RESUMO
Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure-based design of AG-946, an activator of PK isoforms, including red blood cell-specific forms of PK (PKR). This was designed to have a pseudo-C2-symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off-targets observed from the original scaffold. AG-946 (1) demonstrated activation of human wild-type PK (half-maximal activation concentration [AC50 ]=0.005â µM) and a panel of mutated PK proteins (K410E [AC50 =0.0043â µM] and R510Q [AC50 =0.0069â µM]), (2) displayed a significantly longer half-time of activation (>150-fold) compared with 6-(3-methoxybenzyl)-4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thieno[2',3':4,5]pyrrolo[2,3-d]pyridazin-5-one, and (3) stabilized PKR R510Q, an unstable mutant PKR enzyme, and preserved its catalytic activity under increasingly denaturing conditions. As a potent, oral, small-molecule allosteric activator of wild-type and mutant PKR, AG-946 was advanced to human clinical trials.
Assuntos
Trifosfato de Adenosina , Piruvato Quinase , Humanos , Sítio Alostérico , Sítios de Ligação , Ácido PirúvicoRESUMO
Polymerization of deoxygenated hemoglobin S underlies the pathophysiology of sickle cell disease (SCD). In activating red blood cell pyruvate kinase and glycolysis, mitapivat (AG-348) increases adenosine triphosphate (ATP) levels and decreases the 2,3-diphosphoglycerate (2,3-DPG) concentration, an upstream precursor in glycolysis. Both changes have therapeutic potential for patients with SCD. Here, we evaluated the safety and tolerability of multiple ascending doses of mitapivat in adults with SCD with no recent blood transfusions or changes in hydroxyurea or l-glutamine therapy. Seventeen subjects were enrolled; 1 subject was withdrawn shortly after starting the study. Sixteen subjects completed 3 ascending dose levels of mitapivat (5, 20, and 50 mg, twice daily [BID]) for 2 weeks each; following a protocol amendment, the dose was escalated to 100 mg BID in 9 subjects. Mitapivat was well tolerated at all dose levels, with the most common treatment-emergent adverse events (AEs) being insomnia, headache, and hypertension. Six serious AEs (SAEs) included 4 vaso-occlusive crises (VOCs), non-VOC-related shoulder pain, and a preexisting pulmonary embolism. Two VOCs occurred during drug taper and were possibly drug related; no other SAEs were drug related. Mean hemoglobin increase at the 50 mg BID dose level was 1.2 g/dL, with 9 of 16 (56.3%) patients achieving a hemoglobin response of a ≥1 g/dL increase compared with baseline. Mean reductions in hemolytic markers and dose-dependent decreases in 2,3-DPG and increases in ATP were also observed. This study provides proof of concept that mitapivat has disease-modifying potential in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.
Assuntos
Anemia Falciforme , Piruvato Quinase , Adulto , Humanos , Ácido Pirúvico , 2,3-Difosfoglicerato , Anemia Falciforme/tratamento farmacológico , Hemoglobinas , Trifosfato de AdenosinaRESUMO
Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.
Assuntos
Anemia Falciforme , 2,3-Difosfoglicerato/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Modelos Animais de Doenças , Eritrócitos/metabolismo , Hemoglobina Falciforme/metabolismo , Hemoglobinas/análise , Humanos , Camundongos , Mitocôndrias/metabolismo , Estresse Oxidativo , Piperazinas , QuinolinasRESUMO
Anemia in ß-thalassemia is related to ineffective erythropoiesis and reduced red cell survival. Excess free heme and accumulation of unpaired α-globin chains impose substantial oxidative stress on ß-thalassemic erythroblasts and erythrocytes, impacting cell metabolism. We hypothesized that increased pyruvate kinase activity induced by mitapivat (AG-348) in the Hbbth3/+ mouse model for ß-thalassemia would reduce chronic hemolysis and ineffective erythropoiesis through stimulation of red cell glycolytic metabolism. Oral mitapivat administration ameliorated ineffective erythropoiesis and anemia in Hbbth3/+ mice. Increased ATP, reduced reactive oxygen species production, and reduced markers of mitochondrial dysfunction associated with improved mitochondrial clearance suggested enhanced metabolism following mitapivat administration in ß-thalassemia. The amelioration of responsiveness to erythropoietin resulted in reduced soluble erythroferrone, increased liver Hamp expression, and diminished liver iron overload. Mitapivat reduced duodenal Dmt1 expression potentially by activating the pyruvate kinase M2-HIF2α axis, representing a mechanism additional to Hamp in controlling iron absorption and preventing ß-thalassemia-related liver iron overload. In ex vivo studies on erythroid precursors from patients with ß-thalassemia, mitapivat enhanced erythropoiesis, promoted erythroid maturation, and decreased apoptosis. Overall, pyruvate kinase activation as a treatment modality for ß-thalassemia in preclinical model systems had multiple beneficial effects in the erythropoietic compartment and beyond, providing a strong scientific basis for further clinical trials.
