RESUMEN
Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.
Asunto(s)
Sistemas CRISPR-Cas , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Metotrexato/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Ciclo Celular , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.
Asunto(s)
Antimaláricos/química , Antimaláricos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Administración Oral , Animales , Antimaláricos/administración & dosificación , Antimaláricos/farmacocinética , Técnicas de Química Sintética , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Resistencia a Medicamentos/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Femenino , Glicina Hidroximetiltransferasa/química , Glicina Hidroximetiltransferasa/metabolismo , Células Hep G2/efectos de los fármacos , Humanos , Hígado/metabolismo , Hígado/parasitología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Ratones Endogámicos , Ratones SCID , Microsomas Hepáticos/efectos de los fármacos , Organismos Modificados Genéticamente , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/patogenicidad , Plasmodium falciparum/enzimología , Plasmodium falciparum/patogenicidad , Plasmodium vivax/enzimología , Plasmodium vivax/patogenicidad , Pirazoles/química , RatasRESUMEN
Serine hydroxymethyltransferase (SHMT) provides activated one-carbon units required for the biosynthesis of nucleotides, protein, and methyl group by converting serine and tetrahydrofolate to glycine and N(5),N(10)-methylenetetrahydrofolate. It is postulated that SHMT activity is associated with the development of methotrexate resistance and the in vivo activity of SHMT is regulated by the binding of N(5)-CHO-THF, the rescue agent in high-dose methotrexate chemotherapy. The aim of this study is to advance our understanding of the folate-mediated one-carbon metabolism in zebrafish by characterizing zebrafish mitochondrial SHMT. The cDNA encoding zebrafish mitochondrial SHMT was cloned, overexpressed in Escherichia coli, and purified with a three-step purification protocol. Similarities in structural, physical, and kinetic properties were revealed between the recombinant zebrafish mitochondrial SHMT and its mammalian orthologs. Surprisingly, leucovorin significantly inhibits the aldol cleavage of serine catalyzed by zebrafish cytosolic SHMT but inhibits to a lesser extent the reaction catalyzed by the mitochondrial isozyme. This is, to our knowledge, the first report on zebrafish mitochondrial folate enzyme as well as the differential inhibition of leucovorin on these two SHMT isoforms. Western blot analysis revealed tissue-specific distribution with the highest enrichment present in liver for both cytosolic and mitochondrial SHMTs. Intracellular localization was confirmed by confocal microscopy for both mitochondrial and cytosolic SHMTs. Unexpectedly, the cytosolic isoform was observed in both nucleus and cytosol. Together with the previous report on zebrafish cytosolic SHMT, we suggest that zSHMTs can be used in in vitro assays for folate-related investigation and antifolate drug discovery.
Asunto(s)
Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Leucovorina/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas de Pez Cebra/antagonistas & inhibidores , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Metotrexato/metabolismo , Microscopía Confocal , Mitocondrias/enzimología , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Serina/metabolismo , Tetrahidrofolatos/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Glycine specifically induces genes encoding subunits of the glycine decarboxylase complex (GCV1, GCV2, and GCV3), and this is mediated by a fall in cytoplasmic levels of 5,10-methylenetetrahydrofolate caused by inhibition of cytoplasmic serine hydroxymethyltransferase. Here it is shown that this control system extends to genes for other enzymes of one-carbon metabolism and de novo purine biosynthesis. Northern analysis of the response to glycine demonstrated that the induction of the GCV genes and the induction of other amino acid metabolism genes are temporally distinct. The genome-wide response to glycine revealed that several other genes are rapidly co-induced with the GCV genes, including SHM2, which encodes cytoplasmic serine hydroxymethyltransferase. These results were refined by examining transcript levels in an shm2Delta strain (in which cytoplasmic 5,10-methylenetetrahydrofolate levels are reduced) and a met13Delta strain, which lacks the main methylenetetrahydrofolate reductase activity of yeast and is effectively blocked at consumption of 5,10-methylene tetrahydrofolate for methionine synthesis. Glycine addition also caused a substantial transient disturbance to metabolism, including a sequence of changes in induction of amino acid biosynthesis and respiratory chain genes. Analysis of the glycine response in the shm2Delta strain demonstrated that apart from the one-carbon regulon, most of these transient responses were not contingent on a disturbance to one-carbon metabolism. The one-carbon response is distinct from the Bas1p purine biosynthesis regulon and thus represents the first example of transcriptional regulation in response to activated one-carbon status.
Asunto(s)
Carbono/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Aminoácido Oxidorreductasas/fisiología , Northern Blotting , Carbono/química , División Celular , Citoplasma/enzimología , Citoplasma/metabolismo , ADN/metabolismo , ADN Complementario/metabolismo , Inhibidores Enzimáticos/farmacología , Glicina/química , Complejo Glicina-Descarboxilasa , Glicina-Deshidrogenasa (Descarboxilante) , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Modelos Biológicos , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Purinas/química , ARN/química , Proteínas de Saccharomyces cerevisiae , Factores de Tiempo , Transcripción GenéticaRESUMEN
A naturally occurring inhibitor of serine hydroxymethyltransferase (EC 2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98 degrees C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.