The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents

A Novel gene overexpression plasmid library and its application in mapping genetic networks by systematic dosage suppression

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.Ph

p53 Suppresses Metabolic Stress-Induced Ferroptosis in Cancer Cells

How cancer cells respond to nutrient deprivation remains poorly understood. In certain cancer cells, deprivation of cystine induces a non-apoptotic, iron-dependent form of cell death termed ferroptosis. Recent evidence suggests that ferroptosis sensitivity may be modulated by the stress-responsive transcription factor and canonical tumor suppressor protein p53. Using CRISPR/Cas9 genome editing, small-molecule probes, and high-resolution, time-lapse imaging, we find that stabilization of wild-type p53 delays the onset of ferroptosis in response to cystine deprivation. This delay requires the p53 transcriptional target CDKN1A (encoding p21) and is associated with both slower depletion of intracellular glutathione and a reduced accumulation of toxic lipid-reactive oxygen species (ROS). Thus, the p53-p21 axis may help cancer cells cope with metabolic stress induced by cystine deprivation by delaying the onset of non-apoptotic cell death

Ether lipid deficiency disrupts lipid homeostasis leading to ferroptosis sensitivity.

Ferroptosis is an iron-dependent form of regulated cell death associated with uncontrolled membrane lipid peroxidation and destruction. Previously, we showed that dietary dihomo-gamma-linolenic acid (DGLA; 20: 3(n-6)) triggers ferroptosis in the germ cells of the model organism, Caenorhabditis elegans. We also demonstrated that ether lipid-deficient mutant strains are sensitive to DGLA-induced ferroptosis, suggesting a protective role for ether lipids. The vinyl ether bond unique to plasmalogen lipids has been hypothesized to function as an antioxidant, but this has not been tested in animal models. In this study, we used C. elegans mutants to test the hypothesis that the vinyl ether bond in plasmalogens acts as an antioxidant to protect against germ cell ferroptosis as well as to protect from whole-body tert-butyl hydroperoxide (TBHP)-induced oxidative stress. We found no role for plasmalogens in either process. Instead, we demonstrate that ether lipid-deficiency disrupts lipid homeostasis in C. elegans, leading to altered ratios of saturated and monounsaturated fatty acid (MUFA) content in cellular membranes. We demonstrate that ferroptosis sensitivity in both wild type and ether-lipid deficient mutants can be rescued in several ways that change the relative abundance of saturated fats, MUFAs and specific polyunsaturated fatty acids (PUFAs). Specifically, we reduced ferroptosis sensitivity by (1) using mutant strains unable to synthesize DGLA, (2) using a strain carrying a gain-of-function mutation in the transcriptional mediator MDT-15, or (3) by dietary supplementation of MUFAs. Furthermore, our studies reveal important differences in how dietary lipids influence germ cell ferroptosis versus whole-body peroxide-induced oxidative stress. These studies highlight a potentially beneficial role for endogenous and dietary MUFAs in the prevention of ferroptosis

Dietary MUFAs are protective in DGLA-induced ferroptosis through inhibition of lipid peroxidation.

(A) Relative fatty acid composition of worms of the indicated genotype as determined with gas chromatography-mass spectrometry. SA-stearic acid (18:0), Pla-plasmalogen, OA-oleic acid (18:1(n-9)), cVA-cis-vaccenic acid (18:1(n-7)), LA-linoleic acid (18:2(n-6)), ALA-alpha linolenic acid (18:3(n-3)), GLA-gamma linolenic acid (18:3(n-6)), STA-stearidonic acid (18:(4n-3)), DGLA-dihomo-gamma linolenic acid (20:(3n-6)), AA-arachidonic acid (20:(4n-6)), ETA-eicosatetraenoeic acid (20:(4n-3)), EPA-eicosapentaenoic acid (20:(5n-3)) (B and C) Percentage (%) sterility in young adult worms of the indicated genotype raised on the indicated fatty acids. In (B and C) each dot represents an independent experiment of 50 worms for each treatment. Statistical significance was determined using a two-way ANOVA with Tukey’s test for multiple comparisons summarized in S2 Table. Fatty acid composition and sterility for (B, C, and D) are reported in S1 Table. In (D) each data point represents an independent experiment of 1,250 worms for each treatment. Statistical analysis was performed with Student’s t-test and P values are reported in S2 Table. * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.</p

Exogenous 20-carbon PUFAs differentially modulates ferroptosis and peroxide-induced oxidative stress.

(A) Relative fatty acid composition in wild type worms treated with DGLA, AA, and EPA determined using gas chromatography-mass spectrometry. SA-stearic acid (18:0), Pla-plasmalogen, OA-oleic acid (18:1(n-9)), cVA-cis-vaccenic acid (18:1(n-7)), LA-linoleic acid (18:2(n-6)), ALA-alpha linolenic acid (18:3(n-3)), GLA-gamma linolenic acid (18:3(n-6)), STA-stearidonic acid (18:(4n-3)), DGLA-dihomo-gamma linolenic acid (20:(3n-6)), AA-arachidonic acid (20:(4n-6)), ETA-eicosatetraenoeic acid (20:(4n-3)), EPA-eicosapentaenoic acid (20:(5n-3)) (B) Percentage (%) sterility of wild type wormsraised on the indicated fatty acids. (C and D) Survival of young adult worms raised on the indicated fatty acids before exposure to 14.7mM tert-butyl hydroperoxide (TBHP). In (B) each dot represents an independent experiment of 50 worms for each treatment. Statistical significance was determined using a two-way ANOVA with Tukey’s test for multiple comparisons summarized in S2 Table. In (C and D) approximately 100–200 worms were used for each strain per treatment. Statistical significance for survival was determined using log rank tests (Mantel Cox) is shown in S2 Table. In (B, C and D), fatty acid compositions are reported in S1 Table.</p

The mediator complex, MDT-15, plays a protective function through increased expression of Delta-9 desaturases in ether lipid deficiency.

(A) Percentage (%) sterility in young adult worms of the indicated genotype raised on dihommo-gamma linolenic acid (DGLA). (B) Survival of young adult worms exposed to 14.7mM tert-butyl-hydroperoxide (TBHP). (C) Relative fatty acid composition determined with gas chromatography-mass spectrometry of strains used in (A) and (B). (D and E) Fold-change of basal mRNA levels in mutant worms relative to wild type worms grown on standard nematode growth media. mRNA levels were normalized to cdc-42 and Y45F10D.4. In (A) each dot represents an independent experiment of 50 worms for each treatment. Statistical significance was determined using a two-way ANOVA with Tukey’s test for multiple comparisons summarized in S2 Table. In (B) approximately 100–200 worms were used for each strain per treatment. Statistical significance for survival was determined using log rank tests (Mantel Cox) and is shown in S2 Table. In (C), values do not add up to 100% because dietary cyclopropane fatty acids and several others are not displayed in this chart. The complete fatty acid composition and sterility data are reported in S1 Table. In (D and E), student’s t-tests were performed to determine statistical significance and summarized in S2 Table. Statistical differences compared to WT are NS, not significant, * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.</p