Differential expression analysis of RNA-seq from CCLE was conducted using R. 2011), and levels of GSH and its rate-limiting metabolite cysteine have been shown to increase with tumor progression in patients (Hakimi et al., 2016). Furthermore, both primary and metastasized tumors have been shown to utilize the reducing factor nicotinamide adenine dinucleotide phosphate, reduced (NADPH) to Rabbit Polyclonal to RAD51L1 regenerate GSH stores and survive oxidative stress (Jiang et al., 2016; Piskounova et al., 2015). Blocking antioxidant production, including the synthesis of GSH, has long been viewed as a potential mechanism to treat cancers (Arrick et al., 1982; Hirono, 1961). Treatment of patients with l-buthionine-sulfoximine (BSO) (Griffith and Meister, 1979), an MSC1094308 inhibitor of GCLC, is well tolerated and has been used in combination with the alkylating agent melphalan in multiple Phase 1 clinical trials with mixed results (“type”:”clinical-trial”,”attrs”:”text”:”NCT00005835″,”term_id”:”NCT00005835″NCT00005835 and “type”:”clinical-trial”,”attrs”:”text”:”NCT00002730″,”term_id”:”NCT00002730″NCT00002730) (Bailey, 1998; Villablanca et al., 2016). Inhibition of GSH synthesis has been shown to prevent tumor initiation in multiple mouse models of spontaneous tumorigenesis; however, limited effects have been reported in established tumors (Harris et al., 2015). Another major antioxidant pathway, governed by the protein thioredoxin 1 (TXN), has been shown to support survival of cells upon GSH depletion. Treatment of thioredoxin reductase 1 (caused minimal effects on proliferation across cancer cell lines, as indicated by a essentiality score close to zero (Figure 1A). This score contrasted with those from other non-redundant metabolic genes such as those encoding phosphogluconate dehydrogenase (in the human breast cancer cell line HCC-1806 (a cell line with an essentiality score for above the ?0.6 threshold) (Figure 1B). Deletion of caused a drastic reduction in GSH levels without any effect on cellular proliferation (Figures 1C and 1D), mirroring the results observed in the published pooled CRISPR screens. To evaluate the differential sensitivity of cancer cell lines to glutathione depletion more quantitatively, we used an inhibitor of GCLC, L-buthionine-sulfoximine (BSO) (Griffith and Meister, 1979), to evaluate the effects of titratable depletion of GSH across a large panel of cancer cell lines (Figure 1E). The efficacy of BSO was confirmed by assessment of the reduction in GSH levels; BSO induced potent and rapid depletion of GSH within 48 hours (Figures 1F, 1G and S1A). Extending this analysis to a larger panel of breast cancer cell lines revealed near uniform kinetics of GSH depletion by BSO (Figure 1H). The effect of BSO on cell number after 72 hours was determined for 49 cell lines derived from breast cancer (both basal and luminal subtypes), lung cancer and ovarian cancer. Across all tumor types, the majority of cancer cell lines displayed no reduction in cell number after depletion of GSH by BSO (Figures 1I, 1J and S1B-1E). Interestingly, a minority of cell lines (six) was highly sensitive to BSO, with IC50 values ranging from 1 to 6 M (matching the MSC1094308 IC50 values for depletion of intracellular GSH). To identify candidate genes underlying sensitivity to GSH depletion, RNA-seq data obtained from the Cancer Cell Line Encyclopedia (CCLE) was analyzed (Barretina et al., MSC1094308 2012; Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer, 2015). Fewer than 30 genes MSC1094308 were differentially expressed in the six highly sensitive cell lines relative to the other cancer cell lines (Table S1). These genes were not investigated further because the cell lines were derived from diverse tissues and it was not.