Assuntos
Ativadores de Enzimas/farmacologia , Hemólise/efeitos dos fármacos , Piperazinas/farmacologia , Piruvato Quinase/metabolismo , Quinolinas/farmacologia , Talassemia beta/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Transgênicos , Talassemia beta/enzimologia , Talassemia beta/genéticaAssuntos
Anemia Falciforme/enzimologia , Eritrócitos Anormais/enzimologia , Terapia de Alvo Molecular , Piperazinas/farmacologia , Piruvato Quinase/deficiência , Quinolinas/farmacologia , 2,3-Difosfoglicerato/sangue , Adolescente , Adulto , Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Forma Celular/efeitos dos fármacos , Criança , Pré-Escolar , Eritrócitos Anormais/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/uso terapêutico , Estabilidade Proteica , Piruvato Quinase/sangue , Piruvato Quinase/química , Quinolinas/uso terapêutico , Adulto JovemRESUMO
Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red cell (RBC) glycolysis, causing changes in metabolism including a deficiency in ATP. This affects red cell homeostasis, promoting premature removal of RBCs from the circulation. In this study we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBCs and erythroid precursors from PK-deficient patients. In 15 patients ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patient cells after 24 hours (mean increase 1.8-fold, range 1.2-3.4). ATP levels increased (mean increase 1.5-fold, range 1.0-2.2) similar to control cells (mean increase 1.6-fold, range, 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBCs. Ex vivo treatment with AG-348 increased residual activity 1.4 to >10-fold than residual activity of vehicle-treated samples. Protein analyses suggests that a sufficient level of PK protein is required for cells to respond to AG-348 treatment ex-vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG-348 effectively upregulates PK enzymatic activity and increases stability in PK-deficient RBCs over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).
Assuntos
Eritrócitos , Piruvato Quinase , Trifosfato de Adenosina , Eritrócitos/metabolismo , Genótipo , Humanos , Piperazinas , Estabilidade Proteica , Piruvato Quinase/genética , QuinolinasRESUMO
Diagnosis of pyruvate kinase deficiency (PKD), the most common cause of hereditary non-spherocytic haemolytic anaemia, remains challenging in routine practice and no biomarkers for clinical severity have been characterised. This prospective study enrolled 41 patients with molecularly confirmed PKD from nine North American centres to evaluate the diagnostic sensitivity of pyruvate kinase (PK) enzyme activity and PK:hexokinase (HK) enzyme activity ratio, and evaluate the erythrocyte PK (PK-R) protein level and erythrocyte metabolites as biomarkers for clinical severity. In this population not transfused for ≥90 days before sampling, the diagnostic sensitivity of the PK enzyme assay was 90% [95% confidence interval (CI) 77-97%], whereas the PK:HK ratio sensitivity was 98% (95% CI 87-100%). There was no correlation between PK enzyme activity and clinical severity. Transfusion requirements correlated with normalised erythrocyte ATP levels (r = 0·527, P = 0·0016) and PK-R protein levels (r = -0·527, P = 0·0028). PK-R protein levels were significantly higher in the never transfused [median (range) 40·1 (9·8-73·9)%] versus ever transfused [median (range) 7·7 (0·4-15·1)%] patients (P = 0·0014). The PK:HK ratio had excellent sensitivity for PK diagnosis, superior to PKLR exon sequencing. Given that the number of PKLR variants and genotype combinations limits prognostication based on molecular findings, PK-R protein level may be a useful prognostic biomarker of disease severity and merits further study.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/sangue , Eritrócitos/enzimologia , Hexoquinase/sangue , Piruvato Quinase/sangue , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/sangue , Adolescente , Adulto , Anemia Hemolítica Congênita não Esferocítica/genética , Biomarcadores/sangue , Criança , Pré-Escolar , Feminino , Hexoquinase/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Índice de Gravidade de DoençaRESUMO
The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.
Assuntos
Mitocôndrias/metabolismo , Fosfoenolpiruvato/metabolismo , Animais , Homeostase , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piruvato Quinase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.
