RESUMO
Although the kinase CHK1 is a key player in the DNA damage response (DDR), several studies have recently provided evidence of DDR-independent roles of CHK1, in particular following phosphorylation of its S280 residue. Here, we demonstrate that CHK1 S280 phosphorylation is cell cycle-dependent and peaks during mitosis. We found that this phosphorylation was catalyzed by the kinase PIM2, whose protein expression was also increased during mitosis. Importantly, we identified polo-like kinase 1 (PLK1) as a direct target of CHK1 during mitosis. Genetic or pharmacological inhibition of CHK1 reduced the activating phosphorylation of PLK1 on T210, and recombinant CHK1 was able to phosphorylate T210 of PLK1 in vitro Accordingly, S280-phosphorylated CHK1 and PLK1 exhibited similar specific mitotic localizations, and PLK1 was co-immunoprecipitated with S280-phosphorylated CHK1 from mitotic cell extracts. Moreover, CHK1-mediated phosphorylation of PLK1 was dependent on S280 phosphorylation by PIM2. Inhibition of PIM proteins reduced cell proliferation and mitotic entry, which was rescued by expressing a T210D phosphomimetic mutant of PLK1. Altogether, these data identify a new PIM-CHK1-PLK1 phosphorylation cascade that regulates different mitotic steps independently of the CHK1 DDR function.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Células HeLa , Humanos , Camundongos Knockout , Mitose/genética , Fosforilação/genética , Fosforilação/fisiologia , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem , Quinase 1 Polo-LikeRESUMO
Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, single-cell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.
Assuntos
Biomarcadores Tumorais/metabolismo , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Peptidase 7 Específica de Ubiquitina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The checkpoint kinase 1 (CHK1) is a master regulator of genome integrity in vertebrate cells. Despite its important cell cycle functions, its regulation is still incompletely understood. Cassidy et al. provide novel insights on the regulation of the CHK1 abundance by the HECT E3 ligase HUWE1 during unperturbed cell cycle as well as in response to replicative stress. These results may help us to apprehend the underlying mechanism of tumorigenesis.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Quinase 1 do Ponto de Checagem/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.