RESUMO
SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.
Assuntos
Proteínas Nucleares/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/metabolismo , Pirazinas , Pirazóis , Proteína de Replicação A/metabolismoRESUMO
Schlafen-11 (SLFN11) inactivation in â¼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1-CUL4CDT2 ubiquitin ligase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Dano ao DNA/genética , Replicação do DNA , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Mutações Sintéticas Letais/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Cromossomos Humanos/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Estabilidade Enzimática , Genoma Humano , Humanos , Mitose , Modelos Biológicos , Terapia de Alvo Molecular , Fosforilação , Ligação Proteica , Interferência de RNA , Transdução de SinaisRESUMO
PARP inhibitors (PARPis) were initially developed as DNA repair inhibitors that inhibit the catalytic activity of PARP1 and PARP2 and are expected to induce synthetic lethality in BRCA- or homologous recombination (HR)-deficient tumors. However, the clinical indications for PARPis are not necessarily limited to BRCA mutations or HR deficiency; BRCA wild-type and HR-proficient cancers can also derive some benefit from PARPis. These facts are interpretable by an additional primary antitumor mechanism of PARPis named PARP trapping, resulting from the stabilization of PARP-DNA complexes. Favorable response to platinum derivatives (cisplatin and carboplatin) in preceding treatment is used as a clinical biomarker for some PARPis, implying that sensitivity factors for platinum derivatives and PARPis are mainly common. Such common sensitivity factors include not only HR defects (HRD) but also additional factors. One of them is Schlafen 11 (SLFN11), a putative DNA/RNA helicase, that sensitizes cancer cells to a broad type of DNA-damaging agents, including platinum and topoisomerase inhibitors. Mechanistically, SLFN11 induces a lethal replication block in response to replication stress (ie, DNA damage). As SLFN11 acts upon replication stress, trapping PARPis can activate SLFN11. Preclinical models show the importance of SLFN11 in PARPi sensitivity. However, the relevance of SLFN11 in PARPi response is less evident in clinical data compared with the significance of SLFN11 for platinum sensitivity. In this review, we consider the reasons for variable indications of PARPis resulting from clinical outcomes and review the mechanisms of action for PARPis as anticancer agents.
Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , DNA Helicases/genética , Reparo do DNA , Recombinação Homóloga , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutações Sintéticas LetaisRESUMO
The utility of Schlafen 11 (SLFN11) expression as a predictive biomarker for platinum-based chemotherapy has been established for cancers from different histologies. However, the therapeutic relevance of SLFN11 in bladder cancer (BC) is unknown. Here, we examined the clinicopathologic significance of SLFN11 expression across 120 BC cases by immunohistochemistry. We divided the cases into two cohorts, one including 50 patients who received adjuvant or neoadjuvant platinum-based chemotherapy, and the other including 70 BC patients treated by surgical resection without chemotherapy. In the cohort of 50 BC cases treated with platinum-based chemotherapy, the SLFN11-positive group (n = 25) showed significantly better overall survival than the SLFN11-negative group (n = 25, P = .012). Schlafen 11 expression correlated significantly with the expression of luminal subtype marker GATA3. Multivariate analyses identified SLFN11 expression as an independent prognostic predictor (odds ratio, 0.32; 95% confidence interval, 0.11-0.91; P = .033). Conversely, in the cohort of 70 BC cases not receiving platinum-based chemotherapy, the SLFN11-positive group (n = 29) showed significantly worse overall survival than the SLFN11-negative group (n = 41, P = .034). In vitro analyses using multiple BC cell lines confirmed that SLFN11 KO rendered cells resistant to cisplatin. The epigenetic modifying drugs 5-azacytidine and entinostat restored SLFN11 expression and resensitized cells to cisplatin and carboplatin in SLFN11-negative BC cell lines. We conclude that SLFN11 is a predictive biomarker for BC patients who undergo platinum-based chemotherapy and that the combination of epigenetic modifiers could rescue refractory BC patients to platinum derivatives by reactivating SLFN11 expression.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Nucleares/metabolismo , Platina/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Idoso , Antineoplásicos/farmacologia , Azacitidina/farmacologia , Benzamidas/farmacologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Feminino , Fator de Transcrição GATA3/metabolismo , Humanos , Masculino , Proteínas Nucleares/genética , Platina/farmacologia , Prognóstico , Piridinas/farmacologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
BACKGROUND: Although unresectable or recurrent gastric cancers (GC) are frequently treated with platinum-based chemotherapy, response to treatment remains unpredictable. Because Schlafen 11 (SLFN11) is recently identified as a critical determinant of platinum sensitivity, we investigated the potential clinical utility of SLFN11 in the treatment of GC. METHODS: We analysed the correlation between SLFN11 expression and overall survival in 169 GC patients by our established immunohistochemical approach. The impact of SLFN11 expression on the response to platinum and transition of SLFN11 expression upon long-term treatment with platinum were examined using GC cell lines and organoids. RESULTS: GC patients with high-SLFN11 expression exhibited significantly better survival than those with low-SLFN11 expression, and the significance increased when we selected patients treated with platinum-based chemotherapy. Knockout of SLFN11 and reactivation of SLFN11 in GC cells conferred resistance and sensitivity to platinum, respectively. In GC cells and organoids, long-term treatment with oxaliplatin suppressed SLFN11 expression while imparting drug resistance. The acquired resistance to oxaliplatin was reversed by reactivation of SLFN11 with epigenetic modifying drugs. CONCLUSIONS: This is the first report revealing definitive clinical implications of SLFN11 in the treatment of GC patients and providing novel strategies for the drug selection based on SLFN11 expression.
Assuntos
Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Platina/farmacologia , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Platina/uso terapêutico , Prognóstico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Análise de Sobrevida , Resultado do TratamentoRESUMO
BACKGROUND: Schlafen 11 (SLFN11) was recently identified as a dominant determinant of sensitivity to DNA-targeting agents including platinum-based drugs. SLFN11 also reportedly enhances cellular radiosensitivity. In this study, we examined the prognostic value of SLFN11 expression in esophageal squamous cell carcinoma (ESCC) patients treated with definitive chemoradiotherapy (dCRT), including the platinum derivative nedaplatin. METHODS: Seventy-three patients with ESCC who received dCRT were examined. SLFN11 expression was analyzed in pre-dCRT biopsies using immunohistochemistry and evaluated using a histo-score (H-score). Correlation between the H-score and overall survival was analyzed. An H-score ≥ 51 was provisionally defined as indicating high SLFN11 expression. Viability assays were performed using previously established isogenic human cell lines differentially expressing SLFN11 to test the usefulness of SLFN11 as marker of response to the dCRT regimen. RESULTS: High SLFN11 expression was independently associated with better prognosis in ESCC patients (hazard ratio = 0.295, 95% CI = 0.143-0.605, p = 0.001 for multivariate analysis). Kaplan-Meier survival curves showed that the prognostic value of high SLFN11 expression was most evident in patients at clinical stages II and III (p = 0.004). In in vitro study, SLFN11-proficient cells were highly sensitive to platinum derivatives compared to SLFN11-deficient cells. CONCLUSION: SLFN11 expression is an independent prognostic factor for ESCC patients treated with dCRT and a potential biomarker for treatment selection of ESCC. Examination of SLFN11 may be particularly useful for clinical Stage II-III patients who wish to choose dCRT (instead of surgery) to preserve esophageal function.
Assuntos
Quimiorradioterapia/métodos , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/radioterapia , Proteínas Nucleares/metabolismo , Idoso , Feminino , Humanos , Masculino , Prognóstico , Estudos RetrospectivosRESUMO
Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.
Assuntos
Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Processamento Alternativo/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Diester Fosfórico Hidrolases , Isoformas de Proteínas/genética , Fatores de Transcrição/antagonistas & inibidoresRESUMO
The USP1/UAF1 complex deubiquitinates the Fanconi anemia protein FANCD2, thereby promoting homologous recombination and DNA cross-link repair. How USP1/UAF1 is targeted to the FANCD2/FANCI heterodimer has remained unknown. Here we show that UAF1 contains a tandem repeat of SUMO-like domains in its C terminus (SLD1 and SLD2). SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 sequence of UAF1 or mutation of the SIM on FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination and DNA repair. The USP1/UAF1 complex also deubiquitinates PCNA-Ub, and deubiquitination requires the PCNA-binding protein hELG1. The SLD2 sequence of UAF1 binds to a SIM on hELG1, thus targeting the USP1/UAF1 complex to its PCNA-Ub substrate. We propose that the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates homologous recombination and translesion DNA synthesis.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Regulação da Expressão Gênica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis , Linhagem Celular , Galinhas , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Recombinação Genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sequências de Repetição em Tandem/genética , Ubiquitina/metabolismo , Proteases Específicas de UbiquitinaRESUMO
The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.
Assuntos
DNA Polimerase III/metabolismo , Replicação do DNA , DNA/biossíntese , DNA/química , Alelos , Linhagem Celular , Dano ao DNA , DNA Polimerase III/química , DNA Polimerase III/genética , DNA Polimerase III/isolamento & purificação , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/isolamento & purificação , Holoenzimas/metabolismo , Humanos , Imunoglobulinas/genética , Raios UltravioletaRESUMO
SLFN11 is a recently discovered protein with a putative DNA/RNA helicase function. First identified in association with the maturation of thymocytes, SLFN11 was later causally associated, by two independent groups, with the resistance to DNA damaging agents such as topoisomerase I and II inhibitors, platinum compounds, and other alkylators, making it an attractive molecule for biomarker development. Later, SLFN11 was linked to antiviral response in human cells and interferon production, establishing a potential bond between immunity and chemotherapy. Recently, we demonstrated the potential role of SLN11 as a biomarker to predict sensitivity to the carboplatin/taxol combination in ovarian cancer. The present manuscript reports on the first international monothematic workshop on SLFN11. Several researchers from around the world, directly and actively involved in the discovery, functional characterization, and study of SLFN11 for its biomarker and medicinal properties gathered to share their views on the current knowledge advances concerning SLFN11. The aim of the manuscript is to summarize the authors' interventions and the main take-home messages resulting from the workshop.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Consenso , Dano ao DNA , Humanos , Mutagênicos/toxicidade , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , FenótipoRESUMO
Approximately half of high-grade serous epithelial ovarian cancers incur alterations in genes of homologous recombination (BRCA1, BRCA2, RAD51C, Fanconi anemia genes), and the rest incur alterations in other DNA repair pathways at high frequencies. Such cancer-specific gene alterations can confer selective sensitivity to DNA damaging agents such as cisplatin and carboplatin, topotecan, etoposide, doxorubicin, and gemcitabine. Originally presumed to inhibit DNA repair, PARP inhibitors that have recently been approved by the FDA for the treatment of advanced ovarian cancer also act as DNA damaging agents by inducing PARP-DNA complexes. These DNA damaging agents induce different types of DNA lesions that require various DNA repair genes for the repair, but commonly induce replication fork slowing or stalling, also referred to as replication stress. Replication stress activates DNA repair checkpoint proteins (ATR, CHK1), which prevent further DNA damage. Hence, targeting DNA repair genes or DNA repair checkpoint genes augments the anti-tumor activity of DNA damaging agents. This review describes the rational basis for using DNA repair and DNA repair checkpoint inhibitors as single agents. The review also presents the strategies combining these inhibitors with DNA damaging agents for ovarian cancer therapy based on specific gene alterations.
Assuntos
Antineoplásicos/farmacologia , Reparo do DNA/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antineoplásicos/uso terapêutico , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial do Ovário , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Cisplatino/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Topotecan/uso terapêuticoRESUMO
The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.
Assuntos
Dano ao DNA , DNA Polimerase III/metabolismo , Reparo do DNA , Animais , Sequência de Bases , Linhagem Celular , Galinhas , DNA Polimerase III/química , Primers do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase SRESUMO
Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3'-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1-PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/metabolismo , Epistasia Genética , Humanos , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Domínios e Motivos de Interação entre Proteínas , Sumoilação , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
RING finger protein 4 (RNF4) represents a subclass of ubiquitin ligases that target proteins modified by the small ubiquitin-like modifier (SUMO) for ubiquitin-mediated degradation. We disrupted the RNF4 gene in chicken DT40 cells and found that the resulting RNF4(-/-) cells gradually lost proliferation capability. Strikingly, this compromised proliferation was associated with an unprecedented cellular effect: the gradual decrease in the number of intact chromosomes. In the 6 weeks after gene targeting, there was a 25% reduction in the DNA content of the RNF4(-/-) cells. Regarding trisomic chromosome 2, 60% of the RNF4(-/-) cells lost one homologue, suggesting that DNA loss was mediated by whole chromosome loss. To determine the cause of this chromosome loss, we examined cell-cycle checkpoint pathways. RNF4(-/-) cells showed a partial defect in the spindle assembly checkpoint, premature dissociation of sister chromatids, and a marked increase in the number of lagging chromosomes at anaphase. Thus, combined defects in SAC and sister chromatid cohesion may result in increased lagging chromosomes, leading to chromosome loss without accompanying chromosome gain in RNF4(-/-) cells. We therefore propose that RNF4 plays a novel role in preventing the loss of intact chromosomes and ensures the maintenance of chromosome integrity.
Assuntos
Aberrações Cromossômicas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Anáfase , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/genética , Galinhas , Segregação de Cromossomos , Dano ao DNA , Replicação do DNA , Pontos de Checagem da Fase M do Ciclo Celular , Mutação , Ubiquitina-Proteína Ligases/genéticaRESUMO
Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , Antivirais/toxicidade , Dano ao DNA , Reparo do DNA , Diester Fosfórico Hidrolases/metabolismo , Aciclovir/metabolismo , Aciclovir/toxicidade , Animais , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/toxicidade , Antimetabólitos Antineoplásicos/metabolismo , Antivirais/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Citarabina/metabolismo , Citarabina/toxicidade , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Deleção de Genes , Camundongos , Diester Fosfórico Hidrolases/genética , Zalcitabina/metabolismo , Zalcitabina/toxicidade , Zidovudina/metabolismo , Zidovudina/toxicidadeRESUMO
We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors exert their cytotoxicity primarily by trapping PARP-DNA complexes in addition to their NAD(+)-competitive catalytic inhibitory mechanism. PARP trapping is drug-specific, with olaparib exhibiting a greater ability than veliparib, whereas both compounds are potent catalytic PARP inhibitors. Here, we evaluated the combination of olaparib or veliparib with therapeutically relevant DNA-targeted drugs, including the topoisomerase I inhibitor camptothecin, the alkylating agent temozolomide, the cross-linking agent cisplatin, and the topoisomerase II inhibitor etoposide at the cellular and molecular levels. We determined PARP-DNA trapping and catalytic PARP inhibition in genetically modified chicken lymphoma DT40, human prostate DU145, and glioblastoma SF295 cancer cells. For camptothecin, both PARP inhibitors showed highly synergistic effects due to catalytic PARP inhibition, indicating the value of combining either veliparib or olaparib with topoisomerase I inhibitors. On the other hand, for temozolomide, PARP trapping was critical in addition to catalytic inhibition, consistent with the fact that olaparib was more effective than veliparib in combination with temozolomide. For cisplatin and etoposide, olaparib only showed no or a weak combination effect, which is consistent with the lack of involvement of PARP in the repair of cisplatin- and etoposide-induced lesions. Hence, we conclude that catalytic PARP inhibitors are highly effective in combination with camptothecins, whereas PARP inhibitors capable of PARP trapping are more effective with temozolomide. Our study provides insights in combination treatment rationales for different PARP inhibitors.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/farmacologia , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Camptotecina/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Dano ao DNA , Reparo do DNA , Dacarbazina/administração & dosagem , Dacarbazina/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/administração & dosagem , Humanos , Ftalazinas/administração & dosagem , Ftalazinas/farmacologia , Piperazinas/administração & dosagem , Piperazinas/farmacologia , TemozolomidaRESUMO
The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3'- or 5'-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase 'poisons'. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3'-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3'-tyrosyl DNA phosphodiesterase (3'-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1-/- cells, suggesting that Tdp2 contributes to cellular 3'-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1-/-/Tdp2-/-/- DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1-/- DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.
Assuntos
Quebras de DNA , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/fisiologia , Animais , Camptotecina/toxicidade , Células Cultivadas , Proteínas de Ligação a DNA , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Diester Fosfórico Hidrolases/genética , Inibidores da Topoisomerase I/toxicidade , Fatores de Transcrição/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
BRCA1/2 mutations are robust biomarkers for platinum-based chemotherapy in epithelial ovarian cancers. However, BRCA1/2 mutations in clear cell ovarian carcinoma (CCC) are less frequent compared with high-grade serous ovarian cancer (HGSC). The discovery of biomarkers that can be applied to CCC is an unmet need in chemotherapy. Schlafen 11 (SLFN11) has attracted attention as a novel sensitizer for DNA-damaging agents including platinum. In this study, we investigated the utility of SLFN11 in HGSC and CCC for platinum-based chemotherapy. SLFN11 expression was analyzed retrospectively by IHC across 326 ovarian cancer samples. The clinicopathologic significance of SLFN11 expression was analyzed across 57 advanced HGSC as a discovery set, 96 advanced HGSC as a validation set, and 57 advanced CCC cases, all of whom received platinum-based chemotherapy. BRCA1/2 mutation was analyzed using targeted-gene sequencing. In the HGSC cohort, the SLFN11-positive and BRCA mutation group showed significantly longer whereas the SLFN11-negative and BRCA wild-type group showed significantly shorter progression-free survival and overall survival. Moreover, SLFN11-positive HGSC shrunk significantly better than SLFN11-negative HGSC after neoadjuvant chemotherapy. Comparable results were obtained with CCC but without consideration of BRCA1/2 mutation due to a small population. Multivariate analysis identified SLFN11 as an independent factor for better survival in HGSC and CCC. The SLFN11-dependent sensitivity to platinum and PARP inhibitors were validated with genetically modified non-HGSC ovarian cancer cell lines. Our study reveals that SLFN11 predicts platinum sensitivity in HGSC and CCC independently of BRCA1/2 mutation status, indicating that SLFN11 assessment can guide treatment selection in HGSC and CCC.
Assuntos
Adenocarcinoma de Células Claras , Neoplasias Ovarianas , Humanos , Feminino , Proteína BRCA1/genética , Estudos Retrospectivos , Proteína BRCA2/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Proteínas Nucleares/genéticaRESUMO
The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Replicação do DNA , DNA de Cadeia Simples , Proteína Homóloga a MRE11 , Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Feminino , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genética , Cromatina/metabolismo , Ftalazinas/farmacologiaRESUMO
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.