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1.
Mol Cell ; 81(5): 1084-1099.e6, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33450211

RESUMO

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
2.
Int J Mol Sci ; 21(24)2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33352723

RESUMO

Poly (ADP-ribose) polymerase inhibitor (PARPi, olaparib) impairs the repair of DNA single-strand breaks (SSBs), resulting in double-strand breaks (DSBs) that cannot be repaired efficiently in homologous recombination repair (HRR)-deficient cancers such as BRCA1/2-mutant cancers, leading to synthetic lethality. Despite the efficacy of olaparib in the treatment of BRCA1/2 deficient tumors, PARPi resistance is common. We hypothesized that the combination of olaparib with anticancer agents that disrupt HRR by targeting ataxia telangiectasia and Rad3-related protein (ATR) or checkpoint kinase 1 (CHK1) may be an effective strategy to reverse ovarian cancer resistance to olaparib. Here, we evaluated the effect of olaparib, the ATR inhibitor AZD6738, and the CHK1 inhibitor MK8776 alone and in combination on cell survival, colony formation, replication stress response (RSR) protein expression, DNA damage, and apoptotic changes in BRCA2 mutated (PEO-1) and HRR-proficient BRCA wild-type (SKOV-3 and OV-90) cells. Combined treatment caused the accumulation of DNA DSBs. PARP expression was associated with sensitivity to olaparib or inhibitors of RSR. Synergistic effects were weaker when olaparib was combined with CHK1i and occurred regardless of the BRCA2 status of tumor cells. Because PARPi increases the reliance on ATR/CHK1 for genome stability, the combination of PARPi with ATR inhibition suppressed ovarian cancer cell growth independently of the efficacy of HRR. The present results were obtained at sub-lethal doses, suggesting the potential of these inhibitors as monotherapy as well as in combination with olaparib.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Cistadenocarcinoma Seroso/patologia , Recombinação Homóloga , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , RNA Interferente Pequeno/genética , Transdução de Sinais
3.
PLoS One ; 15(12): e0244060, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338048

RESUMO

Ataxia-Telangiectasia (A-T), a pleiotropic chromosomal breakage syndrome, is caused by the loss of the kinase Ataxia-telangiectasia mutated (ATM). ATM is not only involved in the response to DNA damage, but also in sensing and counteracting oxidative stress. Since a disturbed redox balance has been implicated in the pathophysiology of A-T lung disease, we aimed to further explore the interplay between ATM and oxidative stress in lung cells. Using a kinetic trapping approach, we could demonstrate an interaction between the trapping mutant TRX1-CS and ATM upon oxidative stress. We could further show that combined inhibition of thioredoxin reductase (TrxR) and ATM kinase activity, using Auranofin and KU55933 respectively, induced an increase in cellular reactive oxygen species (ROS) levels and protein oxidation in lung cells. Furthermore, ATM inhibition sensitized lung cells to Auranofin-induced cell death that could be rescued by ROS scavengers. As a consequence, targeted reduction of ATM by TRX1 could serve as a regulator of oxidative ATM activation and contribute to the maintenance of the cellular redox homeostasis. These results highlight the importance of the redox-active function of ATM in preventing ROS accumulation and cell death in lung cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Auranofina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células A549 , Animais , Morte Celular/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pironas/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
4.
Nat Commun ; 11(1): 4766, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958778

RESUMO

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inflamação/patologia , Telômero/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antibacterianos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Criança , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Gastroenteropatias/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-18/genética , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transdução de Sinais , Telomerase/genética , Telomerase/metabolismo
5.
PLoS One ; 15(8): e0237669, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810137

