Unraveling ETC complex I function in ferroptosis reveals a potential ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

The role of the mitochondrial electron transport chain (ETC) in regulating ferroptosis is not fully elucidated. Here, we reveal that pharmacological inhibition of the ETC complex I reduces ubiquinol levels while decreasing ATP levels and activating AMP-activated protein kinase (AMPK), the two effects known for their roles in promoting and suppressing ferroptosis, respectively. Consequently, the impact of complex I inhibitors on ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition is limited. The pharmacological inhibition of complex I in LKB1-AMPK-inactivated cells, or genetic ablation of complex I (which does not trigger apparent AMPK activation), abrogates the AMPK-mediated ferroptosis-suppressive effect and sensitizes cancer cells to GPX4-inactivation-induced ferroptosis. Furthermore, complex I inhibition synergizes with radiotherapy (RT) to selectively suppress the growth of LKB1-deficient tumors by inducing ferroptosis in mouse models. Our data demonstrate a multifaceted role of complex I in regulating ferroptosis and propose a ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.

Spatial mapping of mitochondrial networks and bioenergetics in lung cancer.

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer.

Ferroptosis, a form of regulated cell death that is induced by excessive lipid peroxidation, is a key tumour suppression mechanism1-4. Glutathione peroxidase 4 (GPX4)5,6 and ferroptosis suppressor protein 1 (FSP1)7,8 constitute two major ferroptosis defence systems. Here we show that treatment of cancer cells with GPX4 inhibitors results in acute depletion of N-carbamoyl-L-aspartate, a pyrimidine biosynthesis intermediate, with concomitant accumulation of uridine. Supplementation with dihydroorotate or orotate-the substrate and product of dihydroorotate dehydrogenase (DHODH)-attenuates or potentiates ferroptosis induced by inhibition of GPX4, respectively, and these effects are particularly pronounced in cancer cells with low expression of GPX4 (GPX4low). Inactivation of DHODH induces extensive mitochondrial lipid peroxidation and ferroptosis in GPX4low cancer cells, and synergizes with ferroptosis inducers to induce these effects in GPX4high cancer cells. Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity). The DHODH inhibitor brequinar selectively suppresses GPX4low tumour growth by inducing ferroptosis, whereas combined treatment with brequinar and sulfasalazine, an FDA-approved drug with ferroptosis-inducing activity, synergistically induces ferroptosis and suppresses GPX4high tumour growth. Our results identify a DHODH-mediated ferroptosis defence mechanism in mitochondria and suggest a therapeutic strategy of targeting ferroptosis in cancer treatment.

A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria.

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Targeting In Vivo Metabolic Vulnerabilities of Th2 and Th17 Cells Reduces Airway Inflammation.

T effector cells promote inflammation in asthmatic patients, and both Th2 and Th17 CD4 T cells have been implicated in severe forms of the disease. The metabolic phenotypes and dependencies of these cells, however, remain poorly understood in the regulation of airway inflammation. In this study, we show the bronchoalveolar lavage fluid of asthmatic patients had markers of elevated glucose and glutamine metabolism. Further, peripheral blood T cells of asthmatics had broadly elevated expression of metabolic proteins when analyzed by mass cytometry compared with healthy controls. Therefore, we hypothesized that glucose and glutamine metabolism promote allergic airway inflammation. We tested this hypothesis in two murine models of airway inflammation. T cells from lungs of mice sensitized with Alternaria alternata extract displayed genetic signatures for elevated oxidative and glucose metabolism by single-cell RNA sequencing. This result was most pronounced when protein levels were measured in IL-17-producing cells and was recapitulated when airway inflammation was induced with house dust mite plus LPS, a model that led to abundant IL-4- and IL-17-producing T cells. Importantly, inhibitors of the glucose transporter 1 or glutaminase in vivo attenuated house dust mite + LPS eosinophilia, T cell cytokine production, and airway hyperresponsiveness as well as augmented the immunosuppressive properties of dexamethasone. These data show that T cells induce markers to support metabolism in vivo in airway inflammation and that this correlates with inflammatory cytokine production. Targeting metabolic pathways may provide a new direction to protect from disease and enhance the effectiveness of steroid therapy.

Metabolic QTL analysis links chloroquine resistance in Plasmodium falciparum to impaired hemoglobin catabolism.

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations.

Branched tricarboxylic acid metabolism in Plasmodium falciparum.

A central hub of carbon metabolism is the tricarboxylic acid cycle, which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which tricarboxylic acid metabolism plays a minor role. Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen, yet the parasite genome encodes all of the enzymes necessary for a complete tricarboxylic acid cycle. Here, by tracing (13)C-labelled compounds using mass spectrometry we show that tricarboxylic acid metabolism in the human malaria parasite Plasmodium falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture from the canonical textbook pathway. We find that this pathway is not cyclic, but rather is a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction, thereby generating two-carbon units in the form of acetyl-coenzyme A. We further show that glutamine-derived acetyl-coenzyme A is used for histone acetylation, whereas glucose-derived acetyl-coenzyme A is used to acetylate amino sugars. Thus, the parasite has evolved two independent production mechanisms for acetyl-coenzyme A with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments.

