A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity

• Published in: Cell reports (Cambridge) 2019-02, Vol.26 (6), p.1544-1556.e8
• Main Authors: Cao, Jennifer Yinuo, Poddar, Aunoy, Magtanong, Leslie, Lumb, Jennifer H., Mileur, Trevor R., Reid, Michael A., Dovey, Cole M., Wang, Jin, Locasale, Jason W., Stone, Everett, Cole, Susan P.C., Carette, Jan E., Dixon, Scott J.
• Format: Article
• Language: English
• Subjects: cancer; Cell Line, Tumor; collateral sensitivity; Female; ferroptosis; Ferroptosis - genetics; Flow Cytometry - methods; Genome, Human; glutathione; Glutathione - metabolism; Haploidy; Humans; iron; Kelch-Like ECH-Associated Protein 1 - genetics; Kelch-Like ECH-Associated Protein 1 - metabolism; Male; metabolite efflux; multidrug resistance; Multidrug Resistance-Associated Proteins - genetics; Multidrug Resistance-Associated Proteins - metabolism; N-Terminal Acetyltransferase C - genetics; N-Terminal Acetyltransferase C - metabolism; necrosis; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; Ribonucleoprotein, U4-U6 Small Nuclear - genetics; Ribonucleoprotein, U4-U6 Small Nuclear - metabolism; ROS
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2019.01.043

The tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity. [Display omitted] •FACS-based screening can identify genetic regulators of glutathione abundance•MRP1 effluxes glutathione, which accelerates ferroptosis•NAA38 and KEAP1 regulate expression of NRF2 and also MRP1•High MRP1 expression promotes collateral sensitivity to ferroptosis-inducing agents Glutathione suppresses the non-apoptotic cell death process of ferroptosis. Using genome-wide human haploid cell mutagenesis and FACS-based detection, Cao et al. identify negative regulators of intracellular glutathione abundance that affect glutathione efflux and NRF2 protein levels, altering ferroptosis sensitivity.