RESUMO

Pancreatic beta cell death is a hallmark of type 1 and 2 diabetes (T1D/T2D), but the underlying molecular mechanisms are incompletely understood. Key proteins of the DNA damage response (DDR), including tumor protein P53 (P53, also known as TP53 or TRP53 in rodents) and Ataxia Telangiectasia Mutated (ATM), a kinase known to act upstream of P53, have been associated with T2D. Here we test and compare the effect of ATM and P53 ablation on beta cell survival in the rat beta cell line Ins1E. We demonstrate that ATM and P53 differentially regulate beta cell apoptosis induced upon fundamentally different types of diabetogenic beta cell stress, including DNA damage, inflammation, lipotoxicity and endoplasmic reticulum (ER) stress. DNA damage induced apoptosis by treatment with the commonly used diabetogenic agent streptozotocin (STZ) is regulated by both ATM and P53. We show that ATM is a key STZ induced activator of P53 and that amelioration of STZ induced cell death by inhibition of ATM mainly depends on P53. While both P53 and ATM control lipotoxic beta cell apoptosis, ATM but not P53 fails to alter inflammatory beta cell death. In contrast, tunicamycin induced (ER stress associated) apoptosis is further increased by ATM knockdown or inhibition, but not by P53 knockdown. Our results reveal differential roles for P53 and ATM in beta cell survival in vitro in the context of four key pathophysiological types of diabetogenic beta cell stress, and indicate that ATM can use P53 independent signaling pathways to modify beta cell survival, dependent on the cellular insult.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sobrevivência Celular/genética , Células Secretoras de Insulina/patologia , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Diabetes Mellitus/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Estreptozocina/toxicidade , Tunicamicina/toxicidade
6.
Cancer Treat Rev ; 88: 102026, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32592909

RESUMO

Progress in cancer therapy changed the outcome of many patients and moved therapy from chemotherapy agents to targeted drugs. Targeted drugs already changed the clinical practice in treatment of leukemias, such as imatinib (BCR/ABL inhibitor) in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL), ibrutinib (Bruton's tyrosine kinase inhibitor) in chronic lymphocytic leukemia (CLL), venetoclax (BCL2 inhibitor) in CLL and acute myeloid leukemia (AML) or midostaurin (FLT3 inhibitor) in AML. In this review, we focused on DNA damage response (DDR) inhibition, specifically on inhibition of ATR-CHK1 pathway. Cancer cells harbor often defects in different DDR pathways, which render them vulnerable to DDR inhibition. Some DDR inhibitors showed interesting single-agent activity even in the absence of cytotoxic drug especially in cancers with underlying defects in DDR or DNA replication. Almost no mutations were found in ATR and CHEK1 genes in leukemia patients. Together with the fact that ATR-CHK1 pathway is essential for cell development and survival of leukemia cells, it represents a promising therapeutic target for treatment of leukemia. ATR-CHK1 inhibition showed excellent results in preclinical testing in acute and chronic leukemias. However, results in clinical trials are so far insufficient. Therefore, the ongoing and future clinical trials will decide on the success of ATR/CHK1 inhibitors in clinical practice of leukemia treatment.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Leucemia/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Doença Aguda , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Doença Crônica , Dano ao DNA , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Terapia de Alvo Molecular , Ensaios Clínicos Controlados Aleatórios como Assunto
7.
J Med Chem ; 63(13): 7293-7325, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32502336

RESUMO

The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Because DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Disponibilidade Biológica , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Inibidores do Citocromo P-450 CYP2C8/química , Inibidores do Citocromo P-450 CYP2C8/farmacologia , Reparo do DNA/efeitos dos fármacos , Cães , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Humanos , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Ratos Wistar , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Sci Rep ; 10(1): 7488, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366852

RESUMO

Only few drugs have shown activity in patients with advanced soft-tissue and the median overall survival is only 18 months. Alterations of genes involved in the DNA damage repair pathway have been associated with sarcoma risk and prognosis. ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. The objective of this study is to evaluate the pre-clinical activity of ATR inhibition in soft tissue sarcomas (STS). We explored the ability of the ATR inhibitor, VE-822, to prevent chemotherapy-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo in STS cell lines and in a patient-derived xenograft model. The combination of VE-822 and gemcitabine in vitro was synergistic, inhibited cell proliferation, induced apoptosis, and accumulated in the S phase of the cell cycle with higher efficacy than either single agent alone. The combination also resulted in enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. These effects were unrelated to the alternative lengthening of telomeres pathway. In vivo, the combination of VE-822 and gemcitabine significantly enhanced tumor growth inhibition and progression-free survival in an aggressive model of undifferentiated pleomorphic sarcoma. The combination of ATR inhibitor and chemotherapy is beneficial in pre-clinical models of soft-tissue sarcoma and deserves further exploration in the clinical setting.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Isoxazóis/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Pirazinas/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Sarcoma , Homeostase do Telômero/efeitos dos fármacos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Humanos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Sarcoma/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Med Oncol ; 37(5): 47, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32277292

