer Cells

Induction of ferroptosis, a recently defined form of nonapoptotic cell death caused by iron-dependent lipid peroxidation, has emerged as an anticancer strategy. Erastin is a ferroptosis activator that promotes cell death that not only depends on the depletion of cellular cysteine but also relies on mitochondrial oxidative metabolism of glutamine. Here, we demonstrate that ASS1, a key enzyme involved in the urea cycle, plays a crucial role in ferroptosis resistance. Loss of ASS1 increased the sensitivity of non-small cell lung cancer (NSCLC) cells to erastin in vitro and decreased tumor growth in vivo. Metabolomics analysis with stable isotope-labeled glutamine showed that ASS1 promotes reductive carboxylation of cytosolic glutamine and compromises the oxidative tricarboxylic acid cycle from glutamine anaplerosis, reducing mitochondrial-derived lipid reactive oxygen species. Moreover, transcriptome sequencing showed that ASS1 activates the mTORC1-SREBP1-SCD5 axis to promote de novo monounsaturated fatty acid synthesis by using acetyl-CoA derived from the glutamine reductive pathway. Treating ASS1-deficient NSCLC cells with erastin combined with arginine deprivation significantly enhanced cell death compared with either treatment alone. Collectively, these results reveal a previously unknown regulatory role of ASS1 in ferroptosis resistance and provide a potential therapeutic target for ASS1-deficient NSCLC.ASS1 promotes reductive carboxylation of glutamine and confers ferroptosis resistance, providing multiple treatment options for ASS1-deficient non-small cell lung cancer.