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Ferroptosis—an iron-dependent, non-apoptotic cell death process—is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers 1 . The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions 2 – 5 . However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR–Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome–ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis. The cellular organelles peroxisomes contribute to the sensitivity of cells to ferroptosis by synthesizing polyunsaturated ether phospholipids, and changes in the abundances of these lipids are associated with altered sensitivity to ferroptosis during cell-state transitions.

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase 1 , which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis 2 . Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production 3 . Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials 4 , its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy 5 . To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR–Cas9-based screen 6 , 7 . This screen yielded FTCD , which encodes an enzyme—formimidoyltransferase cyclodeaminase—that is required for the catabolism of the amino acid histidine 8 , a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention. Histidine metabolism influences the sensitivity of cancer cells to methotrexate, with mice bearing leukaemia xenografts showing increased response to the drug upon histidine supplementation.

One-carbon (1C) metabolism is a universal metabolic process that is required for purine synthesis and supports the high levels of proliferation in cancer cells. The transport of serine into mitochondria supplies most of the 1C units needed for biosynthesis. Kory et al. used a genetic screen to identify the long-sought-after mitochondrial serine transporter. Elucidating the key step of serine transport is important for our understanding of metabolism and has potential implications for cancer treatment. Science , this issue p. eaat9528 A mitochondrial serine transporter supports one-carbon metabolism, which is important for nucleotide synthesis in both normal and cancer cells. One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.

Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.

Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson’s disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conduct a genome-wide CRISPR screen and uncover an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinson’s disease. By offering a lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development. Polyamines are essential metabolites linked to aging, cancer, and Parkinson’s disease. Here, authors develop a live-cell polyamine reporter and use a genome-wide CRISPR screen to uncover a link between mitochondrial respiration and polyamine import.

Many essential proteins require pyridoxal 5’-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5’-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5’-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5’-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5’-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5’-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5’-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia. Pyridoxal 5’-phosphate (PLP, vitamin B6) is crucial for various metabolic processes. Here, the authors identified SLC25A38 as a key regulator of mitochondrial PLP levels using a genome-wide CRISPRi screen and organellar metabolomics, with insights into congenital sideroblastic anemia.

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN -adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1 , which is adjacent to and frequently co-deleted with PTEN , predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion. Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN , the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1 , individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 – and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1 -deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR–Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance. Mitochondrial energy metabolism regulates proteotoxic stress tolerance, exposing a newly discovered sensitivity to the small molecule elesclomol, which induces FDX1-mediated, copper-dependent cell death.

Epithelial–mesenchymal transition (EMT) programs operate within carcinoma cells, where they generate phenotypes associated with malignant progression. In their various manifestations, EMT programs enable epithelial cells to enter into a series of intermediate states arrayed along the E–M phenotypic spectrum. At present, we lack a coherent understanding of how carcinoma cells control their entrance into and continued residence in these various states, and which of these states favour the process of metastasis. Here we characterize a layer of EMT-regulating machinery that governs E–M plasticity (EMP). This machinery consists of two chromatin-modifying complexes, PRC2 and KMT2D-COMPASS, which operate as critical regulators to maintain a stable epithelial state. Interestingly, loss of these two complexes unlocks two distinct EMT trajectories. Dysfunction of PRC2, but not KMT2D-COMPASS, yields a quasi-mesenchymal state that is associated with highly metastatic capabilities and poor survival of patients with breast cancer, suggesting that great caution should be applied when PRC2 inhibitors are evaluated clinically in certain patient cohorts. These observations identify epigenetic factors that regulate EMP, determine specific intermediate EMT states and, as a direct consequence, govern the metastatic ability of carcinoma cells. Through genome-wide and focused CRISPR screens, Zhang et al. discover that loss of PRC2 or KMT2D-COMPASS enables distinct EMT trajectories, which exert differential effects on the metastatic capability of carcinoma cells.

mTORC1 is shown to regulate a translational program that requires the rapamycin-resistant 4E-BP family of translational repressors and consists almost entirely of mRNAs containing 5′ terminal oligopyrimidine or related motifs. Mechanism of mTOR signalling The mTOR pathway is important in the regulation of protein synthesis and is activated in many human cancers. Two papers in this issue of Nature use ribosome profiling to study the control of messenger RNA translation by mTOR signalling. Hsieh et al . find that in prostate cancer cells and mouse prostate tumours, the translation of several genes involved in cancer invasion is regulated by mTOR by means of the 4EBP1 translational repressor. The experimental drug INK128, currently in clinical trials in people with prostate cancer, inhibits mTOR signalling and reduces the progression of prostate cancers to invasive carcinomas in a mouse model. Thoreen et al . show that through the 4E-BP protein family, the mTORC1 kinase recognizes and regulates a subset of mRNAs with an oligopyrimidine motif at the 5′ end. The mTOR complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses 1 . mTORC1 regulates messenger RNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in mouse cells acutely treated with the mTOR inhibitor Torin 1, which, unlike rapamycin, fully inhibits mTORC1 (ref. 2 ). Our data reveal a surprisingly simple model of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5′ terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1 , with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5′ untranslated region length or complexity 3 . mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown 4 . Remarkably, loss of just the 4E-BP family of translational repressors, arguably the best characterized mTORC1 substrates, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin 1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors.