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Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells.

Ferroptosis is an important mediator of pathophysiological cell death and an emerging target for cancer therapy. Whether ferroptosis sensitivity is governed by a single regulatory mechanism is unclear. Here, based on the integration of 24 published chemical genetic screens combined with targeted follow-up experimentation, we find that the genetic regulation of ferroptosis sensitivity is highly variable and context-dependent. For example, the lipid metabolic gene acyl-coenzyme A (CoA) synthetase long chain family member 4 (ACSL4) appears far more essential for ferroptosis triggered by direct inhibition of the lipid hydroperoxidase glutathione peroxidase 4 (GPX4) than by cystine deprivation. Despite this, distinct pro-ferroptotic stimuli converge upon a common lethal effector mechanism: accumulation of lipid peroxides at the plasma membrane. These results indicate that distinct genetic mechanisms regulate ferroptosis sensitivity, with implications for the initiation and analysis of this process in vivo.

Nonapoptotic cell death is important for human health and disease. Here, we show how various tools and techniques drawn from the chemical biology field have played a central role in the discovery and characterization of nonapoptotic cell death pathways. Focusing on the example of ferroptosis, we describe how phenotypic screening, chemoproteomics, chemical genetic analysis, and other methods enabled the elucidation of this pathway. Synthetic small-molecule inducers and inhibitors of ferroptosis identified in early studies have now been leveraged to identify an even broader set of compounds that affect ferroptosis and to validate new chemical methods and probes for various ferroptosis-associated processes. A number of limitations associated with specific chemical biology tools or techniques have also emerged and must be carefully considered. Nevertheless, the study of ferroptosis provides a roadmap for how chemical biology methods may be used to discover and characterize nonapoptotic cell death mechanisms.