RESUMEN
Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , ADN Glicosilasas/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Poli(ADP-Ribosa) Polimerasa-1/inmunología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/mortalidad , Daño del ADN , ADN Glicosilasas/antagonistas & inhibidores , ADN Glicosilasas/metabolismo , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.
Asunto(s)
ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Bencimidazoles/farmacología , Línea Celular , ADN/metabolismo , Daño del ADN/genética , ADN Glicosilasas/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Guanina/análogos & derivados , Humanos , Cinética , Mutación , Estrés Oxidativo , Piperidinas/farmacología , Estudios Prospectivos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The NUDIX hydrolase NUDT22 converts UDP-glucose into glucose-1-phosphate and the pyrimidine nucleotide uridine monophosphate but a biological significance for this biochemical reaction has not yet been established. Glucose-1-phosphate is an important metabolite for energy and biomass production through glycolysis and nucleotides required for DNA replication are produced through energetically expensive de novo or energy-efficient salvage pathways. Here, we describe p53-regulated pyrimidine salvage through NUDT22-dependent hydrolysis of UDP-glucose to maintain cancer cell growth and to prevent replication stress. NUDT22 expression is consistently elevated in cancer tissues and high NUDT22 expression correlates with worse survival outcomes in patients indicating an increased dependency of cancer cells to NUDT22. Furthermore, we show that NUDT22 transcription is induced after inhibition of glycolysis, MYC-mediated oncogenic stress, and DNA damage directly through p53. NUDT22-deficient cancer cells suffer from growth retardation, S-phase delay, and slower DNA replication fork speed. Uridine supplementation rescues replication fork progression and alleviates replication stress and DNA damage. Conversely, NUDT22 deficiency sensitizes cells to de novo pyrimidine synthesis inhibition in vitro and reduces cancer growth in vivo. In conclusion, NUDT22 maintains pyrimidine supply in cancer cells and depletion of NUDT22 leads to genome instability. Targeting NUDT22 therefore has high potential for therapeutic applications in cancer therapy.
Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Glucosa , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Pirimidinas/farmacología , Uridina/metabolismo , Uridina DifosfatoRESUMEN
Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.
Asunto(s)
Daño del ADN , ADN Glicosilasas , Reparación del ADN , Estrés Oxidativo , Biocatálisis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN Glicosilasas/química , ADN Glicosilasas/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Activación Enzimática , Glicina/química , Humanos , Ligandos , Estrés Oxidativo/genética , Fenilalanina/química , Especificidad por SustratoRESUMEN
The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Bencimidazoles/farmacología , ADN Glicosilasas/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Metotrexato/farmacología , Estrés Oxidativo , Piperidinas/farmacología , Telómero/metabolismo , Ciclo Celular , Línea Celular Tumoral , ADN Glicosilasas/metabolismo , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Inestabilidad Genómica , Humanos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
8-oxoguanine DNA glycosylase (OGG1) is the main DNA glycosylase responsible for the excision of 7,8-dihydro-8-oxoguanine (8-oxoG) from duplex DNA to initiate base excision repair. This glycosylase activity is relevant in many pathological conditions including cancer, inflammation, and neurodegenerative diseases. To have a better understanding of the role of OGG1, we previously reported TH5487, a potent active site inhibitor of OGG1. Here, we further investigate the consequences of inhibiting OGG1 with TH5487. TH5487 treatment induces accumulation of genomic 8-oxoG lesions. Furthermore, it impairs the chromatin binding of OGG1 and results in lower recruitment of OGG1 to regions of DNA damage. Inhibiting OGG1 with TH5487 interferes with OGG1's incision activity, resulting in fewer DNA double-strand breaks in cells exposed to oxidative stress. This study validates TH5487 as a potent OGG1 inhibitor that prevents the repair of 8-oxoG and alters OGG1-chromatin dynamics and OGG1's recruitment kinetics.
Asunto(s)
Bencimidazoles/farmacología , Cromatina/efectos de los fármacos , ADN Glicosilasas/antagonistas & inhibidores , Piperidinas/farmacología , Cromatina/metabolismo , ADN Glicosilasas/metabolismo , Humanos , Células Tumorales CultivadasRESUMEN
The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.