Phospholipids with two polyunsaturated fatty acyl tails promote ferroptosis.

Phospholipids containing a single polyunsaturated fatty acyl tail (PL-PUFA1s) are considered the driving force behind ferroptosis, whereas phospholipids with diacyl-PUFA tails (PL-PUFA2s) have been rarely characterized. Dietary lipids modulate ferroptosis, but the mechanisms governing lipid metabolism and ferroptosis sensitivity are not well understood. Our research revealed a significant accumulation of diacyl-PUFA phosphatidylcholines (PC-PUFA2s) following fatty acid or phospholipid treatments, correlating with cancer cell sensitivity to ferroptosis. Depletion of PC-PUFA2s occurred in aging and Huntington's disease brain tissue, linking it to ferroptosis. Notably, PC-PUFA2s interacted with the mitochondrial electron transport chain, generating reactive oxygen species (ROS) for initiating lipid peroxidation. Mitochondria-targeted antioxidants protected cells from PC-PUFA2-induced mitochondrial ROS (mtROS), lipid peroxidation, and cell death. These findings reveal a critical role for PC-PUFA2s in controlling mitochondria homeostasis and ferroptosis in various contexts and explain the ferroptosis-modulating mechanisms of free fatty acids. PC-PUFA2s may serve as diagnostic and therapeutic targets for modulating ferroptosis.

Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones.

Ferroptosis, a cell death process driven by iron-dependent phospholipid peroxidation, has been implicated in various diseases. There are two major surveillance mechanisms to suppress ferroptosis: one mediated by glutathione peroxidase 4 (GPX4) that catalyzes the reduction of phospholipid peroxides and the other mediated by enzymes, such as FSP1, that produce metabolites with free radical-trapping antioxidant activity. In this study, through a whole-genome CRISPR activation screen, followed by mechanistic investigation, we identified phospholipid-modifying enzymes MBOAT1 and MBOAT2 as ferroptosis suppressors. MBOAT1/2 inhibit ferroptosis by remodeling the cellular phospholipid profile, and strikingly, their ferroptosis surveillance function is independent of GPX4 or FSP1. MBOAT1 and MBOAT2 are transcriptionally upregulated by sex hormone receptors, i.e., estrogen receptor (ER) and androgen receptor (AR), respectively. A combination of ER or AR antagonist with ferroptosis induction significantly inhibited the growth of ER+ breast cancer and AR+ prostate cancer, even when tumors were resistant to single-agent hormonal therapies.

Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications.

Ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, was identified as a distinct phenomenon and named a decade ago. Ferroptosis has been implicated in a broad set of biological contexts, from development to aging, immunity, and cancer. This review describes key regulators of this form of cell death within a framework of metabolism, ROS biology, and iron biology. Key concepts and major unanswered questions in the ferroptosis field are highlighted. The next decade promises to yield further breakthroughs in the mechanisms governing ferroptosis and additional ways of harnessing ferroptosis for therapeutic benefit.

SnapShot: Ferroptosis.

Ferroptosis is a regulated form of cell death that occurs when phospholipids with polyunsaturated fatty acyl tails are oxidized in an iron-dependent manner. Research in recent years has uncovered complex cellular networks that induce and suppress lethal lipid peroxidation. This SnapShot provides an overview of ferroptosis-related pathways, including relevant biomolecules and small-molecule modulators regulating them.

Ironing out the role of ferroptosis in immunity.

Ferroptosis is a type of regulated cell death that drives the pathophysiology of many diseases. Oxidative stress is detectable in many types of regulated cell death, but only ferroptosis involves lipid peroxidation and iron dependency. Ferroptosis originates and propagates from several organelles, including the mitochondria, endoplasmic reticulum, Golgi, and lysosomes. Recent data have revealed that immune cells can both induce and undergo ferroptosis. A mechanistic understanding of how ferroptosis regulates immunity is critical to understanding how ferroptosis controls immune responses and how this is dysregulated in disease. Translationally, more work is needed to produce ferroptosis-modulating immunotherapeutics. This review focuses on the role of ferroptosis in immune-related diseases, including infection, autoimmune diseases, and cancer. We discuss how ferroptosis is regulated in immunity, how this regulation contributes to disease pathogenesis, and how targeting ferroptosis may lead to novel therapies.

Ferroptosis: mechanisms, biology and role in disease.

The research field of ferroptosis has seen exponential growth over the past few years, since the term was coined in 2012. This unique modality of cell death, driven by iron-dependent phospholipid peroxidation, is regulated by multiple cellular metabolic pathways, including redox homeostasis, iron handling, mitochondrial activity and metabolism of amino acids, lipids and sugars, in addition to various signalling pathways relevant to disease. Numerous organ injuries and degenerative pathologies are driven by ferroptosis. Intriguingly, therapy-resistant cancer cells, particularly those in the mesenchymal state and prone to metastasis, are exquisitely vulnerable to ferroptosis. As such, pharmacological modulation of ferroptosis, via both its induction and its inhibition, holds great potential for the treatment of drug-resistant cancers, ischaemic organ injuries and other degenerative diseases linked to extensive lipid peroxidation. In this Review, we provide a critical analysis of the current molecular mechanisms and regulatory networks of ferroptosis, the potential physiological functions of ferroptosis in tumour suppression and immune surveillance, and its pathological roles, together with a potential for therapeutic targeting. Importantly, as in all rapidly evolving research areas, challenges exist due to misconceptions and inappropriate experimental methods. This Review also aims to address these issues and to provide practical guidelines for enhancing reproducibility and reliability in studies of ferroptosis. Finally, we discuss important concepts and pressing questions that should be the focus of future ferroptosis research.

Transforming Lipoxygenases: PE-Specific Enzymes in Disguise.

In this issue of Cell, Wenzel et al. solve a long-standing mystery regarding how damage to cell membranes occurs during ferroptosis, an iron-dependent form of regulated cell death. They found that lipoxygenases are like Transformer toys, being converted from one enzyme type to another in the presence of the protein PEBP1.

Multivalent Small-Molecule Pan-RAS Inhibitors.

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.

Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

ALOX5-mediated ferroptosis acts as a distinct cell death pathway upon oxidative stress in Huntington's disease.

Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine-expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, we showed that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mice. Moreover, by using RNAi-mediated screening, we identified ALOX5 as a major factor required for the ACSL4-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.

Blended Learning Improves Science Education.

Blended learning is an emerging paradigm for science education but has not been rigorously assessed. We performed a randomized controlled trial of blended learning. We found that in-class problem solving improved exam performance, and video assignments increased attendance and satisfaction. This validates a new model for science communication and education.

Elucidating Compound Mechanism of Action by Network Perturbation Analysis.

Genome-wide identification of the mechanism of action (MoA) of small-molecule compounds characterizing their targets, effectors, and activity modulators represents a highly relevant yet elusive goal, with critical implications for assessment of compound efficacy and toxicity. Current approaches are labor intensive and mostly limited to elucidating high-affinity binding target proteins. We introduce a regulatory network-based approach that elucidates genome-wide MoA proteins based on the assessment of the global dysregulation of their molecular interactions following compound perturbation. Analysis of cellular perturbation profiles identified established MoA proteins for 70% of the tested compounds and elucidated novel proteins that were experimentally validated. Finally, unknown-MoA compound analysis revealed altretamine, an anticancer drug, as an inhibitor of glutathione peroxidase 4 lipid repair activity, which was experimentally confirmed, thus revealing unexpected similarity to the activity of sulfasalazine. This suggests that regulatory network analysis can provide valuable mechanistic insight into the elucidation of small-molecule MoA and compound similarity.

Vitamin K: A new guardian against ferroptosis.

The dietary factor vitamin K has been found to protect against ferroptosis, a form of cell death driven by lipid peroxidation. This reveals new dietary links to cancers and degenerative conditions and a key factor involved in warfarin poisoning.

Regulation of ferroptotic cancer cell death by GPX4.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

A cell state-specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma.

Glioma cells hijack developmental programs to control cell state. Here, we uncover a glioma cell state-specific metabolic liability that can be therapeutically targeted. To model cell conditions at brain tumor inception, we generated genetically engineered murine gliomas, with deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling astrocyte differentiation during brain development. N1IC tumors harbored quiescent astrocyte-like transformed cell populations while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. Further, N1IC transformed cells exhibited increased mitochondrial lipid peroxidation, high ROS production and depletion of reduced glutathione. This altered mitochondrial phenotype rendered the astrocyte-like, quiescent populations more sensitive to pharmacologic or genetic inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Treatment of patient-derived early-passage cell lines and glioma slice cultures generated from surgical samples with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles. Collectively, these findings reveal a specific therapeutic vulnerability to ferroptosis linked to mitochondrial redox imbalance in a subpopulation of quiescent astrocyte-like glioma cells resistant to standard forms of treatment.

An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules.

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene ß-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.

MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Protein folding drives disulfide formation.

PDI catalyzes the oxidative folding of disulfide-containing proteins. However, the sequence of reactions leading to a natively folded and oxidized protein remains unknown. Here we demonstrate a technique that enables independent measurements of disulfide formation and protein folding. We find that non-native disulfides are formed early in the folding pathway and can trigger misfolding. In contrast, a PDI domain favors native disulfides by catalyzing oxidation at a late stage of folding. We propose a model for cotranslational oxidative folding wherein PDI acts as a placeholder that is relieved by the pairing of cysteines caused by substrate folding. This general mechanism can explain how PDI catalyzes oxidative folding in a variety of structurally unrelated substrates.

Ferroptosis: an iron-dependent form of nonapoptotic cell death.

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.