RESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ferroptosis , Inmunoterapia , Neoplasias/inmunología , Neoplasias/terapia , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Cisteína/metabolismo , Femenino , Ferroptosis/efectos de los fármacos , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Humanos , Interferón gamma/inmunología , Peroxidación de Lípido , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Ratones , Neoplasias/metabolismo , Nivolumab/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Catálisis , Enzimas , Evolución Molecular , Sustitución de Aminoácidos , Dominio Catalítico , Enzimas/genética , Enzimas/metabolismo , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Inmunoterapia , Cinética , Neoplasias/terapiaRESUMEN
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.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Carcinoma de Células Renales , Cistina , Ferroptosis , Glutatión , Neoplasias Renales , Sistema de Transporte de Aminoácidos y+/metabolismo , Transporte Biológico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Cistina/metabolismo , Glutatión/metabolismo , Disulfuro de Glutatión/deficiencia , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Terapia Molecular Dirigida , gamma-Glutamiltransferasa/metabolismoRESUMEN
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.
Asunto(s)
Cistationina gamma-Liasa/farmacología , Metionina/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/aislamiento & purificación , Cistationina gamma-Liasa/uso terapéutico , Daño del ADN/efectos de los fármacos , Pruebas de Enzimas , Humanos , Masculino , Metionina/sangre , Metionina/metabolismo , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/sangre , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Pruebas de Toxicidad Aguda , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Linfocitos T/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Purina-Nucleósido Fosforilasa/genética , Inmunoterapia , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
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.
Asunto(s)
Carcinoma de Células Renales , Quistes , Ferroptosis , Neoplasias Renales , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Cisteína/metabolismo , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Leiomiomatosis , Masculino , Ratones , Síndromes Neoplásicos Hereditarios , Sirolimus/farmacología , Neoplasias Cutáneas , Neoplasias UterinasRESUMEN
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.
RESUMEN
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.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Cisteína/deficiencia , Ferroptosis , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Animales , Transportador de Aminoácidos Catiónicos 1/genética , Línea Celular Tumoral , Cistationina gamma-Liasa/administración & dosificación , Cistationina gamma-Liasa/farmacología , Cistina/metabolismo , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Eliminación de Gen , Humanos , Ratones , Ratones MutantesRESUMEN
Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Hidrolasas/uso terapéutico , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/metabolismo , Neoplasias/tratamiento farmacológico , Animales , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Humanos , Neoplasias/enzimología , Neoplasias/inmunología , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.
Asunto(s)
Neoplasias de la Mama/metabolismo , Cistationina gamma-Liasa/farmacología , Cisteína/efectos de los fármacos , Cistina/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/metabolismo , Polietilenglicoles/farmacología , Neoplasias de la Próstata/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Cisteína/metabolismo , Cistina/metabolismo , Femenino , Glutatión/metabolismo , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Trasplante de Neoplasias , Estrés Oxidativo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Taurine, the most abundant free amino acid in mammals, with many critical roles such as neuronal development, had so far only been reported to be synthetized in eukaryotes. Taurine is the major product of cysteine metabolism in mammals, and its biosynthetic pathway consists of cysteine dioxygenase and cysteine sulfinic acid decarboxylase (hCSAD). Sequence, structural, and mutational analyses of the structurally and sequentially related hCSAD and human glutamic acid decarboxylase (hGAD) enzymes revealed a three residue substrate recognition motif (X1aa19X2aaX3), within the active site that is responsible for coordinating their respective preferred amino acid substrates. Introduction of the cysteine sulfinic acid (CSA) motif into hGAD (hGAD-S192F/N212S/F214Y) resulted in an enzyme with a >700 fold switch in selectivity toward the decarboxylation of CSA over its preferred substrate, l-glutamic acid. Surprisingly, we found this CSA recognition motif in the genome sequences of several marine bacteria, prompting us to evaluate the catalytic properties of bacterial amino acid decarboxylases that were predicted by sequence motif to decarboxylate CSA but had been annotated as GAD enzymes. We show that CSAD from Synechococcus sp. PCC 7335 specifically decarboxylated CSA and that the bacteria accumulated intracellular taurine. The fact that CSAD homologues exist in certain bacteria and are frequently found in operons containing the recently discovered bacterial cysteine dioxygenases that oxidize l-cysteine to CSA supports the idea that a bona fide bacterial taurine biosynthetic pathway exists in prokaryotes.
Asunto(s)
Bacterias/enzimología , Carboxiliasas/química , Carboxiliasas/metabolismo , Descubrimiento de Drogas , Taurina/biosíntesis , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Carboxiliasas/genética , Células Cultivadas , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Estructura Molecular , Transducción de Señal , Especificidad por Sustrato , Taurina/química , Taurina/genéticaRESUMEN
Arginases catalyze the divalent cation-dependent hydrolysis of L-arginine to urea and L-ornithine. There is significant interest in using arginase as a therapeutic antineogenic agent against L-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.