One-carbon unit supplementation fuels purine synthesis in tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate contributions to purine nucleotides from salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic or lymph node T cells) synthesize purines de novo. Shortage of 1C units for T cell purine synthesis is accordingly a potential bottleneck for anti-tumor immunity. Supplementing 1C units by infusing formate drives formate assimilation into purines in tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling kinetic control of formate production. Safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade in MC38 tumors, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

Cell cycle arrest induces lipid droplet formation and confers ferroptosis resistance.

How cells coordinate cell cycling with cell survival and death remains incompletely understood. Here, we show that cell cycle arrest has a potent suppressive effect on ferroptosis, a form of regulated cell death induced by overwhelming lipid peroxidation at cellular membranes. Mechanistically, cell cycle arrest induces diacylglycerol acyltransferase (DGAT)-dependent lipid droplet formation to sequester excessive polyunsaturated fatty acids (PUFAs) that accumulate in arrested cells in triacylglycerols (TAGs), resulting in ferroptosis suppression. Consequently, DGAT inhibition orchestrates a reshuffling of PUFAs from TAGs to phospholipids and re-sensitizes arrested cells to ferroptosis. We show that some slow-cycling antimitotic drug-resistant cancer cells, such as 5-fluorouracil-resistant cells, have accumulation of lipid droplets and that combined treatment with ferroptosis inducers and DGAT inhibitors effectively suppresses the growth of 5-fluorouracil-resistant tumors by inducing ferroptosis. Together, these results reveal a role for cell cycle arrest in driving ferroptosis resistance and suggest a ferroptosis-inducing therapeutic strategy to target slow-cycling therapy-resistant cancers.

Proteomic analysis of ferroptosis pathways reveals a role of CEPT1 in suppressing ferroptosis.

Ferroptosis has been recognized as a unique cell death modality driven by excessive lipid peroxidation and unbalanced cellular metabolism. In this study, we established a protein interaction landscape for ferroptosis pathways through proteomic analyses, and identified choline/ethanolamine phosphotransferase 1 (CEPT1) as a lysophosphatidylcholine acyltransferase 3 (LPCAT3)-interacting protein that regulates LPCAT3 protein stability. In contrast to its known role in promoting phospholipid synthesis, we showed that CEPT1 suppresses ferroptosis potentially by interacting with phospholipases and breaking down certain pro-ferroptotic polyunsaturated fatty acid (PUFA)-containing phospholipids. Together, our study reveals a previously unrecognized role of CEPT1 in suppressing ferroptosis.

Concurrent loss of LKB1 and KEAP1 enhances SHMT-mediated antioxidant defence in KRAS-mutant lung cancer.

Non-small-cell lung cancer (NSCLC) with concurrent mutations in KRAS and the tumour suppressor LKB1 (KL NSCLC) is refractory to most therapies and has one of the worst predicted outcomes. Here we describe a KL-induced metabolic vulnerability associated with serine-glycine-one-carbon (SGOC) metabolism. Using RNA-seq and metabolomics data from human NSCLC, we uncovered that LKB1 loss enhanced SGOC metabolism via serine hydroxymethyltransferase (SHMT). LKB1 loss, in collaboration with KEAP1 loss, activated SHMT through inactivation of the salt-induced kinase (SIK)-NRF2 axis and satisfied the increased demand for one-carbon units necessary for antioxidant defence. Chemical and genetic SHMT suppression increased cellular sensitivity to oxidative stress and cell death. Further, the SHMT inhibitor enhanced the in vivo therapeutic efficacy of paclitaxel (first-line NSCLC therapy inducing oxidative stress) in KEAP1-mutant KL tumours. The data reveal how this highly aggressive molecular subtype of NSCLC fulfills their metabolic requirements and provides insight into therapeutic strategies.

One-carbon unit supplementation fuels tumor-infiltrating T cells and augments checkpoint blockade.

Nucleotides perform important metabolic functions, carrying energy and feeding nucleic acid synthesis. Here, we use isotope tracing-mass spectrometry to quantitate the contributions to purine nucleotides of salvage versus de novo synthesis. We further explore the impact of augmenting a key precursor for purine synthesis, one-carbon (1C) units. We show that tumors and tumor-infiltrating T cells (relative to splenic T cells) synthesize purines de novo. Purine synthesis requires two 1C units, which come from serine catabolism and circulating formate. Shortage of 1C units is a potential bottleneck for anti-tumor immunity. Elevating circulating formate drives its usage by tumor-infiltrating T cells. Orally administered methanol functions as a formate pro-drug, with deuteration enabling control of formate-production kinetics. In MC38 tumors, safe doses of methanol raise formate levels and augment anti-PD-1 checkpoint blockade, tripling durable regressions. Thus, 1C deficiency can gate antitumor immunity and this metabolic checkpoint can be overcome with pharmacological 1C supplementation.

SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells.

The cystine transporter solute carrier family 7 member 11 (SLC7A11; also called xCT) protects cancer cells from oxidative stress and is overexpressed in many cancers. Here we report a surprising finding that, whereas moderate overexpression of SLC7A11 is beneficial for cancer cells treated with H2O2, a common oxidative stress inducer, its high overexpression dramatically increases H2O2-induced cell death. Mechanistically, high cystine uptake in cancer cells with high overexpression of SLC7A11 in combination with H2O2 treatment results in toxic buildup of intracellular cystine and other disulfide molecules, NADPH depletion, redox system collapse, and rapid cell death (likely disulfidptosis). We further show that high overexpression of SLC7A11 promotes tumor growth but suppresses tumor metastasis, likely because metastasizing cancer cells with high expression of SLC7A11 are particularly susceptible to oxidative stress. Our findings reveal that SLC7A11 expression level dictates cancer cells' sensitivity to oxidative stress and suggests a context-dependent role for SLC7A11 in tumor biology.

Actin cytoskeleton vulnerability to disulfide stress mediates disulfidptosis.

SLC7A11-mediated cystine uptake suppresses ferroptosis yet promotes cell death under glucose starvation; the nature of the latter cell death remains unknown. Here we show that aberrant accumulation of intracellular disulfides in SLC7A11high cells under glucose starvation induces a previously uncharacterized form of cell death distinct from apoptosis and ferroptosis. We term this cell death disulfidptosis. Chemical proteomics and cell biological analyses showed that glucose starvation in SLC7A11high cells induces aberrant disulfide bonds in actin cytoskeleton proteins and F-actin collapse in a SLC7A11-dependent manner. CRISPR screens and functional studies revealed that inactivation of the WAVE regulatory complex (which promotes actin polymerization and lamellipodia formation) suppresses disulfidptosis, whereas constitutive activation of Rac promotes disulfidptosis. We further show that glucose transporter inhibitors induce disulfidptosis in SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results reveal that the susceptibility of the actin cytoskeleton to disulfide stress mediates disulfidptosis and suggest a therapeutic strategy to target disulfidptosis in cancer treatment.

Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite.

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum.

A targetable CoQ-FSP1 axis drives ferroptosis- and radiation-resistance in KEAP1 inactive lung cancers.

Targeting ferroptosis, a unique cell death modality triggered by unrestricted lipid peroxidation, in cancer therapy is hindered by our incomplete understanding of ferroptosis mechanisms under specific cancer genetic contexts. KEAP1 (kelch-like ECH associated protein 1) is frequently mutated or inactivated in lung cancers, and KEAP1 mutant lung cancers are refractory to most therapies, including radiotherapy. In this study, we identify ferroptosis suppressor protein 1 (FSP1, also known as AIFM2) as a transcriptional target of nuclear factor erythroid 2-related factor 2 (NRF2) and reveal that the ubiquinone (CoQ)-FSP1 axis mediates ferroptosis- and radiation- resistance in KEAP1 deficient lung cancer cells. We further show that pharmacological inhibition of the CoQ-FSP1 axis sensitizes KEAP1 deficient lung cancer cells or patient-derived xenograft tumors to radiation through inducing ferroptosis. Together, our study identifies CoQ-FSP1 as a key downstream effector of KEAP1-NRF2 pathway and as a potential therapeutic target for treating KEAP1 mutant lung cancers.

Inhibition of glucose transport synergizes with chemical or genetic disruption of mitochondrial metabolism and suppresses TCA cycle-deficient tumors.

Efforts to target glucose metabolism in cancer have been limited by the poor potency and specificity of existing anti-glycolytic agents and a poor understanding of the glucose dependence of cancer subtypes in vivo. Here, we present an extensively characterized series of potent, orally bioavailable inhibitors of the class I glucose transporters (GLUTs). The representative compound KL-11743 specifically blocks glucose metabolism, triggering an acute collapse in NADH pools and a striking accumulation of aspartate, indicating a dramatic shift toward oxidative phosphorylation in the mitochondria. Disrupting mitochondrial metabolism via chemical inhibition of electron transport, deletion of the malate-aspartate shuttle component GOT1, or endogenous mutations in tricarboxylic acid cycle enzymes, causes synthetic lethality with KL-11743. Patient-derived xenograft models of succinate dehydrogenase A (SDHA)-deficient cancers are specifically sensitive to KL-11743, providing direct evidence that TCA cycle-mutant tumors are vulnerable to GLUT inhibitors in vivo.