RESUMO

Pancreatic cancer is a highly progressive malignant tumor for which there is a critical unmet need for novel therapeutic strategies. A previous study of the authors indicated that VE-821, a selective inhibitor of the ataxia-telangiectasia-mutated and rad3-related protein (ATR), has antitumor efficacy. In this study, the effect of programmed death ligand 1 (PD-L1) on the sensitivity to VE-821 was investigated in p53 mutant pancreatic cancer cells. These results show that BxPC-3 cells exhibited higher sensitivity to VE-821 than mesenchymal PANC-1 cells, which were more migratory and had higher expressions of PD-L1 and CD44. When VE-821 was applied to two cells, epithelial-to-mesenchymal transition (EMT) was induced in PANC-1 cells with concomitant upregulation of PD-L1 and CD44, while BxPC-3 cells did not manifest these changes. Attenuation of PD-L1 expression suppressed VE-821-induced EMT, inhibited cell migration, and downregulated CD44 expression. Furthermore, PD-L1 inhibition partially reversed the activation of AKT/ERK, enhanced DNA damage, and increased VE-821 sensitivity in PANC-1 cells. Analysis of GEPIA data showed positive correlation of PD-L1 expression with EMT-related transcription factors. Taken together, these results suggest a novel function of PD-L1 in regulating response to ATR inhibition. These data highlight PD-L1 inhibition as a promising target to enhance sensitivity to ATR inhibitors in mesenchymal pancreatic cancer.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Antígeno B7-H1/genética , Transição Epitelial-Mesenquimal , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Técnicas de Silenciamento de Genes , Humanos , Receptores de Hialuronatos/genética , Neoplasias Pancreáticas/metabolismo , Pirazinas/farmacologia , Sulfonas/farmacologia , Proteína Supressora de Tumor p53/deficiência , Regulação para Cima/efeitos dos fármacos
10.
Sci Rep ; 10(1): 4441, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157166

RESUMO

The DNA damage response after kidney injury induces cell cycle arrest in renal tubular epithelial cells, resulting in the secretion of pro-fibrotic cytokines, thereby promoting interstitial fibrosis in a paracrine manner. Phosphorylation of ataxia-telangiectasia mutated (ATM) is the initial step in the DNA damage response and subsequent cell cycle arrest; however, the effects of ATM inhibition on the injured kidney have not been explored. Pharmacological ATM inhibition by KU55933 in cisplatin-treated mice did not ameliorate, but instead exacerbated cisplatin-induced DNA damage and tubular injury, thereby increasing mortality. Analysis of isolated tubular epithelia by FACS from bigenic SLC34a1-CreERt2; R26tdTomato proximal tubular-specific reporter mice revealed that KU55933 upregulated p53 and subsequent pro-apoptotic signaling in tubular epithelia of cisplatin-treated mice, leading to marked mitochondrial injury and apoptosis. In addition, KU55933 attenuated several DNA repair processes after cisplatin treatment, including single-strand DNA repair and Fanconi anemia pathways, suggesting that DNA repair after dual treatment of cisplatin and KU55933 was not sufficient to prevent the cisplatin-induced tubular injury. Our study suggested that ATM inhibition does not increase DNA repair after cisplatin-induced DNA damage and exacerbates tubular injury through the upregulation of p53-dependent pro-apoptotic signaling. Acute kidney injury must be carefully monitored when ATM inhibitors become available in clinical practice in the future.


