Ferroptosis regulation by the NGLY1/NFE2L1 pathway.

SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

A compendium of kinetic modulatory profiles identifies ferroptosis regulators.

Cell death can be executed by regulated apoptotic and nonapoptotic pathways, including the iron-dependent process of ferroptosis. Small molecules are essential tools for studying the regulation of cell death. Using time-lapse imaging and a library of 1,833 bioactive compounds, we assembled a large compendium of kinetic cell death modulatory profiles for inducers of apoptosis and ferroptosis. From this dataset we identify dozens of ferroptosis suppressors, including numerous compounds that appear to act via cryptic off-target antioxidant or iron chelating activities. We show that the FDA-approved drug bazedoxifene acts as a potent radical trapping antioxidant inhibitor of ferroptosis both in vitro and in vivo. ATP-competitive mechanistic target of rapamycin (mTOR) inhibitors, by contrast, are on-target ferroptosis inhibitors. Further investigation revealed both mTOR-dependent and mTOR-independent mechanisms that link amino acid metabolism to ferroptosis sensitivity. These results highlight kinetic modulatory profiling as a useful tool to investigate cell death regulation.

GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis.

Oxygen is necessary for aerobic metabolism but can cause the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe2+ )-dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to lipid peroxidation and can result in the induction of ferroptosis, an iron-dependent, non-apoptotic form of cell death. This review describes the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation, and the induction of ferroptosis.

Stelliosphaerols A and B, Sesquiterpene-Polyol Conjugates from an Ecuadorian Fungal Endophyte.

Endophytic fungi are plant tissue-associated fungi that represent a rich resource of unexplored biological and chemical diversity. As part of an ongoing effort to characterize Amazon rainforest-derived endophytes, numerous fungi were isolated and cultured from plants collected in the Yasuní National Park in Ecuador. Of these samples, phylogenetic and morphological data revealed a previously undescribed fungus in the order Pleosporales that was cultured from the tropical tree Duroia hirsuta. Extracts from this fungal isolate displayed activity against Staphylococcus aureus and were thus subjected to detailed chemical studies. Two compounds with modest antibacterial activity were isolated, and their structures were elucidated using a combination of NMR spectroscopic analysis, LC-MS studies, and chemical degradation. These efforts led to the identification of stelliosphaerols A (1) and B (2), new sesquiterpene-polyol conjugates that are responsible, at least in part, for the S. aureus inhibitory activity of the fungal extract.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells.

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Kinetic Heterogeneity of Cancer Cell Fractional Killing.

Lethal drugs can induce incomplete cell death in a population of cancer cells, a phenomenon referred to as fractional killing. Here, we show that high-throughput population-level time-lapse imaging can be used to quantify fractional killing in response to hundreds of different drug treatments in parallel. We find that stable intermediate levels of fractional killing are uncommon, with many drug treatments resulting in complete or near-complete eradication of all cells, if given enough time. The kinetics of fractional killing over time vary substantially as a function of drug, drug dose, and genetic background. At the molecular level, the antiapoptotic protein MCL1 is an important determinant of the kinetics of fractional killing in response to MAPK pathway inhibitors but not other lethal stimuli. These studies suggest that fractional killing is governed by diverse lethal stimulus-specific mechanisms.

Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State.

The initiation and execution of cell death can be regulated by various lipids. How the levels of environmental (exogenous) lipids impact cell death sensitivity is not well understood. We find that exogenous monounsaturated fatty acids (MUFAs) potently inhibit the non-apoptotic, iron-dependent, oxidative cell death process of ferroptosis. This protective effect is associated with the suppression of lipid reactive oxygen species (ROS) accumulation at the plasma membrane and decreased levels of phospholipids containing oxidizable polyunsaturated fatty acids. Treatment with exogenous MUFAs reduces the sensitivity of plasma membrane lipids to oxidation over several hours. This effect requires MUFA activation by acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) and is independent of lipid droplet formation. Exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by the accumulation of saturated fatty acids, but in an ACSL3-independent manner. Our work demonstrates that ACSL3-dependent MUFA activation promotes a ferroptosis-resistant cell state.

Systematic Quantification of Population Cell Death Kinetics in Mammalian Cells.

Cytotoxic compounds are important drugs and research tools. Here, we introduce a method, scalable time-lapse analysis of cell death kinetics (STACK), to quantify the kinetics of compound-induced cell death in mammalian cells at the population level. STACK uses live and dead cell markers, high-throughput time-lapse imaging, and mathematical modeling to determine the kinetics of population cell death over time. We used STACK to profile the effects of over 1,800 bioactive compounds on cell death in two human cancer cell lines, resulting in a large and freely available dataset. 79 potent lethal compounds common to both cell lines caused cell death with widely divergent kinetics. 13 compounds triggered cell death within hours, including the metallophore zinc pyrithione. Mechanistic studies demonstrated that this rapid onset lethal phenotype was caused in human cancer cells by metabolic disruption and ATP depletion. These results provide the first comprehensive survey of cell death kinetics and analysis of rapid-onset lethal compounds.