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1.
Nucleic Acids Res ; 48(19): 10940-10952, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010150

RESUMO

ATR functions as a master regulator of the DNA-damage response. ATR activation requires the ATR activator, topoisomerase IIß-binding protein 1 (TopBP1). However, the underlying mechanism of TopBP1 regulation and how its regulation affects DNA replication remain unknown. Here, we report a specific interaction between TopBP1 and the histone demethylase PHF8. The TopBP1/PHF8 interaction is mediated by the BRCT 7+8 domain of TopBP1 and phosphorylation of PHF8 at Ser854. This interaction is cell-cycle regulated and phosphorylation-dependent. PHF8 is phosphorylated by CK2, which regulates binding of PHF8 to TopBP1. Importantly, PHF8 regulates TopBP1 protein level by preventing its ubiquitination and degradation mediated by the E3 ligase UBR5. Interestingly, PHF8pS854 is likely to contribute to regulation of TopBP1 stability and DNA replication checkpoint. Further, both TopBP1 and PHF8 are required for efficient replication fork restart. Together, these data identify PHF8 as a TopBP1-binding protein and provide mechanistic insight into how PHF8 regulates TopBP1 stability to maintain DNA replication.


Assuntos
Proteínas de Transporte/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Histona Desmetilases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Fosforilação , Ligação Proteica , Domínios Proteicos
2.
Anticancer Res ; 40(11): 6237-6246, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33109561

RESUMO

BACKGROUND/AIM: The topoisomerase 1 catalytic inhibitor 3EZ, 20Ac-ingenol specifically induces apoptosis through the activation of ATR and the up-regulation of PTEN by enhancing the DNA damage response (DDR) in human B lymphoma (BALL-1) cells. The accumulation of cyclin D1 in cancer is known to be related to chemoresistance to DNA damage agents and nuclei of BALL-1 cells exhibit high levels of cyclin D1. However, 3EZ, 20Ac-ingenol effectively induced apoptosis of BALL-1 cells. MATERIALS AND METHODS: Cell growth, protein levels, and apoptosis were determined by an MTT assay, immunoblotting and DNA fragmentation assay, respectively. RESULTS: 3EZ, 20Ac-ingenol strongly induced inhibition of cell proliferation and apoptosis in Jeko-1 and Panc-1 cell lines through the activation of tumor suppressor proteins and caspase 3. CONCLUSION: 3EZ, 20Ac-ingenol-induced apoptosis might occur in cells with cyclin D1 accumulation through enhancing DDR, regardless of the cancer cell type.


Assuntos
Apoptose/efeitos dos fármacos , Ciclina D1/metabolismo , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina A/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Histonas/metabolismo , Humanos , Irinotecano/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
3.
Mol Cell ; 80(2): 327-344.e8, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32966758

RESUMO

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA-deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2-deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA-deficient tumors and identifying epigenetic modulators of chemotherapeutic responses.


Assuntos
Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Exodesoxirribonucleases/metabolismo , Histonas/metabolismo , Proteína Homóloga a MRE11/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lisina/metabolismo , Modelos Biológicos , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/genética
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.
Nat Commun ; 11(1): 4828, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973141

RESUMO

ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Núcleo Celular/metabolismo , Estresse Mecânico , Citoesqueleto de Actina , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Encéfalo , Cromatina , Citoplasma , Citoesqueleto/metabolismo , Dano ao DNA , Camundongos Knockout , Metástase Neoplásica , Neurogênese , Membrana Nuclear/metabolismo
6.
Nucleic Acids Res ; 48(17): 9710-9723, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890395

RESUMO

Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Proteína Quinase Ativada por DNA/metabolismo , Autoantígeno Ku/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Camptotecina/farmacologia , Linhagem Celular , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA de Cadeia Simples , Proteína Quinase Ativada por DNA/genética , Humanos , Autoantígeno Ku/genética , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Fosforilação , Inibidores da Topoisomerase I/farmacologia
7.
Nat Commun ; 11(1): 3940, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769985

RESUMO

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo
8.
Nat Commun ; 11(1): 4083, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796829

RESUMO

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.