Assuntos
Lesão Renal Aguda/etiologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Cisplatino/toxicidade , Morfolinas/farmacologia , Proteínas Mutantes/antagonistas & inibidores , Mutação , Pironas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/patologia , Animais , Antineoplásicos/toxicidade , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/genética , Pontos de Checagem do Ciclo Celular , Reparo do DNA , Camundongos , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor p53/genética
11.
PLoS Biol ; 18(3): e3000666, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203529

RESUMO

Ataxia-telangiectasia mutated (ATM) is an apical kinase of the DNA damage response following DNA double-strand breaks (DSBs); however, the mechanisms of ATM activation are not completely understood. Long noncoding RNAs (lncRNAs) are a class of regulatory molecules whose significant roles in DNA damage response have started to emerge. However, how lncRNA regulates ATM activity remains unknown. Here, we identify an inhibitor of ATM activation, lncRNA HITT (HIF-1α inhibitor at translation level). Mechanistically, HITT directly interacts with ATM at the HEAT repeat domain, blocking MRE11-RAD50-NBS1 complex-dependent ATM recruitment, leading to restrained homologous recombination repair and enhanced chemosensitization. Following DSBs, HITT is elevated mainly by the activation of Early Growth Response 1 (EGR1), resulting in retarded and restricted ATM activation. A reverse association between HITT and ATM activity was also detected in human colon cancer tissues. Furthermore, HITTs sensitize DNA damaging agent-induced cell death both in vitro and in vivo. These findings connect lncRNA directly to ATM activity regulation and reveal potential roles for HITT in sensitizing cancers to genotoxic treatment.


Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , RNA Longo não Codificante/metabolismo , Reparo de DNA por Recombinação/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células HCT116 , Células HeLa , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , RNA Longo não Codificante/genética , Transcrição Genética/efeitos dos fármacos
12.
Cancer Res ; 80(8): 1669-1680, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32060145

RESUMO

The role of the ataxia-telangiectasia-mutated (ATM) gene in human malignancies, especially in solid tumors, remains poorly understood. In the present study, we explored the involvement of ATM in transforming primary human cells into cancer stem cells. We show that ATM plays an unexpected role in facilitating oncogene-induced malignant transformation through transcriptional reprogramming. Exogenous expression of an oncogene cocktail induced a significant amount of DNA double-strand breaks in human fibroblasts that caused persistent activation of ATM, which in turn enabled global transcriptional reprogramming through chromatin relaxation, allowing oncogenic transcription factors to access chromatin. Consistently, deficiencies in ATM significantly attenuated oncogene-induced transformation of human cells. In addition, ATM inhibition significantly reduced tumorigenesis in a mouse model of mammary cancer. ATM and cellular DNA damage response therefore play a previously unknown role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells. SIGNIFICANCE: These findings uncover a novel pro-oncogenic role for ATM and show that contrary to established theory, ATM does not always function as a tumor suppressor; its function is however dependent on cell type.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Transformação Celular Neoplásica/genética , Reprogramação Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Células-Tronco Neoplásicas/patologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/patologia , Cromatina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Fibroblastos/patologia , Técnicas de Inativação de Genes , Marcação de Genes/métodos , Genes p53 , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Ativação Transcricional , Transcriptoma/fisiologia , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Ensaio Tumoral de Célula-Tronco/métodos
13.
Leukemia ; 34(8): 2150-2162, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32060401

RESUMO

To target mechanisms critical for multiple myeloma (MM) plasma cell adaptations to genomic instabilities and further sustain MM cell killing, we here specifically trigger DNA damage response (DDR) in MM cells by a novel BCMA antibody-drug conjugate (ADC) delivering the DNA cross-linking PBD dimer tesirine, MEDI2228. MEDI2228, more effectively than its anti-tubulin MMAF-ADC homolog, induces cytotoxicity against MM cells regardless of drug resistance, BCMA levels, p53 status, and the protection conferred by bone marrow stromal cells and IL-6. Distinctly, prior to apoptosis, MEDI2228 activates DDRs in MM cells via phosphorylation of ATM/ATR kinases, CHK1/2, CDK1/2, and H2AX, associated with expression of DDR-related genes. Significantly, MEDI2228 synergizes with DDR inhibitors (DDRi s) targeting ATM/ATR/WEE1 checkpoints to induce MM cell lethality. Moreover, suboptimal doses of MEDI2228 and bortezomib (btz) synergistically trigger apoptosis of even drug-resistant MM cells partly via modulation of RAD51 and accumulation of impaired DNA. Such combination further induces superior in vivo efficacy than monotherapy via increased nuclear γH2AX-expressing foci, irreversible DNA damages,  and tumor cell death, leading to significantly prolonged host survival. These results indicate leveraging MEDI2228 with DDRi s or btz as novel combination strategies, further supporting ongoing clinical development of MEDI2228 in patients with relapsed and refractory MM.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Antígeno de Maturação de Linfócitos B/imunologia , Bortezomib/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Imunoconjugados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Benzodiazepinas/administração & dosagem , Linhagem Celular Tumoral , Dano ao DNA , Sinergismo Farmacológico , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Pirróis/administração & dosagem
14.
Arch Virol ; 165(3): 683-690, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32002668