Assuntos
Genoma/genética , Genômica , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Animais , Autofagia , Linhagem Celular Tumoral , Feminino , Genes Neoplásicos/genética , Humanos , Interferon gama/imunologia , Masculino , Camundongos , NF-kappa B/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure. Instead, we show that cyclin-dependent kinases (CDKs) are activated by the HFD and responsible for PKL phosphorylation at position S113 in addition to other targets. These CDKs control PKL nuclear retention, alter cytosolic PKL activity, and ultimately influence glucose production. These results change our view of PKL regulation and highlight a previously unrecognized pathway of hepatic CDK activity and metabolic control points that may be important in insulin resistance and type 2 diabetes.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Gluconeogênese , Hepatócitos/metabolismo , Piruvato Quinase/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Células Cultivadas , Dieta Hiperlipídica , Glucose/metabolismo , Resistência à Insulina , Masculino , Fosforilação , Piruvato Quinase/química , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Pyruvate kinase deficiency is caused by mutations in PKLR and leads to congenital hemolytic anemia. Mitapivat is an oral, small-molecule allosteric activator of pyruvate kinase in red cells. METHODS: In this uncontrolled, phase 2 study, we evaluated the safety and efficacy of mitapivat in 52 adults with pyruvate kinase deficiency who were not receiving red-cell transfusions. The patients were randomly assigned to receive either 50 mg or 300 mg of mitapivat twice daily for a 24-week core period; eligible patients could continue treatment in an ongoing extension phase. RESULTS: Common adverse events, including headache and insomnia, occurred at the time of drug initiation and were transient; 92% of the episodes of headache and 47% of the episodes of insomnia resolved within 7 days. The most common serious adverse events, hemolytic anemia and pharyngitis, each occurred in 2 patients (4%). A total of 26 patients (50%) had an increase of more than 1.0 g per deciliter in the hemoglobin level. Among these patients, the mean maximum increase was 3.4 g per deciliter (range, 1.1 to 5.8), and the median time until the first increase of more than 1.0 g per deciliter was 10 days (range, 7 to 187); 20 patients (77%) had an increase of more than 1.0 g per deciliter in the hemoglobin level at more than 50% of visits during the core study period, with improvement in markers of hemolysis. The response was sustained in all 19 patients remaining in the extension phase, with a median follow-up of 29 months (range, 22 to 35). Hemoglobin responses were observed only in patients who had at least one missense PKLR mutation and were associated with the red-cell pyruvate kinase protein level at baseline. CONCLUSIONS: The administration of mitapivat was associated with a rapid increase in the hemoglobin level in 50% of adults with pyruvate kinase deficiency, with a sustained response during a median follow-up of 29 months during the extension phase. Adverse effects were mainly low-grade and transient. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02476916.).
Assuntos
Anemia Hemolítica Congênita não Esferocítica/tratamento farmacológico , Hemoglobinas/metabolismo , Piperazinas/administração & dosagem , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/tratamento farmacológico , Quinolinas/administração & dosagem , Administração Oral , Adolescente , Adulto , Anemia Hemolítica Congênita não Esferocítica/sangue , Anemia Hemolítica Congênita não Esferocítica/genética , Catecóis , Esquema de Medicação , Feminino , Seguimentos , Cefaleia/induzido quimicamente , Humanos , Masculino , Mutação , Piperazinas/efeitos adversos , Piruvato Quinase/sangue , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/sangue , Erros Inatos do Metabolismo dos Piruvatos/genética , Quinolinas/efeitos adversos , Distúrbios do Início e da Manutenção do Sono/induzido quimicamente , Tirfostinas , Adulto JovemRESUMO
Pyruvate kinase deficiency is a chronic hemolytic anemia caused by mutations in PK-R, a key glycolytic enzyme in erythrocytes. These 2 phase 1 randomized, placebo-controlled, double-blind healthy-volunteer studies assessed the safety, tolerability, and pharmacokinetics/pharmacodynamics of AG-348, a first-in-class allosteric PK-R activator. Twelve sequential cohorts were randomized 2:6 to receive oral placebo or AG-348, respectively, as a single dose (30-2500 mg) in the single-ascending-dose (SAD) study (ClinicalTrials.gov: NCT02108106) or 15-700 mg every 12 hours or 120 mg every 24 hours, for 14 days in the multiple-ascending-dose (MAD) study (ClinicalTrials.gov: NCT02149966). All 48 subjects completed the fasted SAD part; 44 of 48 completed the MAD (2 discontinued because of adverse events [AEs], 2 withdrew consent). The most common treatment-related AEs in AG-348-treated subjects were headache (16.7% [SAD] and 13.9% [MAD]) and nausea (13.9%, both studies). AE frequency increased at AG-348 doses ≥ 700 mg (SAD) and at 700 mg every 12 hours (MAD); 1 grade ≥ 3 AE occurred in the latter cohort. Pharmacokinetics were favorable with low variability. Dose-dependent changes in blood glycolytic intermediates consistent with glycolytic pathway activation were observed at all MAD doses, supporting future trials investigating the potential of AG-348 for treating PK deficiency or other anemias.
