Murray, M.; Upadhyay, R.; Szylo, K.; Gautam, A.; Goncalves, J.; Forcina, G.; Vinayak, A.; Brandmann, O.; Dixon, S. J.

The proteasome is an essential multiprotein complex whose inhibition can lead to apoptosis. Ferroptosis is a non-apoptotic cell death mechanism whose fundamental regulation continues to be elucidated. How proteasome function regulates ferroptosis sensitivity is poorly understood and difficult to study given the essential nature of the proteasome. Here, we isolated the effects of proteasome inhibition on ferroptosis by combining direct cell death imaging, cell death pathway-specific inhibitors, and mathematical modeling. We find that proteasome inhibition enhances sensitivity to ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition while simultaneously promoting resistance to ferroptosis induced by system xc- inhibition. Sensitization to GPX4 inhibition requires protein synthesis but not the apoptosis execution machinery and is opposed by the activating transcription factor 4 (ATF4) stress response pathway. This work demonstrates a complex role for proteasome function in ferroptosis regulation and establishes new methods to dissect cross-talk between ferroptosis and essential cellular processes.