RESUMO

In the search for new antiviral therapies against human immunodeficiency virus type 1 (HIV-1), several cellular targets are being investigated. Ataxia telangiectasia and Rad3-related protein (ATR) has been implicated in HIV-1 replication, namely during retroviral DNA integration. We studied the effect of the ATR inhibitor ETP-46464 on HIV-1 replication in peripheral blood mononuclear cells (PBMCs) and in the persistently HIV-1-infected cell line H61-D. After treatment with ETP-46464, a significant decrease in virus production was observed in both cell systems. Quantification of viral DNA forms in the acutely infected PBMCs suggests that inhibition could take place in the early phase of the viral life cycle before viral DNA integration. Moreover, after treatment of H61-D cells with 3'-azido-3'-deoxythymidine (AZT), which blocks new reverse transcription events, ETP-46464 decreased viral production, suggesting that inhibition of viral replication occurred in the late phase of the life cycle after viral DNA integration. A decrease in virus production after transfection of 293T cells with an HIV-1 infectious molecular clone also suggested that the effect of ETP-46464 is exerted at a post-integration step. We propose that ETP-46464 produces its inhibitory effect on HIV-1 replication by acting in both the early and late phases of the retroviral replication cycle. Thus, ATR could represent a new target for inhibition of HIV-1 replication.


Assuntos
Antivirais/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Oxazinas/farmacologia , Quinolinas/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Humanos
15.
Int J Radiat Biol ; 96(4): 461-468, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31859574

RESUMO

Purpose: Although EGFR inhibitor (EGFRi) is used in cancer therapy to suppress tumor growth and resistance to treatment including radiotherapy, EGFRi resistance frequently developed, which significantly reduced treatment outcomes. Therefore, developing alternative approaches for EGFRi is of great importance. Based on our recent observation that ATM inhibitor (ATMi) efficiently inhibited ionizing radiation (IR)-induced EGFR activation in mouse embryo fibroblasts (MEF), the main purpose of this study is to determine whether ATMi could inhibit IR-induced EGFR activation in human tumor cell lines and explore its potential in EGFRi-alternative therapies.Materials and methods: We compared the effects of ATMi, EGFRi individually or in combination on IR-induced EGFR phosphorylation, cell growth and radio-sensitization in nine human tumor cell lines including lung adenocarcinoma (A549 and H358), glioblastoma (LN229), cervical cancer (HeLa), colorectal carcinoma (SW480 and HCT116) and nasopharygeal carcinoma (5-8 F, 6-10B and HK1) cell lines. In addition, we detected the effects of ATMi, EGFRi alone or both on the efficiency of non-homologous end-joining (NHEJ) and homologous recombination (HR) using I-SceI -GFP based NHEJ or HR reporter cell lines.Results: Compared to EGFRi treatment, ATMi treatment decreased IR-induced EGFR phosphorylation, suppressed growth and increased IR sensitization in tested cell lines at a similar or even more efficient level. Combining ATMi and EGFRi did not significantly increased the effects on these phenotypes as ATMi treatment alone. Also, similar to ATMi, EGFRi mainly reduced the efficiency of HR but not NHEJ although combining ATMi and EGFRi further inhibited the HR efficiency.Conclusions: Our study demonstrates that ATMi can function like EGFRi in human tumor cells to inhibit tumor cell growth and sensitize the tumor cells to IR, suggesting that ATMi treatment as an alternative approach may exert anticancer effects on EGFRi-resistant tumor cells and facilitate radiotherapy.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Neoplasias/radioterapia , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Receptores ErbB/fisiologia , Receptores ErbB/efeitos da radiação , Recombinação Homóloga/efeitos dos fármacos , Humanos , Neoplasias/patologia , Fosforilação , Tolerância a Radiação
16.
Mol Cancer Res ; 18(1): 91-104, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31649026

