RESUMEN
Pediatric high-grade gliomas are among the deadliest of childhood cancers due to limited knowledge of early driving events in their gliomagenesis and the lack of effective therapies available. In this study, we investigate the oncogenic role of PPM1D, a protein phosphatase often found truncated in pediatric gliomas such as DIPG, and uncover a synthetic lethal interaction between PPM1D mutations and nicotinamide phosphoribosyltransferase (NAMPT) inhibition. Specifically, we show that mutant PPM1D drives hypermethylation of CpG islands throughout the genome and promotes epigenetic silencing of nicotinic acid phosphoribosyltransferase (NAPRT), a key gene involved in NAD biosynthesis. Notably, PPM1D mutant cells are shown to be sensitive to NAMPT inhibitors in vitro and in vivo, within both engineered isogenic astrocytes and primary patient-derived model systems, suggesting the possible application of NAMPT inhibitors for the treatment of pediatric gliomas. Overall, our results reveal a promising approach for the targeting of PPM1D mutant tumors, and define a critical link between oncogenic driver mutations and NAD metabolism, which can be exploited for tumor-specific cell killing.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Tronco Encefálico/genética , Glioma Pontino Intrínseco Difuso/genética , Nicotinamida Fosforribosiltransferasa/genética , Proteína Fosfatasa 2C/genética , Animales , Antineoplásicos/uso terapéutico , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/patología , Línea Celular Tumoral , Niño , Citocinas/antagonistas & inhibidores , Metilación de ADN , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/patología , Represión Epigenética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Puente/citología , Puente/patología , Cultivo Primario de Células , Proteína Fosfatasa 2C/metabolismo , Mutaciones Letales Sintéticas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Sleeve gastrectomy with ileal transposition has been shown to be superior to sleeve gastrectomy alone for promoting weight loss in rat and porcine models. The absence of a mouse model for this procedure has impeded efforts to understand the molecular physiology underlying its efficacy. This study demonstrates the long-term survivability of sleeve gastrectomy with ileal transposition in mice. MATERIALS AND METHODS: In this study of technical feasibility, a sleeve gastrectomy with ileal transposition (SGIT), sleeve gastrectomy (SG), or sham surgery (SH) was performed on 7- to 8-week-old C57Bl/6J mice (n = 8 for each). To evaluate long-term survivability, mice were placed on an obesogenic diet and weighed weekly for 10 weeks. The intestinal identity of the transposed segment was assessed with gene expression analysis of duodenal-, jejunal-, and ileal-specific hormones using quantitative polymerase chain reaction. RESULTS: Overall, SGIT better prevented weight gain than the SG or sham procedures (10-week post-operative weight: SH 45.3 ± 1.0 g, SG 41.25 ± 1.6 g, SGIT 35.4 ± 0.8 g). Gene expression pattern analysis of three markers of intestinal identity (gastrin, cholecystokinin, and peptide YY) suggests that the ileal identity of the transposed segment is maintained 10 weeks after transposition. CONCLUSIONS: We demonstrate for the first time a reproducible mouse model of sleeve gastrectomy with ileal transposition. Future studies utilizing this model will expand our understanding of the molecular pathways through which the hindgut regulates satiety.
Asunto(s)
Gastrectomía/métodos , Íleon/cirugía , Animales , Biomarcadores , Glucemia/análisis , Colecistoquinina/genética , Colecistoquinina/metabolismo , Modelos Animales de Enfermedad , Estudios de Factibilidad , Gastrinas/genética , Gastrinas/metabolismo , Expresión Génica , Ratones Endogámicos C57BL , Péptido YY/genética , Péptido YY/metabolismo , ARN/metabolismo , Distribución Aleatoria , Pérdida de PesoRESUMEN
2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.
Asunto(s)
Glioma/tratamiento farmacológico , Glutaratos/farmacología , Recombinación Homóloga , Isocitrato Deshidrogenasa/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Animales , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Glioma/genética , Humanos , Isocitrato Deshidrogenasa/farmacología , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Drosophila melanogaster/genética , Eucromatina/genética , Heterocromatina/genética , Animales , Técnicas Citológicas , Drosophila melanogaster/citología , Recombinación Homóloga , LarvaRESUMEN
Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multiple sub-pathways, some of which still remain poorly understood. As such, there is great interest in the development of novel assays to interrogate these key pathways, which could lead to the development of novel therapeutics, and a better understanding of how DSBs are repaired. Furthermore, assays which can measure repair specifically at endogenous chromosomal loci are of particular interest, because of an emerging understanding that chromatin interactions heavily influence DSB repair pathway choice. Here, we present the design and validation of a novel, next-generation sequencing-based approach to study DSB repair at chromosomal loci in cells. We demonstrate that NHEJ repair "fingerprints" can be identified using our assay, which are dependent on the status of key DSB repair proteins. In addition, we have validated that our system can be used to detect dynamic shifts in DSB repair activity in response to specific perturbations. This approach represents a unique alternative to many currently available DSB repair assays, which typical rely on the expression of reporter genes as an indirect read-out for repair. As such, we believe this tool will be useful for DNA repair researchers to study NHEJ repair in a high-throughput and sensitive manner, with the capacity to detect subtle changes in DSB repair patterns that was not possible previously.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Cromatina/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Sitios Genéticos , Humanos , Mutación INDEL , Mamíferos , Reparación del ADN por RecombinaciónRESUMEN
BACKGROUND: Evidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function. METHODOLOGY/PRINCIPAL FINDINGS: Effects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O(2)) or hypoxic (2% O(2)) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR-/-) had elevated heart rates compared to A1AR+/- littermates, A1AR-/- heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR-/- embryos. CONCLUSIONS/SIGNIFICANCE: These data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia.