Title: Global Survey of Cell Death Mechanisms Reveals Metabolic Regulation of GPX4-Dependent Ferroptosis
Description: Cells die not merely as a consequence of catastrophic failure of homeostasis but when programmed cell death is activated. The existence of non-apoptotic modes of regulated cell death is increasingly appreciated. However, the full extent and diversity of these alternative cell death mechanisms remains uncharted. In this thesis, we developed a systematic framework to discover and characterize lethal compounds that induce distinct cell death phenotypes. In the first part, we investigated the landscape of pharmacologically-accessible non-apoptotic cell death mechanisms. This effort resulted in the discovery of a novel ferroptosis inducer, FIN56. The rest of my work focused on characterizing the mechanism of action of FIN56. Technologies used here should be generally applicable for the systematic study of various cell death mechanisms. First, to globally survey pharmacologically-accessible cell death mechanisms, we used 3,169 uncharacterized lethal compounds as cell death probes. We found that 451 compounds (14%) were lethal without activating caspase activity. 56 most potent and structurally diverse compounds were more closely studied using the 'modulatory profiling' approach, which involves examining changes in potency of lethal compounds by co-treatment with chemical modulators.

We discovered that caspase-independent lethals induced three types of regulated non-apoptotic cell death: metal ion-dependent cell death, necrostatin-1-dependent cell death, and ferroptosis, a regulated form of iron-dependent oxidative cell death. With further structural optimization, we discovered a specific ferroptosis inducer, FIN56. Ferroptosis is induced when the lipid repair enzyme glutathione peroxide 4 (GPX4) is inhibited or inactivated by depletion of glutathione. We found that, in contrast, FIN56 induced ferroptosis through decreasing the abundance of GPX4. Second, we developed a technology that identifies proteins responsible for cell death mechanisms of interest utilizing chemical library screening. The technology consists of three steps: (i) binding targets of each molecule in the chemical library were predicted using Similarity Ensemble Approach, a chemoinformatic ligand-based target prediction algorithm; (ii) the chemical library was screened for enhancers/suppressors of the cell death; (iii) incorporating the screening data into the prediction to make the prediction more reliable.

This approach, termed `Target Enrichment Analysis', resulted in the discovery of two features of FIN56-induced ferroptosis: calcium ion influx and activation of lipoxygenases, enzymes that peroxidize fatty acids. Inhibiting either of them suppressed FIN56-induced ferroptosis. Third, we tried to capture metabolic changes induced upon FIN56 treatment that were relevant to the mechanism of action of FIN56 and identify protein targets of FIN56 using chemoproteomics. Metabolomic profiling experiments discovered that non-steroidogenic intermediates in the mevalonate pathway regulated cellular sensitivity to FIN56-induced ferroptosis. Although none of the proteins identified through target identification effort has yet been fully confirmed as responsible for induction of ferroptosis triggered by FIN56, we found through the analysis that inhibition of squalene synthase, an enzyme in the mevalonate pathway, suppressed FIN56-induced ferroptosis consistently. Finally, to define biomarkers that predict sensitivity to ferroptosis inducers including FIN56, we investigated the molecular determinants of sensitivity in the NCI60 panel and identified nicotinamide adenine dinucleotide phosphate (NADPH) levels as a global predictor of sensitivity to ferroptosis.

These studies demonstrate that sensitivity to ferroptosis is regulated by metabolic pathways, suggesting that it may be a relevant form of cell death in cases of dysregulated metabolism. This systematic approach using a combination of modulatory profiling and cell line selectivity analysis is an effective means to explore, discover and characterize cellular phenotypes induced by unknown small molecules.