RESUMO

Inhibition of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides, causes DNA replication stress and activates the ataxia telangiectasia and rad3-related protein (ATR)-checkpoint kinase 1 (CHK1) pathway. Notably, a number of different cancers, including Ewing sarcoma tumors, are sensitive to the combination of RNR and ATR-CHK1 inhibitors. However, multiple, overlapping mechanisms are reported to underlie the toxicity of ATR-CHK1 inhibitors, both as single agents and in combination with RNR inhibitors, toward cancer cells. Here, we identified a feedback loop in Ewing sarcoma cells in which inhibition of the ATR-CHK1 pathway depletes RRM2, the small subunit of RNR, and exacerbates the DNA replication stress and DNA damage caused by RNR inhibitors. Mechanistically, we identified that the inhibition of ATR-CHK1 activates CDK2, which targets RRM2 for degradation via the proteasome. Similarly, activation of CDK2 by inhibition or knockdown of the WEE1 kinase also depletes RRM2 and causes DNA damage and apoptosis. Moreover, we show that the concurrent inhibition of ATR and WEE1 has a synergistic effect in Ewing sarcoma cells. Overall, our results provide novel insight into the response to DNA replication stress, as well as a rationale for targeting the ATR, CHK1, and WEE1 pathways, in Ewing sarcoma tumors. IMPLICATIONS: Targeting the ATR, CHK1, and WEE1 kinases in Ewing sarcoma cells activates CDK2 and increases DNA replication stress by promoting the proteasome-mediated degradation of RRM2.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Ribonucleosídeo Difosfato Redutase/metabolismo , Sarcoma de Ewing/tratamento farmacológico , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA , Células HEK293 , Humanos , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transfecção
17.
Biochem Biophys Res Commun ; 521(2): 303-309, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31668372

RESUMO

Circular RNAs (circRNAs) are a new class of noncoding RNAs, play a crucial role in tumor initiation and development. Hsa_circ_0001546 is a novel circular RNA that was downregulated in gastric cancer (GC) tissues, however its function and mechanism in GC has not been studied. Our study verified that circ_0001546 was decreased in GC and correlated with the poor prognosis. Next, Pull-down assay and dual-luciferase reporter assay verified that miR-421 was a target of circ_0001546 while ATM (Ataxia telangiectasia mutated) was target by miR-421. Overexpression of circ_0001546 inhibited the proliferation and chemoresistance of HGC-27 cells, and increased the expression of ATM. In addition, circ_0001546 overexpression reversed the effect of miR-421 overexpression. What is more, circ_0001546 inhibits the chemoresistance of HGC-27 cells to L-OPH (Oxaliplatin) may through the activation of the ATM/checkpoint kinase 2 (Chk2)/p53-dependent signaling pathway. In summary, our study proved that circ_0001546 sponges miR-421 to upregulate the expression level of ATM and inhibit the proliferation and chemoresistance through the activation of the ATM/Chk2/p53-dependent pathway.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MicroRNAs/metabolismo , RNA Circular/fisiologia , Neoplasias Gástricas/patologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase do Ponto de Checagem 2/metabolismo , Humanos , MicroRNAs/antagonistas & inibidores , RNA Circular/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
18.
Cancer Res Treat ; 52(1): 149-166, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31291716

