Loss of cysteinyl-tRNA synthetase (CARS) induces the transsulfuration pathway and inhibits ferroptosis induced by cystine deprivation

Ferroptosis is a form of regulated non-apoptotic cell death that has been implicated in several disease contexts. A better understanding of the ferroptotic death mechanism could lead to the development of new therapeutics for degenerative diseases, and a better understanding of how to induce ferroptosis in specific tumor contexts. We performed an unbiased genome-wide siRNA screen to find genetic suppressors of ferroptosis. We determined that loss of CARS, the cysteinyl-tRNA synthetase, suppresses ferroptosis induced by erastin, which inhibits the cystine–glutamate antiporter known as system x_c⁻. Knockdown of CARS inhibited erastin-induced death by preventing the induction of lipid reactive oxygen species, without altering iron homeostasis. Knockdown of CARS led to the accumulation of cystathionine, a metabolite on the transsulfuration pathway, and upregulated genes associated with serine biosynthesis and transsulfuration. In addition, inhibition of the transsulfuration pathway resensitized cells to erastin, even after CARS knockdown. These studies demonstrate a new mechanism of resistance to ferroptosis and may lead to strategies for inducing and suppressing ferroptosis in diverse contexts.Precise regulation of cell death is essential for tissue homeostasis. Dysregulation of cell death processes is implicated in a variety of pathological conditions, such as ischemia and neurodegenerative diseases, providing a rationale for exploring cell-death-modulating compounds as potential therapeutics.¹ However, an incomplete understanding of cell death mechanisms in specific disease contexts has hindered efforts to develop therapeutics. Mechanistic analyses of cell death processes in disease contexts may uncover new strategies for drug discovery. Ferroptosis, a form of oxidative, non-apoptotic cell death, has recently been described and implicated in several pathological conditions, including Huntington''s disease (HD), periventricular leukomalacia (PVL) and kidney dysfunction.^{2, 3, 4} Ferroptotic cell death can be induced through perturbation of redox homeostasis maintained by glutathione, a key regulator of the intracellular redox state.Glutathione (GSH) is a tripeptide, the synthesis of which is dependent on the availability of the amino acid cysteine. A substantial fraction of extracellular cysteine exists as its oxidized disulfide form, cystine, because of the oxidative extracellular environment.⁵ Some cells primarily obtain cysteine by importing extracellular cystine through system x_c⁻, the cystine–glutamate antiporter. Cystine is then reduced to cysteine inside cells, fueling GSH synthesis. GSH maintains redox homeostasis by acting as a reductive substrate for reactive oxygen species (ROS)-detoxifying enzymes. As one example, glutathione peroxidase 4 (GPX4) uses GSH to reduce lipid hydroperoxides and organic hydroperoxides to alcohols, serving a critical role in lipid repair and detoxification. GPX4 was recently shown to be a central regulator of ferroptosis.⁶Ferroptosis can be induced by two classes of compounds, exemplified by erastin and (1 S, 3 R)-RSL3.^{6, 7, 8, 9} These two compounds target different parts of the ferroptotic pathway. Erastin inhibits system x_c⁻ to deplete GSH, which effectively inactivates all cellular glutathione peroxidases, including GPX4. RSL3, on the other hand, acts downstream, inhibiting GPX4 directly. In both cases, the loss of GPX4 activity causes accumulation of lipid peroxides, and ultimately, cell death. Recently, the FDA-approved drugs sorafenib and sulfasalazine were also found to induce ferroptosis through inhibition of system x_c⁻ activity,^{10, 11} although these lower-potency compounds may also activate other competing processes at similar or slightly higher concentrations. A specific inhibitor of ferroptosis, ferrostatin-1, and its analogs have been shown to suppress cell death in several degenerative disease models, including HD, PVL and kidney dysfunction, as well as in a model of glutamate toxicity, suggesting the involvement of ferroptosis in these conditions.^{4, 12} Collectively, these findings suggest that modulation of ferroptosis is of potential therapeutic relevance in several pathological conditions.Given the involvement of ferroptosis in these different contexts, we sought to identify specific features and regulators of ferroptosis. Ferroptosis is biochemically and morphologically distinct from necrosis and apoptosis.¹² Genetic analysis of ferroptosis has been performed using a limited set of genes related to mitochondrial function.¹² This previous analysis revealed that ferroptosis requires a distinct set of genes compared with apoptosis. However, this analysis cast a relatively narrow net; therefore, we sought to extend our understanding of the genetic regulation of ferroptosis further to identify essential genes and pathways using a genome-wide siRNA screen. Such genes may illuminate novel targets whose inhibition could be therapeutic in disease conditions involving aberrant activation of ferroptosis, or suggest strategies for inducing ferroptosis in specific tumor contexts.