Assuntos
Piperazinas/administração & dosagem , Piperazinas/farmacocinética , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Glicólise , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/efeitos adversos , Quinolinas/efeitos adversosRESUMO
Pyruvate kinase (PK) deficiency is a rare genetic disease that causes chronic hemolytic anemia. There are currently no targeted therapies for PK deficiency. Here, we describe the identification and characterization of AG-348, an allosteric activator of PK that is currently in clinical trials for the treatment of PK deficiency. We demonstrate that AG-348 can increase the activity of wild-type and mutant PK enzymes in biochemical assays and in patient red blood cells treated ex vivo. These data illustrate the potential for AG-348 to restore the glycolytic pathway activity in patients with PK deficiency and ultimately lead to clinical benefit.
Assuntos
Ativadores de Enzimas/farmacologia , Ativadores de Enzimas/uso terapêutico , Eritrócitos/enzimologia , Piruvato Quinase/deficiência , Piruvato Quinase/metabolismo , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Anemia Hemolítica Congênita não Esferocítica , Animais , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/química , Eritrócitos/efeitos dos fármacos , Humanos , Cinética , Camundongos , Piperazinas , Piruvato Quinase/efeitos dos fármacos , Erros Inatos do Metabolismo dos Piruvatos , Quinolinas/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/química , Doadores de TecidosRESUMO
Many hemolytic anemias results in major metabolic abnormalities: two common metabolite abnormalities include increased levels of 2,3-diphosphoglycerate (2,3-DPG) and decreased levels of adenosine triphosphate (ATP). To better monitor the concentration changes of these metabolites, the development of a reliable LC-MS/MS method to quantitatively profile the concentrations of 2, 3-DPG and ATP in whole blood is essential to understand the effects of investigational therapeutics. Accurate quantification of both compounds imposes great challenges to bioanalytical scientists due to their polar, ionic and endogenous nature. Here we present an LC-MS/MS method for the reliable quantification of 2,3-DPG and ATP from K2EDTA human whole blood (WB) simultaneously. Whole blood samples were spiked with stable isotope labeled internal standards, processed by protein precipitation extraction, and analyzed using zwitterionic ion chromatography-hydrophilic interaction chromatography (ZIC-HILIC) coupled with tandem mass spectrometry. The linear analytical range of the assay was 50-3000µg/mL. The fit-for-purpose method demonstrated excellent accuracy and precision. The overall accuracy was within ±10.5% (%RE) for both analytes and the intra- and inter-assay precision (%CV) were less than 6.7% and 6.2% for both analytes, respectively. ATP and 2,3-DPG were found to be stable in human K2EDTA blood for at least 8h at 4°C, 96days when stored at -70°C and after three freeze/thaw cycles. The assay has been successfully applied to K2EDTA human whole blood samples to support clinical studies.
Assuntos
2,3-Difosfoglicerato/sangue , Trifosfato de Adenosina/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , 2,3-Difosfoglicerato/química , Trifosfato de Adenosina/química , Ácido Edético , Humanos , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos TestesRESUMO
Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Anemia Hemolítica Congênita não Esferocítica/terapia , Terapia Genética , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/genética , Erros Inatos do Metabolismo dos Piruvatos/terapia , Anemia Hemolítica Congênita não Esferocítica/metabolismo , Animais , Células Sanguíneas/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoese , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/genética , Glicólise , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lentivirus/genética , Redes e Vias Metabólicas , Metaboloma , Metabolômica , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Erros Inatos do Metabolismo dos Piruvatos/metabolismo , Transdução GenéticaRESUMO
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Anemia Hemolítica Congênita não Esferocítica/terapia , Células Eritroides/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Erros Inatos do Metabolismo dos Piruvatos/terapia , Alelos , Sequência de Bases , Contagem de Células , DNA Complementar/genética , Células Eritroides/metabolismo , Marcação de Genes , Terapia Genética , Humanos , Leucócitos Mononucleares/metabolismo , Recombinação GenéticaRESUMO
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.
Assuntos
Aldeído Desidrogenase/genética , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Mutação/genética , Intoxicação Alcoólica/enzimologia , Intoxicação Alcoólica/patologia , Aldeído-Desidrogenase Mitocondrial , Substituição de Aminoácidos , Animais , Sequência de Bases , Carcinogênese/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Etanol/efeitos adversos , Técnicas de Introdução de Genes , Técnicas de Genotipagem , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Hiperpigmentação/patologia , Imuno-Histoquímica , Fígado/enzimologia , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Polimorfismo Genético , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Pele/patologia , Análise de SobrevidaRESUMO
Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Serina/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Hormônios Tireóideos/metabolismo , Sítio Alostérico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteínas de Ligação a Hormônio da TireoideRESUMO
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.
Assuntos
Biopolímeros/metabolismo , Transformação Celular Neoplásica , Ativadores de Enzimas/farmacologia , Piruvato Quinase/metabolismo , Animais , Biopolímeros/química , Western Blotting , Proliferação de Células , Humanos , Camundongos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Piruvato Quinase/químicaRESUMO
Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.