MTA-cooperative PRMT5 inhibitors enhance T cell-mediated antitumor activity in MTAP-loss tumors.

BACKGROUND: Hyperactivated protein arginine methyltransferases (PRMTs) are implicated in human cancers. Inhibiting tumor intrinsic PRMT5 was reported to potentiate antitumor immune responses, highlighting the possibility of combining PRMT5 inhibitors (PRMT5i) with cancer immunotherapy. However, global suppression of PRMT5 activity impairs the effector functions of immune cells. Here, we sought to identify strategies to specifically inhibit PRMT5 activity in tumor tissues and develop effective PRMT5i-based immuno-oncology (IO) combinations for cancer treatment, particularly for methylthioadenosine phosphorylase (MTAP)-loss cancer. METHODS: Isogeneic tumor lines with and without MTAP loss were generated by CRISPR/Cas9 knockout. The effects of two PRMT5 inhibitors (GSK3326595 and MRTX1719) were evaluated in these isogenic tumor lines and T cells in vitro and in vivo. Transcriptomic and proteomic changes in tumors and T cells were characterized in response to PRMT5i treatment. Furthermore, the efficacy of MRTX1719 in combination with immune checkpoint blockade was assessed in two syngeneic murine models with MTAP-loss tumor. RESULTS: GSK3326595 significantly suppresses PRMT5 activity in tumors and T cells regardless of the MTAP status. However, MRTX1719, a methylthioadenosine-cooperative PRMT5 inhibitor, exhibits tumor-specific PRMT5 inhibition in MTAP-loss tumors with limited immunosuppressive effects. Mechanistically, transcriptomic and proteomic profiling analysis reveals that MRTX1719 successfully reduces the activation of the PI3K pathway, a well-documented immune-resistant pathway. It highlights the potential of MRTX1719 to overcome immune resistance in MTAP-loss tumors. In addition, MRTX1719 sensitizes MTAP-loss tumor cells to the killing of tumor-reactive T cells. Combining MRTX1719 and anti-PD-1 leads to superior antitumor activity in mice bearing MTAP-loss tumors. CONCLUSION: Collectively, our results provide a strong rationale and mechanistic insights for the clinical development of MRTX1719-based IO combinations in MTAP-loss tumors.

Enzyme-mediated depletion of methylthioadenosine restores T cell function in MTAP-deficient tumors and reverses immunotherapy resistance.

Chromosomal region 9p21 containing tumor suppressors CDKN2A/B and methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic deletions in cancer. 9p21 loss is correlated with reduced tumor-infiltrating lymphocytes (TILs) and resistance to immune checkpoint inhibitor (ICI) therapy. Previously thought to be caused by CDKN2A/B loss, we now show that it is loss of MTAP that leads to poor outcomes on ICI therapy and reduced TIL density. MTAP loss causes accumulation of methylthioadenosine (MTA) both intracellularly and extracellularly and profoundly impairs T cell function via the inhibition of protein arginine methyltransferase 5 (PRMT5) and by adenosine receptor agonism. Administration of MTA-depleting enzymes reverses this immunosuppressive effect, increasing TILs and drastically impairing tumor growth and importantly, synergizes well with ICI therapy. As several studies have shown ICI resistance in 9p21/MTAP null/low patients, we propose that MTA degrading therapeutics may have substantial therapeutic benefit in these patients by enhancing ICI effectiveness.

Enzymatic depletion of l-Met using an engineered human enzyme as a novel therapeutic strategy for melanoma.

Many cancers, including melanoma, have a higher requirement for l-methionine in comparison with noncancerous cells. In this study, we show that administration of an engineered human methionine-γ-lyase (hMGL) significantly reduced the survival of both human and mouse melanoma cells in vitro. A multiomics approach was utilized to identify global changes in gene expression and in metabolite levels with hMGL treatment in melanoma cells. There was considerable overlap in the perturbed pathways identified in the two data sets. Common pathways were flagged for further investigation to understand their mechanistic importance. In this regard, hMGL treatment induced S and G2 phase cell cycle arrest, decreased nucleotide levels, and increased DNA double-strand breaks suggesting an important role for replication stress in the mechanism of hMGL effects on melanoma cells. Further, hMGL treatment resulted in increased cellular reactive oxygen species levels and increased apoptosis as well as uncharged transfer RNA pathway upregulation. Finally, treatment with hMGL significantly inhibited the growth of both mouse and human melanoma cells in orthotopic tumor models in vivo. Overall, the results of this study provide a strong rationale for further mechanistic evaluation and clinical development of hMGL for the treatment of melanoma skin cancer and other cancers.