Assuntos
Proteínas de Ciclo Celular/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Estruturas R-Loop/efeitos dos fármacos , Fatores de Transcrição/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HeLa , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Fase S , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
9.
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
10.
Nat Commun ; 11(1): 4154, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32814778

RESUMO

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53CHK1/CHK2 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21NPAT on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1ATM and Rpd3HDAC activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Glucose/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Dano ao DNA , Reparo do DNA , Inativação Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Serina/metabolismo , Telômero/genética , Fatores de Transcrição/genética
11.
Nat Commun ; 11(1): 3726, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709856

RESUMO

Ovarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1-amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.


Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Ciclinas/metabolismo , Combinação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/genética , Rad51 Recombinase/metabolismo , Células-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nature ; 582(7813): 586-591, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494005

RESUMO

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Recombinação Homóloga , Neoplasias/metabolismo , Transdução de Sinais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Lisina Acetiltransferase 5/metabolismo , Metilação/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais/efeitos dos fármacos
14.
PLoS Pathog ; 16(6): e1008514, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479542

RESUMO

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Células Gigantes , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Vírus da Doença de Newcastle/fisiologia , Vírus Oncolíticos/fisiologia , Replicação Viral , Células A549 , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrião de Galinha , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Gigantes/metabolismo , Células Gigantes/virologia , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/virologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética
15.
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
16.
Nat Commun ; 11(1): 3256, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591500

RESUMO

BRCA1 mutation carriers have a higher risk of developing triple-negative breast cancer (TNBC), which is a refractory disease due to its non-responsiveness to current clinical targeted therapies. Using the Sleeping Beauty transposon system in Brca1-deficient mice, we identified 169 putative cancer drivers, among which Notch1 is a top candidate for accelerating TNBC by promoting the epithelial-mesenchymal transition (EMT) and regulating the cell cycle. Activation of NOTCH1 suppresses mitotic catastrophe caused by BRCA1 deficiency by restoring S/G2 and G2/M cell cycle checkpoints, which may through activation of ATR-CHK1 signalling pathway. Consistently, analysis of human breast cancer tissue demonstrates NOTCH1 is highly expressed in TNBCs, and the activated form of NOTCH1 correlates positively with increased phosphorylation of ATR. Additionally, we demonstrate that inhibition of the NOTCH1-ATR-CHK1 cascade together with cisplatin synergistically kills TNBC by targeting the cell cycle checkpoint, DNA damage and EMT, providing a potent clinical option for this fatal disease.


Assuntos
Proteína BRCA1/deficiência , Carcinogênese/patologia , Receptor Notch1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Alelos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Morte Celular , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Elementos de DNA Transponíveis/genética , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Knockout , Mitose , Mutação/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética
17.
Nucleic Acids Res ; 48(13): 7252-7264, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542389

RESUMO

The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.


Assuntos
Replicação do DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Mutações Sintéticas Letais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Humanos , Poli(ADP-Ribose) Polimerases/genética
18.
Breast Cancer Res ; 22(1): 49, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414408

RESUMO

BACKGROUND: Most breast cancer-associated fibroblasts (CAFs) are active and important cancer-promoting cells, with significant impact on patient prognosis. Therefore, we investigated here the role of the protein kinase ATR in breast stromal fibroblasts in the prognosis of locally advanced breast cancer patients. METHODS: We have used immunohistochemistry to assess the level of ATR in breast cancer tissues and their adjacent normal tissues. Immunoblotting as well as quantitative RT-PCR were utilized to show the role of breast cancer cells and IL-6 as well as AUF-1 in downregulating ATR in breast stromal fibroblasts. Engineered human breast tissue model was also used to show that ATR-deficient breast stromal fibroblasts enhance the growth of breast cancer cells. RESULTS: We have shown that the protein kinase ATR is downregulated in cancer cells and their neighboring CAFs in breast cancer tissues as compared to their respective adjacent normal tissues. The implication of cancer cells in ATR knockdown in CAFs has been proven in vitro by showing that breast cancer cells downregulate ATR in breast fibroblasts in an IL-6/STAT3-dependent manner and via AUF-1. In another cohort of 103 tumors from locally advanced breast cancer patients, we have shown that absence or reduced ATR expression in tumoral cells and their adjacent stromal fibroblasts is correlated with poor overall survival as well as disease-free survival. Furthermore, ATR expression in CAFs was inversely correlated with tumor recurrence and progression. CONCLUSION: ATR downregulation in breast CAFs is frequent, procarcinogenic, and correlated with poor patient survival.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/biossíntese , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Recidiva Local de Neoplasia/patologia , Células Estromais/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patologia , Estudos de Coortes , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Células Estromais/patologia
19.
Proc Natl Acad Sci U S A ; 117(23): 12806-12816, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32444488

