UC Berkeley

Cancer is a leading cause of death worldwide and has propensity for rapid mutation,which constantly challenges our ability to treat aggressive forms. There is an unmet need for next generation cancer medicine capable of overcoming chemotherapeutic resistance in cancer. 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 been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by 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, suggesting that additional factors govern resistance to ferroptosis. Here in this dissertation, we employ a synthetic lethal CRISPR/Cas9 screen, and 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), generating 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 tumor 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 new ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutics. In addition, we screened more than 120,000 compounds using an in vitro FSP1 activity assay and identified 168 compounds that directly inhibit FSP1 oxidoreductase activity. We further characterized 19 FSP1 inhibitors, belonging to 7 structurally distinct groups, and find that these compounds exhibit synthetic lethality with loss of GPX4 in H460 lung cancer cells. The most potent of these FSP1 inhibitors, ferroptosis sensitizer 1 (FSEN1),

acts specifically through inhibition of FSP1 to sensitize cancer cells of different tissueorigins to ferroptosis induced by multiple compounds targeting the GSH-GPX4 pathway. Together, our results define several structurally unique FSP1 inhibitors that sensitize cancer cells to ferroptosis, providing tools for the study of FSP1 biology and enabling further exploration of the potential of FSP1 as a therapeutic target.