Cysteine depletion sensitizes prostate cancer cells to agents that enhance DNA damage and to immune checkpoint inhibition.

BACKGROUND: Prostate Cancer (PCa) represents one of the most commonly diagnosed neoplasms in men and is associated with significant morbidity and mortality. Therapy resistance and significant side effects of current treatment strategies indicate the need for more effective agents to treat both androgen-dependent and androgen-independent PCa. In earlier studies, we demonstrated that depletion of L-cysteine/cystine with an engineered human enzyme, Cyst(e)inase, increased intracellular ROS levels and inhibited PCa growth in vitro and in vivo. The current study was conducted to further explore the mechanisms and potential combinatorial approaches with Cyst(e)inase for treatment of PCa. METHODS: DNA single strand breaks and clustered oxidative DNA damage were evaluated by alkaline comet assay and pulsed field gel electrophoresis, respectively. Neutral comet assay and immunofluorescence staining was used to measure DNA double strand breaks. Cell survival and reactive oxygen species level were measured by crystal violet assay and DCFDA staining, respectively. Western blot was used to determine protein expression. FACS analyses were preformed for immune cell phenotyping. Allograft and xenograft tumor models were used for assessing effects on tumor growth. RESULTS: PCa cells treated with Cyst(e)inase lead to DNA single and double strand breaks resulted from clustered oxidative DNA damage (SSBs and DSBs). Cyst(e)inase in combination with Auranofin, a thioredoxin reductase inhibitor, further increased intracellular ROS and DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo. A combination of Cyst(e)inase with a PARP inhibitor (Olaparib) also increased DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo without additional ROS induction. Knockdown of BRCA2 in PCa cells increased DSBs and enhanced sensitivity to Cyst(e)inase. Finally, Cyst(e)inase treatment altered tumor immune infiltrates and PD-L1 expression and sensitized PCa cells to anti-PD-L1 treatment. CONCLUSIONS: The current results demonstrate the importance of oxidative DNA damage either alone or in combination for Cyst(e)inase-induced anticancer activity. Furthermore, cysteine/cystine depletion alters the tumor immune landscape favoring enhanced immune checkpoint inhibition targeting PD-L1. Thus, combinatorial approaches with Cyst(e)inase could lead to novel therapeutic strategies for PCa.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

Hypersensitivity to ferroptosis in chromophobe RCC is mediated by a glutathione metabolic dependency and cystine import via solute carrier family 7 member 11.

Chromophobe (Ch) renal cell carcinoma (RCC) arises from the intercalated cell in the distal nephron. There are no proven treatments for metastatic ChRCC. A distinguishing characteristic of ChRCC is strikingly high levels of reduced (GSH) and oxidized (GSSG) glutathione. Here, we demonstrate that ChRCC-derived cells exhibit higher sensitivity to ferroptotic inducers compared with clear-cell RCC. ChRCC-derived cells are critically dependent on cystine via the cystine/glutamate antiporter xCT to maintain high levels of glutathione, making them sensitive to inhibitors of cystine uptake and cyst(e)inase. Gamma-glutamyl transferase 1 (GGT1), a key enzyme in glutathione homeostasis, is markedly suppressed in ChRCC relative to normal kidney. Importantly, GGT1 overexpression inhibits the proliferation of ChRCC cells in vitro and in vivo, suppresses cystine uptake, and decreases levels of GSH and GSSG. Collectively, these data identify ferroptosis as a metabolic vulnerability in ChRCC, providing a potential avenue for targeted therapy for these distinctive tumors.

Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre­steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5'-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants.

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1-4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides 'substitutants' to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity.

Cyst(e)inase-Rapamycin Combination Induces Ferroptosis in Both In Vitro and In Vivo Models of Hereditary Leiomyomatosis and Renal Cell Cancer.

Renal cell carcinomas associated with hereditary leiomyomatosis and renal cell cancer (HLRCC) are notoriously aggressive and represent the leading cause of death among patients with HLRCC. To date, a safe and effective standardized therapy for this tumor type is lacking. Here we show that the engineered synthetic therapeutic enzyme, Cyst(e)inase, when combined with rapamycin, can effectively induce ferroptosis in HLRCC cells in vivo. The drug combination promotes lipid peroxidation to a greater degree than cysteine deprivation or Cyst(e)inase treatment alone, while rapamycin treatment alone does not induce ferroptosis. Mechanistically, Cyst(e)inase induces ferroptosis by depleting the exogenous cysteine/cystine supply, while rapamycin reduces cellular ferritin level by promoting ferritins' destruction via ferritinophagy. Since both Cyst(e)inase and rapamycin are well tolerated clinically, the combination represents an opportunity to exploit ferroptosis induction as a cancer management strategy. Accordingly, using a xenograft mouse model, we showed that the combination treatment resulted in tumor growth suppression without any notable side effects. In contrast, both Cyst(e)inase only and rapamycin only treatment groups failed to induce a significant change when compared with the vehicle control group. Our results demonstrated the effectiveness of Cyst(e)inase-rapamycin combination in inducing ferroptotic cell death in vivo, supporting the potential translation of the combination therapy into clinical HLRCC management.

Combinatorial Approaches to Enhance DNA Damage following Enzyme-Mediated Depletion of L-Cys for Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest forms of cancer with very few available therapeutic options. We previously reported that an engineered human enzyme, cyst(e)inase, which degrades L-cysteine (L-Cys) and cystine, inhibits growth of multiple cancer cells, including PDAC both in vitro and in vivo. Here, we show that cyst(e)inase treatment leads to increased clustered oxidative DNA damage, DNA single-strand breaks, apurinic/apyrimidinic sites, and DNA double-strand breaks (DSBs) in PDAC cells sensitive to intracellular depletion of L-Cys that is associated with reduced survival. BRCA2-deficient PDAC cells exhibited increased DSBs and enhanced sensitivity to cyst(e)inase. The blocking of a second antioxidant pathway (thioredoxin/thioredoxin reductase) using auranofin or inhibiting DNA repair using the poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, led to significant increases in DSBs following cyst(e)inase treatment in all PDAC cells examined. Cyst(e)inase plus olaparib also synergistically inhibited growth of sensitive and resistant PDAC cells in both xenograft and allograft tumor models. Collectively, these results demonstrate an important role for oxidative DNA damage and ultimately DNA DSBs in the anticancer action of cyst(e)inase. The data further show the potential for combining agents that target alternate antioxidant pathways or by targeting DNA repair pathways or genetic liabilities in DNA repair pathways to enhance the therapeutic action of cyst(e)inase for PDAC.

Non-canonical Glutamate-Cysteine Ligase Activity Protects against Ferroptosis.

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are poorly characterized. Here, we find that cystine starvation of non-small-cell lung cancer cell lines induces an unexpected accumulation of γ-glutamyl-peptides, which are produced due to a non-canonical activity of glutamate-cysteine ligase catalytic subunit (GCLC). This activity is enriched in cell lines with high levels of NRF2, a key transcriptional regulator of GCLC, but is also inducible in healthy murine tissues following cysteine limitation. γ-glutamyl-peptide synthesis limits the accumulation of glutamate, thereby protecting against ferroptosis. These results indicate that GCLC has a glutathione-independent, non-canonical role in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation.

Conformational Dynamics Contribute to Substrate Selectivity and Catalysis in Human Kynureninase.

Kynureninases (KYNases) are enzymes that play a key role in tryptophan catabolism through the degradation of intermediate kynurenine and 3'-hydroxy-kynurenine metabolites (KYN and OH-KYN, respectively). Bacterial KYNases exhibit high catalytic efficiency toward KYN and moderate activity toward OH-KYN, whereas animal KYNases are highly selective for OH-KYN, exhibiting only minimal activity toward the smaller KYN substrate. These differences reflect divergent pathways for KYN and OH-KYN utilization in the respective kingdoms. We examined the Homo sapiens and Pseudomonas fluorescens KYNases (HsKYNase and PfKYNase respectively) using pre-steady-state and hydrogen-deuterium exchange mass spectrometry (HDX-MS) methodologies. We discovered that the activity of HsKYNase critically depends on formation of hydrogen bonds with the hydroxyl group of OH-KYN to stabilize the entire active site and allow productive substrate turnover. With the preferred OH-KYN substrate, stabilization is observed at the substrate-binding site and the region surrounding the PLP cofactor. With the nonpreferred KYN substrate, less stabilization occurs, revealing a direct correlation with activity. This correlation holds true for PfKYNases; however there is only a modest stabilization at the substrate-binding site, suggesting that substrate discrimination is simply achieved by steric hindrance. We speculate that eukaryotic KYNases use dynamic mobility as a mechanism of substrate specificity to commit OH-KYN to nicotinamide synthesis and avoid futile hydrolysis of KYN. These findings have important ramifications for the engineering of HsKynase with high KYN activity as required for clinical applications in cancer immunotherapy. Our study shows how homologous enzymes with conserved active sites can use dynamics to discriminate between two highly similar substrates.

Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

Cysteine depletion induces pancreatic tumor ferroptosis in mice.

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

Radiotherapy and Immunotherapy Promote Tumoral Lipid Oxidation and Ferroptosis via Synergistic Repression of SLC7A11.

A challenge in oncology is to rationally and effectively integrate immunotherapy with traditional modalities, including radiotherapy. Here, we demonstrate that radiotherapy induces tumor-cell ferroptosis. Ferroptosis agonists augment and ferroptosis antagonists limit radiotherapy efficacy in tumor models. Immunotherapy sensitizes tumors to radiotherapy by promoting tumor-cell ferroptosis. Mechanistically, IFNγ derived from immunotherapy-activated CD8+ T cells and radiotherapy-activated ATM independently, yet synergistically, suppresses SLC7A11, a unit of the glutamate-cystine antiporter xc-, resulting in reduced cystine uptake, enhanced tumor lipid oxidation and ferroptosis, and improved tumor control. Thus, ferroptosis is an unappreciated mechanism and focus for the development of effective combinatorial cancer therapy. SIGNIFICANCE: This article describes ferroptosis as a previously unappreciated mechanism of action for radiotherapy. Further, it shows that ferroptosis is a novel point of synergy between immunotherapy and radiotherapy. Finally, it nominates SLC7A11, a critical regulator of ferroptosis, as a mechanistic determinant of synergy between radiotherapy and immunotherapy.This article is highlighted in the In This Issue feature, p. 1631.

Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction.

BACKGROUND: Smoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction. RESULTS: This paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2- to 19-fold enhanced catalytic activity compared to wt at 10 µM nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen's access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity. CONCLUSIONS: Systematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme's immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.

Enzyme-mediated depletion of l-cyst(e)ine synergizes with thioredoxin reductase inhibition for suppression of pancreatic tumor growth.

Perturbing redox homeostasis potentially constitutes a selective cancer-killing strategy. An engineered human enzyme, cyst(e)inase that degrades extracellular cysteine (l-Cys) and cystine (CSSC) leading to depletion of intracellular l-Cys and glutathione (GSH) was evaluated for its effects on pancreatic cancer cell lines. Cyst(e)inase caused oxidative stress and apoptosis in only Panc1 cells, whereas MIA-PaCa2 and BxPC3 cells demonstrated survival under conditions of cyst(e)inase-mediated l-Cys depletion through maintenance of mitochondrial metabolism and lower levels of reactive oxygen species (ROS). A correlation was also observed between thioredoxin 1 protein levels and resistance to cyst(e)inase treatment. Notably, cyst(e)inase in combination with auranofin, a thioredoxin reductase inhibitor, caused a synergistic increase in mitochondrial ROS and apoptosis and inhibition of mitophagy in the more resistant cells. In addition, auranofin treatment sensitized the more resistant pancreatic cancer xenografts to cyst(e)inase without systemic toxicity. These data provide strong rationale to further investigate therapeutic strategies that target multiple antioxidant pathways for treatment of pancreatic ductal adenocarcinoma.

CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy.

Cancer immunotherapy restores or enhances the effector function of CD8+ T cells in the tumour microenvironment1,2. CD8+ T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways3,4. Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide5,6. Although it has been investigated in vitro7,8, there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios9,10. It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8+ T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNγ) released from CD8+ T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc-, impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc- was negatively associated, in cancer patients, with CD8+ T cell signature, IFNγ expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.

A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity.

The tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity.