Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 182
Filtrar
1.
Nat Cell Biol ; 23(2): 160-171, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462394

RESUMO

The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.


Assuntos
Cromatina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína BRCA1/genética , Proteína BRCA2/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Aberrações Cromossômicas , DNA Helicases/química , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Epistasia Genética/efeitos dos fármacos , Instabilidade Genômica , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Metanossulfonato de Metila , Mutação/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios Proteicos
2.
Mol Cell ; 81(4): 767-783.e11, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33333017

RESUMO

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.


Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Nucleossomos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética
3.
Proc Natl Acad Sci U S A ; 117(32): 19415-19424, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719125

RESUMO

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.


Assuntos
Antineoplásicos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/genética , Instabilidade Genômica/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Proc Natl Acad Sci U S A ; 117(30): 17785-17795, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32651270

RESUMO

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.


Assuntos
Recombinação Homóloga/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Biologia Computacional , Metilases de Modificação do DNA/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Anemia de Fanconi/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Fator de Necrose Tumoral alfa/metabolismo
5.
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
6.
Cancer Chemother Pharmacol ; 85(4): 661-672, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32072218

RESUMO

PURPOSE: We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-ß-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. METHODS: Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. RESULTS: The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CONCLUSION: CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.


Assuntos
Citarabina/análogos & derivados , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Leucemia de Células T/tratamento farmacológico , Sobrevivência Celular , Citarabina/química , Citarabina/farmacologia , Humanos , Leucemia de Células T/patologia , Fosforilação , Células Tumorais Cultivadas
7.
DNA Repair (Amst) ; 88: 102805, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32062581

RESUMO

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products.


Assuntos
Cafeína/farmacologia , Recombinação Homóloga/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA2/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Dano ao DNA , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Nucleares , Rad51 Recombinase/metabolismo
8.
Mol Pharmacol ; 97(4): 237-249, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32063580

RESUMO

Dysfunction of p53 and resistance to cancer drugs can arise through mutually exclusive overexpression of MDM2 or MDM4. Cisplatin-resistant cells, however, can demonstrate increased binding of both MDM2 and MDM4 to p53 but in absence of cellular overexpression. Whether MDM2 inhibitors alone can activate p53 in these resistant cells was investigated with the goal to establish the mechanism for potential synergy with cisplatin. Thus, growth inhibition by individual drugs and combinations was assessed by a colorimetric assay. Drug-treated parental A2780 and resistant tumor cells were also examined for protein expression using immunoblot and reverse phase protein array (RPPA) and then subjected to Ingenuity Pathway Analysis (IPA). Gene expression was assessed by real-time polymerase chain reaction, DNA damage by confocal microscopy, cell cycle by flow cytometry, and homologous recombination (HR) by a GFP reporter assay. Our results demonstrate that Nutlin-3 but not RITA (reactivation of p53 and induction of tumor cell apoptosis) effectively disrupted the p53-MDM2-MDM4 complex to activate p53, which increased robustly with cisplatin/Nutlin-3 combination and enhanced antitumor effects more than either agent alone. RPPA, IPA, and confocal microscopy provided evidence for an "apparent" increase in DNA damage resulting from HR inhibition by cisplatin/Nutlin-3. Molecularly, the specific HR protein Rad51 was severely downregulated by the combination via two mechanisms: p53-dependent transrepression and p53/MDM2-mediated proteasomal degradation. In conclusion, Nutlin-3 fully destabilizes the p53-MDM2-MDM4 complex and synergizes with cisplatin to intensify p53 function, which then downregulates Rad51 through a bimodal mechanism. As a result, HR is inhibited and antitumor activity enhanced in otherwise HR-proficient sensitive and resistant tumor cells. SIGNIFICANCE STATEMENT: Rad51 downregulation by the combination of cisplatin and Nutlin-3 inhibits homologous recombination (HR), which leads to persistence in DNA damage but not an increase. Thus, inhibition of HR enhances antitumor activity in otherwise HR-proficient sensitive and resistant tumor cells.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Rad51 Recombinase/genética , Proteína Supressora de Tumor p53/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Furanos/farmacologia , Furanos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Neoplasias/genética , Neoplasias/patologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/genética
9.
J Med Chem ; 63(5): 2588-2619, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32037829

RESUMO

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Proteína BRCA2/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Antineoplásicos/química , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Descoberta de Drogas , Sinergismo Farmacológico , Recombinação Homóloga/efeitos dos fármacos , Humanos , Modelos Moleculares , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Ftalazinas/química , Piperazinas/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacos
10.
Oncogene ; 39(14): 2905-2920, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32029902

RESUMO

PARP1 and PARP2 play critical roles in regulating DNA repair and PARP inhibitors have been approved for the treatment of BRCA1/2-mutated ovarian and breast cancers. It has long been known that PARP inhibition sensitizes cancer cells to DNA-damaging cytotoxic agents independent of BRCA status, however, clinical use of PARP inhibitors in combination with DNA-damaging chemotherapy is limited by the more-than-additive cytotoxicity. The natural compound alantolactone (ATL) inhibits the thioredoxin reductase to induce ROS accumulation and oxidative DNA damage selectively in cancer cells. Here, we showed that nontoxic doses of ATL markedly synergized with the PARP inhibitor olaparib to result in synthetic lethality irrespective of homologous recombination status. Synergistic cytotoxicity was seen in cancer but not noncancerous cells and was reduced by the ROS inhibitor NAC or knockdown of OGG1, demonstrating that the cytotoxicity resulted from the repair of ATL-induced oxidative DNA damage. PARP1 knockdown suppressed the synergistic lethality and olaparib was much more toxic than veliparib when combined with ATL, suggesting PARP-trapping as the primary inducer of cytotoxicity. Consistently, combined use of ATL and olaparib caused intense signs of replication stress and formation of double strand DNA breaks, leading to S and G2 arrest followed by apoptosis. In vivo, the combination effectively induced regression of tumor xenografts, while either agent alone had no effect. Hence, PARP trapping combined with specific pro-oxidative agents may provide safe and effective ways to broaden the therapeutic potential of PARP inhibitors.


Assuntos
Dano ao DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Lactonas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Sesquiterpenos de Eudesmano/farmacologia , Células A549 , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Fase G2/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células PC-3 , Fase S/efeitos dos fármacos
11.
Cancer Cell ; 37(2): 157-167.e6, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32004442

RESUMO

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antineoplásicos/uso terapêutico , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Neoplasias Ovarianas/genética , Proteína-Arginina N-Metiltransferases/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos dos fármacos
12.
Cancer Cell ; 37(3): 340-353.e6, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32109375

RESUMO

Inhibition of the cell-cycle kinases CDK4 and CDK6 is now part of the standard treatment in advanced breast cancer. CDK4/6 inhibitors, however, are not expected to cooperate with DNA-damaging or antimitotic chemotherapies as the former prevent cell-cycle entry, thus interfering with S-phase- or mitosis-targeting agents. Here, we report that sequential administration of CDK4/6 inhibitors after taxanes cooperates to prevent cellular proliferation in pancreatic ductal adenocarcinoma (PDAC) cells, patient-derived xenografts, and genetically engineered mice with Kras G12V and Cdkn2a-null mutations frequently observed in PDAC. This effect correlates with the repressive activity of CDK4/6 inhibitors on homologous recombination proteins required for the recovery from chromosomal damage. CDK4/6 inhibitors also prevent recovery from multiple DNA-damaging agents, suggesting broad applicability for their sequential administration after available chemotherapeutic agents.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Albuminas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Reparo do DNA/efeitos dos fármacos , Esquema de Medicação , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Nus , Camundongos Transgênicos , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/patologia , Piperazinas/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridinas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cancer Sci ; 111(4): 1375-1384, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31958182

RESUMO

BRCAness is considered a predictive biomarker to platinum and poly(ADP-ribose) polymerase (PARP) inhibitors. However, recent trials showed that its predictive value was limited in triple-negative breast cancer (TNBC) treated with platinum. Moreover, tumors with mutations of DNA damage response (DDR) genes, such as homologous recombination (HR) genes, could be sensitive to platinum and PARP inhibitors. Thus, we aim to explore the relationship between mutation status of DDR genes and BRCAness in TNBC. We sequenced 56 DDR genes in 120 TNBC and identified BRCAness by array comparative genomic hybridization. The sequencing results showed that 13, 14, and 14 patients had BRCA, non-BRCA HR, and non-HR DDR gene mutations, respectively. Array comparative genomic hybridization revealed that BRCA-mutated and HR gene-mutated TNBC shared similar BRCAness features, both having higher numbers and longer length of large-scale structural aberration (LSA, >10 Mb) and similar altered chromosomal regions of LSA. These suggested non-BRCA HR gene-mutated TNBC shared similar characteristics with BRCA-mutated TNBC, indicating non-BRCA HR gene-mutated TNBC sensitive to platinum and PARP inhibitors. Among tumors with mutation of non-HR DDR genes, 3 PTEN and 1 MSH6 mutation also contained significant LSAs (BRCAness); however, they had different regions of genomic alteration to BRCA and HR gene-mutated tumors, might explain prior findings that PTEN- and MSH6-mutated cancer cells not sensitive to PARP inhibitors. Therefore, we hypothesize that the heterogeneous genomic background of BRCAness indicates different responsiveness to platinum and PARP inhibitors. Direct sequencing DDR genes in TNBC should be applied to predict their sensitivity toward platinum and PARP inhibitors.


Assuntos
Dano ao DNA/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Recombinação Homóloga/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteína BRCA1/genética , Proteína BRCA2/genética , Dano ao DNA/genética , Feminino , Humanos , Pessoa de Meia-Idade , Mutação/genética , Platina/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Neoplasias de Mama Triplo Negativas/epidemiologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/genética
14.
Methods Mol Biol ; 2102: 441-457, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989571

RESUMO

A fully optimized staining method for detecting sister chromatid exchanges in cultured cells is presented. The method gives reproducibly robust quantitative results. Sister chromatid exchange is a classic toxicology assay for genotoxicity and for detecting alterations to the biochemistry underlying cellular homologous recombination. Growth of cells in the presence of 5'-bromo-deoxyuridine for two rounds of DNA replication followed by collecting metaphase spreads on glass slides, treatment with the UV-sensitive dye Hoechst 33258, long-wave UV light exposure, and Giemsa staining gives a permanent record of the exchanges.


Assuntos
Metáfase , Testes de Mutagenicidade/métodos , Troca de Cromátide Irmã , Corantes Azur , Bioensaio/métodos , Bisbenzimidazol , Bromodesoxiuridina/metabolismo , Células Cultivadas , Cromátides/efeitos dos fármacos , Cromátides/metabolismo , Cromátides/efeitos da radiação , Cromossomos/efeitos dos fármacos , Cromossomos/metabolismo , Cromossomos/efeitos da radiação , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Humanos , Metáfase/efeitos dos fármacos , Metáfase/efeitos da radiação , Fluxo de Trabalho
15.
PLoS One ; 15(1): e0221681, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923208

RESUMO

DNA repair inhibition has been described as an essential event leading to the initiation of carcinogenesis. In a previous study, we observed that the exposure to metal mixture induces changes in the miR-nome of the cells that was correlated with the sub-expression of mRNA involved in processes and diseases associated with metal exposure. From this analysis, one of the miRNAs that shows changes in its expression is miR-222, which is overexpressed in various cancers associated with exposure to metals. In silico studies showed that a possible target for the microRNA-222 could be Rad 51c, a gene involved in the double-stranded DNA repair. We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. A luciferase assay was performed to validate Rad51c as miR-222 target. Neutral comet assay was performed in order to evaluate DNA double-strand breaks under experimental conditions. Here, we demonstrate that miR-222 up-regulation, directly regulates Rad51c expression negatively, and impairs homologous recombination of double-strand break DNA repair during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process.


Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Neoplasias/genética , Células 3T3 , Animais , Simulação por Computador , DNA/efeitos dos fármacos , DNA/genética , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Metais/metabolismo , Camundongos
16.
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
17.
Biochem Biophys Res Commun ; 522(1): 121-126, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31753490

RESUMO

Lung cancer is the leading cause of cancer death worldwide. PARP inhibitors have become a new line of cancer therapy and a successful demonstration of the synthetic lethality concept. The mechanism and efficacy of PARP inhibitors have been well studied in some cancers, especially homologous recombination (HR)-deficient ovarian cancer and breast cancer, yet such studies are still relatively fewer in lung cancer. Here we found that HR genes are frequently mutated in lung cancer patients, exposing a window for targeted therapies by PARP inhibitors. We depleted BRCA1 and BRCA2 in non-small cell lung cancer (NSCLC) cancer cells and found these cells are hypersensitive to the PARP inhibitor olaparib in cell viability and clonogenic survival assays. Olaparib specifically induces apoptosis in A549 cells with BRCA1 or BRCA2 depletion, as determined by positive Annexin-V staining. In addition, we show that A549 cells with ATM shRNA knockdown are also hypersensitive to Olaparib. In summary, our data support the potential use of PARP inhibitors in NSCLC with HR deficiency.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Células A549 , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular , Recombinação Homóloga/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Proteína Homóloga a MRE11/genética , Mutação , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética
18.
Mol Cell ; 77(1): 26-38.e7, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31653568

RESUMO

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.


Assuntos
Recombinação Homóloga/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Envelhecimento/efeitos dos fármacos , Envelhecimento/genética , Animais , Proteína BRCA1/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Recombinação Homóloga/efeitos dos fármacos , Camundongos , Mutação/efeitos dos fármacos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/genética , Ubiquitina-Proteína Ligases/genética
19.
Cancer Cell ; 36(5): 545-558.e7, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31668947

RESUMO

Epigenetic regulation enables tumors to respond to changing environments during tumor progression and metastases and facilitates treatment resistance. Targeting chromatin modifiers or catalytic effectors of transcription is an emerging anti-cancer strategy. The cyclin-dependent kinases (CDKs) 12 and 13 phosphorylate the C-terminal domain of RNA polymerase II, regulating transcription and co-transcriptional processes. Here we report the development of SR-4835, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells. Mechanistically, inhibition or loss of CDK12/CDK13 triggers intronic polyadenylation site cleavage that suppresses the expression of core DNA damage response proteins. This provokes a "BRCAness" phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA/efeitos dos fármacos , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Íntrons/efeitos dos fármacos , Íntrons/genética , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poliadenilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Proc Natl Acad Sci U S A ; 116(45): 22609-22618, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31591209

RESUMO

A minority of cancers have breast cancer gene (BRCA) mutations that confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis), but the role for PARPis in BRCA-proficient cancers is not well established. This suggests the need for novel combination therapies to expand the use of these drugs. Recent reports that low doses of DNA methyltransferase inhibitors (DNMTis) plus PARPis enhance PARPi efficacy in BRCA-proficient AML subtypes, breast, and ovarian cancer open up the possibility that this strategy may apply to other sporadic cancers. We identify a key mechanistic aspect of this combination therapy in nonsmall cell lung cancer (NSCLC): that the DNMTi component creates a BRCAness phenotype through downregulating expression of key homologous recombination and nonhomologous end-joining (NHEJ) genes. Importantly, from a translational perspective, the above changes in DNA repair processes allow our combinatorial PARPi and DNMTi therapy to robustly sensitize NSCLC cells to ionizing radiation in vitro and in vivo. Our combinatorial approach introduces a biomarker strategy and a potential therapy paradigm for treating BRCA-proficient cancers like NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Animais , Antineoplásicos , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Metilases de Modificação do DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Quimioterapia Combinada , Feminino , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Ftalazinas/administração & dosagem , Radiação Ionizante
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...