RESUMO

The most prevalent human carcinogen is sunlight-associated ultraviolet (UV), a physiologic dose of which generates thousands of DNA lesions per cell, mostly of two types: cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). It has not been possible, in living cells, to precisely characterize the respective contributions of these two lesion types to the signals that regulate cell cycle progression, DNA replication, and cell survival. Here we coupled multiparameter flow cytometry with lesion-specific photolyases that eliminate either CPDs or 6-4PPs and determined their respective contributions to DNA damage responses. Strikingly, only 6-4PP lesions activated the ATR-Chk1 DNA damage response pathway. Mechanistically, 6-4PPs, but not CPDs, impeded DNA replication across the genome as revealed by microfluidic-assisted replication track analysis. Furthermore, single-stranded DNA accumulated preferentially at 6-4PPs during DNA replication, indicating selective and prolonged replication blockage at 6-4PPs. These findings suggest that 6-4PPs, although eightfold fewer in number than CPDs, are the trigger for UV-induced DNA damage responses.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Replicação do DNA , Dímeros de Pirimidina/genética , Raios Ultravioleta , Animais , Células Cultivadas , Quinase 1 do Ponto de Checagem/metabolismo , Reparo do DNA , Células HCT116 , Humanos
20.
Br J Radiol ; 93(1115): 20200067, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32462882

RESUMO

Cancer-specific metabolic changes support the anabolic needs of the rapidly growing tumor, maintain a favorable redox balance, and help cells adapt to microenvironmental stresses like hypoxia and nutrient deprivation. Radiation is extensively applied in a large number of cancer treatment protocols but despite its curative potential, radiation resistance and treatment failures pose a serious problem. Metabolic control of DNA integrity and genomic stability can occur through multiple processes, encompassing cell cycle regulation, nucleotide synthesis, epigenetic regulation of gene activity, and antioxidant defenses. Given the important role of metabolic pathways in oxidative damage responses, it is necessary to assess the potential for tumor-specific radiosensitization by novel metabolism-targeted therapies. Additionally, there are opportunities to identify molecular and functional biomarkers of vulnerabilities to combination treatments, which could then inform clinical decisions. Here, we present a curated list of metabolic pathways in the context of ionizing radiation responses. Glutamine metabolism influences DNA damage responses by mechanisms such as synthesis of nucleotides for DNA repair or of glutathione for ROS detoxification. Repurposed oxygen consumption inhibitors have shown promising radiosensitizing activity against murine model tumors and are now in clinical trials. Production of 2-hydroxy glutarate by isocitrate dehydrogenase1/2 neomorphic oncogenic mutants interferes with the function of α-ketoglutarate-dependent enzymes and modulates Ataxia Telangiectasia Mutated (ATM) signaling and glutathione pools. Radiation-induced oxidative damage to membrane phospholipids promotes ferroptotic cell loss and cooperates with immunotherapies to improve tumor control. In summary, there are opportunities to enhance the efficacy of radiotherapy by exploiting cell-inherent vulnerabilities and dynamic microenvironmental components of the tumor.


Assuntos
Neoplasias/metabolismo , Neoplasias/radioterapia , Tolerância a Radiação/fisiologia , Adaptação Fisiológica , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Instabilidade Genômica , Glutamina/metabolismo , Glutaratos/metabolismo , Glutationa/metabolismo , Humanos , Imunoterapia , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Peroxidação de Lipídeos , Camundongos , Neoplasias/terapia , Nucleotídeos/biossíntese , Consumo de Oxigênio/efeitos dos fármacos , Fosfolipídeos/efeitos da radiação , Radiossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Resultado do Tratamento , Microambiente Tumoral/fisiologia
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