br Material and methods br Acknowledgements and funding

Material and methods
Acknowledgements and funding The authors thank Katharina Elsässer for technical support and Emma Esser for careful editing of the manuscript. We also thank Marcus Conrad for providing Liproxstatin-1 and advisory comments and discussion on the manuscript. This work was partly supported by a DFG grant to CC (DFG-FOR2107).
Introduction Cellular metabolism supports the growth, proliferation, and survival of all GSK1210151A and is altered in diseases such as cancer (Beadle and Tatum, 1941, DeBerardinis and Thompson, 2012, Vander Heiden and DeBerardinis, 2017). The intracellular abundance of specific metabolites is regulated by the expression and activity levels of enzymes and transport proteins (Ducker and Rabinowitz, 2017, Metallo and Vander Heiden, 2013). Genome-wide gene deletion analysis in the single-celled eukaryote Saccharomyces cerevisiae has identified hundreds of genes that can regulate the abundance of individual metabolites (Cooper et al., 2010, Mülleder et al., 2016). Human haploid cell genetic screening technology has recently been developed and applied to identify regulators of viral entry, cell death, and other processes (Carette et al., 2011a, Carette et al., 2011b, Dixon et al., 2015, Dovey et al., 2018). We envisioned that this technology could be combined with a metabolite-specific fluorescent reporter and fluorescence-activated cell sorting (FACS) to identify genes that regulate metabolite abundance in human cells. As proof-of-concept, we focused in this work on genes regulating the abundance of glutathione, an essential intracellular thiol-containing tripeptide. Glutathione functions as an electron donor or acceptor by cycling between reduced (GSH) and oxidized (GSSG) forms and is important for xenobiotic detoxification, protein folding, antioxidant defense, and other processes (Deponte, 2013). As such, glutathione is especially important for the growth and survival of many cancer cells in vitro and in vivo (Harris et al., 2015, Lien et al., 2016, Piskounova et al., 2015). When intracellular GSH levels drop below a critical threshold, the GSH-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) cannot function, which can lead to a fatal buildup of lipid reactive oxygen species (ROS) and cell death via the iron-dependent, non-apoptotic process of ferroptosis (Dixon et al., 2012, Ingold et al., 2018, Yang et al., 2014). De novo GSH synthesis requires cysteine, which is typically found outside cells in the oxidized form as cystine. Small molecule inhibitors of cystine import via the cystine/glutamate antiporter system xc−, such as erastin, cause GSH depletion, lipid ROS accumulation, and ferroptosis induction (Dixon et al., 2012, Dixon et al., 2014). Whether inhibition of de novo GSH synthesis alone accounts for the rapid induction of ferroptosis following system xc− inhibition, or whether other mechanisms contribute to GSH depletion is unclear. Here, using genome-wide human haploid cell genetic screening, we identify negative regulators of intracellular glutathione levels that also alter ferroptosis sensitivity, including multidrug resistance protein 1 (MRP1), whose disruption reduces glutathione efflux from the cell (Cole, 2014a). High levels of MRP1-mediated glutathione efflux promote multidrug resistance and collaterally sensitize cancer cells to ferroptosis-inducing agents. Increased expression of the NRF2 antioxidant transcription factor can also elevate intracellular glutathione but has weak effects on ferroptosis sensitivity, in part because NRF2 upregulates MRP1 expression and therefore simultaneously increases both GSH synthesis and efflux.
Results
Discussion The regulation of metabolite abundance is poorly understood, especially in mammalian cells (Metallo and Vander Heiden, 2013). This work provides a proof-of-concept that fluorescent probes, FACS, and genome-wide haploid cell retroviral mutagenesis can be combined to directly identify regulators of metabolite abundance. Given the unbiased nature of this approach, it could be applied to any metabolite for which a suitable reporter is available. Our study also suggests the importance of confirming primary screening results using orthogonal assays. One limitation of the FACS-based genome-wide analysis is that it requires prolonged incubation of the large cell population under non-standard conditions followed by passage through the FACS instrument, which itself likely alters cellular metabolism (Llufrio et al., 2018). Importantly, the results obtained here using MCB to detect intracellular GSH in the genome-wide FACS screen were validated in follow-up experiments by traditional biochemical methods and an additional GSH-sensitive probe.