RESUMO
Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/metabolismo , Sarcoma de Ewing/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos SCID , RNA Interferente Pequeno/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Damaged DNA has a profound impact on mammalian health and overall survival. In addition to being the source of mutations that initiate cancer, the accumulation of toxic amounts of DNA damage can cause severe developmental diseases and accelerate aging. Therefore, understanding how cells respond to DNA damage has become one of the most intense areas of biomedical research in the recent years. However, whereas most mechanistic studies derive from in vitro or in cellulo work, the impact of a given mutation on a living organism is largely unpredictable. For instance, why BRCA1 mutations preferentially lead to breast cancer whereas mutations compromising mismatch repair drive colon cancer is still not understood. In this context, evaluating the specific physiological impact of mutations that compromise genome integrity has become crucial for a better dimensioning of our knowledge. We here describe the various technologies that can be used for modeling mutations in mice and provide a review of the genes and pathways that have been modeled so far in the context of DNA damage responses.
Assuntos
Dano ao DNA , Modelos Animais , Animais , Marcação de Genes , Técnicas de Transferência de Genes , Instabilidade Genômica , Humanos , Camundongos , FenótipoRESUMO
The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Switching de Imunoglobulina , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Linfócitos B/metabolismo , Proteína BRCA1/metabolismo , Proteínas Cromossômicas não Histona/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Instabilidade Genômica , Camundongos , Mutação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.
