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.
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•Metabolomic profiling revealed GSH depletion as one mechanism of ferroptosis•Chemoproteomics identified GPX4 as a target for RSL3, a second ferroptosis inducer•GPX4 is a central regulator of ferroptosis, akin to bcl-2 in apoptosis•DLBCLs and renal cell carcinomas are sensitive to ferroptotic cell death
A chemoproteomic screen identifies a glutathione peroxidase as an essential regulator of a nonapoptotic form of cell death known as ferroptosis.
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 xc−), 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.
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► Iron-dependent cell death is similar to glutamate-induced excitotoxicity ► Ferroptosis is distinct from apoptosis, necrosis, and autophagy ► Ferroptosis is triggered by inhibition of cystine uptake ► Reduced cystine uptake leads to the production of lethal lipid ROS
The mechanism of drug-induced iron-dependent cell death in RAS-driven cancers relies on cystine/glutamate antiport and the accumulation of hydroxylated lipids. This type of death is similar to glutamate-induced excitotoxicity in neurons, and both types of cell death can be blocked with a specific small-molecule inhibitor called ferrostatin-1.
Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause ...lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown.We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.
Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of ...alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.
Ferroptosis is a distinctive form of regulated cell death that is driven by lethal accumulation of lipid peroxides in plasma membranes. Failure to control ferroptosis has been implicated in multiple ...pathological conditions including cancer development, neurodegeneration, renal injury, ischemia/reperfusion injury, and T-cell immunity. Here we describe a method to detect ferroptosis by determining the amount of lipid peroxides in cellular membranes using BODIPY-C11 probe and flow cytometry. Putative role of ferroptosis in immune modulatory cells can be determined using the same method.
Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human ...tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.
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.
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•DeMAND—a method to predict genes involved in mechanism of action of a compound•DeMAND predictions can be used to identify compound similarity•Known MoA genes are identified with high precision, sensitivity, and specificity•Novel predictions of both MoA and similarity were experimentally validated
The mechanism of action (MoA) of small-molecule compounds is elucidated by analyzing regulatory networks to identify proteins whose interactions are affected following compound perturbation. Experimental validation of novel MoA predictions revealed that the anticancer drug altretamine acts as an inhibitor of GPX4 lipid repair activity, revealing unexpected similarity to the activity of sulfasalazine.
Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA ...interference have enabled the realization of this goal.
We screened 5,760 short hairpin RNA clones targeting the human kinome to detect human kinases on which cancer cells are more dependent than normal cells. We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells. Analysis of gene-expression data revealed that CSNK1E is overexpressed in several cancer tissue samples examined compared to non-tumorigenic normal tissue, suggesting a positive role of CSNK1E in neogenesis or maintenance. Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition. In a search for substrates of CK1epsilon that mediate IC261-induced growth inhibition, we discovered that knocking down PER2, another clock gene involved in circadian rhythm control, rescues IC261-induced growth inhibition.
We identified CK1epsilon as a potential target for developing anticancer reagents with a high therapeutic index. These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.
Background:
Injection of intra-articular corticosteroids is effective for improving the recovery of range of motion (ROM) and pain in various conditions of the shoulder but its use is limited after ...rotator cuff repair owing to concern over the possible harmful effects of steroids on the repaired tendon.
Purpose:
To evaluate the effect of intra-articular corticosteroid injections on the clinical outcomes and cuff integrity of patients after rotator cuff repair.
Study Design:
Randomized controlled trial; Level of evidence, 1.
Methods:
Between March 2011 and April 2014, 80 patients with a small- to medium-sized rotator cuff tear were enrolled in this study and underwent arthroscopic rotator cuff repair. Forty patients received an injection of triamcinolone (40 mg) and lidocaine (1.5 mL) into the glenohumeral joint 8 weeks after surgery (group 1), while the remaining 40 patients received normal saline injection (group 2). Outcome measures—including ROM, American Shoulder and Elbow Surgeons (ASES) score, Constant score, pain visual analog scale, and Simple Shoulder Test score—were evaluated at 3, 6, and 12 months after surgery and at the last follow-up. The integrity of the repaired tendon was evaluated by magnetic resonance imaging (MRI) and classified per Sugaya classification at 8 weeks (before injection) and 12 months after surgery.
Results:
The mean follow-up period was 25.7 months. At 3 months postoperatively, patients in group 1 had a significantly higher ROM with respect to forward flexion (P = .05), external rotation at the side (P = .05), and external rotation at abduction (P = .04) as compared with group 2, whereas no significant difference was noted between the groups for internal rotation behind the back (P = .65). Patients in group 1 had significantly lower visual analog scale pain scores (P = .02) and higher ASES scores (group 1, 68.90; group 2, 60.28; P = .02) at 3-month follow-up. However, there was no significant difference after 6 months with respect to ROM and ASES scores (group 1, 77.80; group 2, 75.88; P = .33). Retears (Sugaya classification IV and V) were determined by MRI at 12 months and observed in a total of 7 patients (8.8%): 3 from group 1 (7.5%) and 4 from group 2 (10%). No retears were observed on MRI in the remaining 73 patients (91.2%): 37 patients from group 1 and 36 patients from group 2. There was no statistically significant difference in rate of retears between groups (P = .69).
Conclusion:
Intra-articular injection of corticosteroids after rotator cuff repair does not increase the risk of retears and is thus an effective and safe treatment method for increasing ROM (forward flexion, external rotation) and improving clinical score (ASES) during the early postoperative period of patients undergoing rotator cuff repair.
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