RESUMO

PURPOSE: Pancreatic cancer (PC) is one of the most lethal cancers worldwide, but there are currently no effective treatments. The DNA damage response (DDR) is under investigation for the development of novel anti-cancer drugs. Since DNA repair pathway alterations have been found frequently in PC, the purpose of this study was to test the DDR-targeting strategy in PC using WEE1 and ATM inhibitors. Materials and Methods: We performed in vitro experiments using a total of ten human PC cell lines to evaluate antitumor effect of AZD1775 (WEE1 inhibitor) alone or combination with AZD0156 (ATM inhibitor). We established Capan-1-mouse model for in vivo experiments to confirm our findings. RESULTS: In our research, we found that WEE1 inhibitor (AZD1775) as single agent showed anti-tumor effects in PC cells, however, targeting WEE1 upregulated p-ATM level. Here, we observed that co-targeting of WEE1 and ATM acted synergistically to reduce cell proliferation and migration, and to induce DNA damage in vitro. Notably, inhibition of WEE1 or WEE1/ATM downregulated programmed cell death ligand 1 expression by blocking glycogen synthase kinase-3ß serine 9 phosphorylation and decrease of CMTM6 expression. In Capan-1 mouse xenograft model, AZD1775 plus AZD0156 (ATM inhibitor) treatment reduced tumor growth and downregulated tumor expression of programmed cell death ligand 1, CMTM6, CD163, and CXCR2, all of which contribute to tumor immune evasion. CONCLUSION: Dual blockade of WEE1 and ATM might be a potential therapeutic strategy for PC. Taken toget.


Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Antígeno B7-H1/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Citocinas , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Imunofenotipagem , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pontos de Checagem da Fase S do Ciclo Celular , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801300

RESUMO

Mechanistic approaches to modeling the effects of ionizing radiation on cells are on the rise, promising a better understanding of predictions and higher flexibility concerning conditions to be accounted for. In this work we modified and extended a previously published mechanistic model of cell survival after photon irradiation under hypoxia to account for radiosensitization caused by deficiency or inhibition of DNA damage repair enzymes. The model is shown to be capable of describing the survival data of cells with DNA damage repair deficiency, both under norm- and hypoxia. We find that our parameterization of radiosensitization is invariant under change of oxygen status, indicating that the relevant parameters for both mechanisms can be obtained independently and introduced freely to the model to predict their combined effect.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Fótons , Inibidores de Proteínas Quinases/farmacologia , Radiossensibilizantes/farmacologia , Células A549 , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células CHO , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cricetulus , DNA/genética , DNA/metabolismo , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/deficiência , Relação Dose-Resposta à Radiação , Expressão Gênica , Humanos , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Oxigênio/farmacologia
20.
Sci Transl Med ; 11(519)2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748230

RESUMO

Patients with rheumatoid arthritis (RA) may display atypical CD21-/lo B cells in their blood, but the implication of this observation remains unclear. We report here that the group of patients with RA and elevated frequencies of CD21-/lo B cells shows decreased ataxia telangiectasia-mutated (ATM) expression and activation in B cells compared with other patients with RA and healthy donor controls. In agreement with ATM involvement in the regulation of V(D)J recombination, patients with RA who show defective ATM function displayed a skewed B cell receptor (BCR) Igκ repertoire, which resembled that of patients with ataxia telangiectasia (AT). This repertoire was characterized by increased Jκ1 and decreased upstream Vκ gene segment usage, suggesting improper secondary recombination processes and selection. In addition, altered ATM function in B cells was associated with decreased osteoprotegerin and increased receptor activator of nuclear factor κB ligand (RANKL) production. These changes favor bone loss and correlated with a higher prevalence of erosive disease in patients with RA who show impaired ATM function. Using a humanized mouse model, we also show that ATM inhibition in vivo induces an altered Igκ repertoire and RANKL production by immature B cells in the bone marrow, leading to decreased bone density. We conclude that dysregulated ATM function in B cells promotes bone erosion and the emergence of circulating CD21-/lo B cells, thereby contributing to RA pathophysiology.


Assuntos
Artrite Reumatoide/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/metabolismo , Reabsorção Óssea/imunologia , Animais , Artrite Reumatoide/fisiopatologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Densidade Óssea , Reabsorção Óssea/fisiopatologia , Sobrevivência Celular/imunologia , Humanos , Imunoglobulinas/imunologia , Articulações/patologia , Contagem de Linfócitos , Camundongos , Pessoa de Meia-Idade , Osteogênese , Osteoprotegerina/metabolismo , Fenótipo , Ligante RANK/metabolismo , Receptores de Complemento 3d/metabolismo , Recombinação Genética/genética
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