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1.
Clin Cancer Res ; 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409614

RESUMO

Purpose: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance.Experimental Design: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed.Results: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability.Conclusions: Prexasertib exhibits monotherapy activity in PARP inhibitor-resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.

2.
Nat Commun ; 10(1): 2910, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266951

RESUMO

PARP inhibitors (PARPis) have clinical efficacy in BRCA-deficient cancers, but not BRCA-intact tumors, including glioblastoma (GBM). We show that MYC or MYCN amplification in patient-derived glioblastoma stem-like cells (GSCs) generates sensitivity to PARPi via Myc-mediated transcriptional repression of CDK18, while most tumors without amplification are not sensitive. In response to PARPi, CDK18 facilitates ATR activation by interacting with ATR and regulating ATR-Rad9/ATR-ETAA1 interactions; thereby promoting homologous recombination (HR) and PARPi resistance. CDK18 knockdown or ATR inhibition in GSCs suppressed HR and conferred PARPi sensitivity, with ATR inhibitors synergizing with PARPis or sensitizing GSCs. ATR inhibitor VE822 combined with PARPi extended survival of mice bearing GSC-derived orthotopic tumors, irrespective of PARPi-sensitivity. These studies identify a role of CDK18 in ATR-regulated HR. We propose that combined blockade of ATR and PARP is an effective strategy for GBM, even for low-Myc GSCs that do not respond to PARPi alone, and potentially other PARPi-refractory tumors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinases Ciclina-Dependentes/genética , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Camundongos , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Cell Rep ; 28(2): 394-407.e6, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31291576

RESUMO

DNA damage activates checkpoints that limit the replicative potential of stem cells, including differentiation. These checkpoints protect against cancer development but also promote tissue aging. Because mice lacking Slug/Snai2 exhibit limited stem cell activity, including luminobasal differentiation, and are protected from mammary cancer, we reasoned that Slug might regulate DNA damage checkpoints in mammary epithelial cells. Here, we show that Slug facilitates efficient execution of RPA32-mediated DNA damage response (DDR) signaling. Slug deficiency leads to delayed phosphorylation of ataxia telangiectasia mutated and Rad3-related protein (ATR) and its effectors RPA32 and CHK1. This leads to impaired RAD51 recruitment to DNA damage sites and persistence of unresolved DNA damage. In vivo, Slug/Snai2 loss leads to increased DNA damage and premature aging of mammary epithelium. Collectively, our work demonstrates that the mammary stem cell regulator Slug controls DDR checkpoints by dually inhibiting differentiation and facilitating DDR repair, and its loss causes unresolved DNA damage and accelerated aging.

4.
Science ; 364(6447)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31249028

RESUMO

Cancer drivers require statistical modeling to distinguish them from passenger events, which accumulate during tumorigenesis but provide no fitness advantage to cancer cells. The discovery of driver genes and mutations relies on the assumption that exact positional recurrence is unlikely by chance; thus, the precise sharing of mutations across patients identifies drivers. Examining the mutation landscape in cancer genomes, we found that many recurrent cancer mutations previously designated as drivers are likely passengers. Our integrated bioinformatic and biochemical analyses revealed that these passenger hotspot mutations arise from the preference of APOBEC3A, a cytidine deaminase, for DNA stem-loops. Conversely, recurrent APOBEC-signature mutations not in stem-loops are enriched in well-characterized driver genes and may predict new drivers. This demonstrates that mesoscale genomic features need to be integrated into computational models aimed at identifying mutations linked to diseases.

5.
Mol Cell ; 74(6): 1103-1105, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226273

RESUMO

In this issue, Li et al. (2019) report a previously unknown Ca2+-CaMKK2-AMPK signaling cascade that protects stalled forks from degradation by phosphorylating and inhibiting the EXO1 nuclease, revealing a surprising role for Ca2+ influx in the maintenance of genomic stability.

6.
Oncologist ; 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040255

RESUMO

BACKGROUND: Alterations in the DNA damage response (DDR) pathway confer sensitivity to certain chemotherapies, radiation, and other DNA damage repair targeted therapies. BRCA1/2 are the most well-studied DDR genes, but recurrent alterations are described in other DDR pathway members across cancers. Deleterious DDR alterations may sensitize tumor cells to poly (ADP-ribose) polymerase inhibition, but there are also increasing data suggesting that there may also be synergy with immune checkpoint inhibitors. The relevance of DDR defects in gastrointestinal (GI) cancers is understudied. We sought to characterize DDR-defective GI malignancies and to explore genomic context and tumor mutational burden (TMB) to provide a platform for future rational investigations. MATERIALS AND METHODS: Tumor samples from 17,486 unique patients with advanced colorectal, gastroesophageal, or small bowel carcinomas were assayed using hybrid-capture-based comprehensive genomic profiling including sequencing of 10 predefined DDR genes: ARID1A, ATM, ATR, BRCA1, BRCA2, CDK12, CHEK1, CHEK2, PALB2, and RAD51. TMB (mutations per megabase [mut/Mb]) was calculated from up to 1.14 Mb of sequenced DNA. Clinicopathologic features were extracted and descriptive statistics were used to explore genomic relationships among identified subgroups. RESULTS: DDR alterations were found in 17% of cases: gastric adenocarcinoma 475/1,750 (27%), small bowel adenocarcinoma 148/666 (22%), esophageal adenocarcinoma 467/2,501 (19%), and colorectal cancer 1,824/12,569 (15%). ARID1A (9.2%) and ATM (4.7%) were the most commonly altered DDR genes in this series, followed by BRCA2 (2.3%), BRCA1 (1.1%), CHEK2 (1.0%), ATR (0.8%), CDK12 (0.7%), PALB2 (0.6%), CHEK1 (0.1%) and RAD51 (0.1%). More than one DDR gene alteration was found in 24% of cases. High microsatellite instability (MSI-H) and high TMB (TMB-H, ≥20 mut/Mb) were found in 19% and 21% of DDR-altered cases, respectively. Of DDR-altered/TMB-H cases, 87% were also MSI-H. However, even in the microsatellite stable (MSS)/DDR-wild-type (WT) versus MSS/DDR-altered, TMB-high was seen more frequently (0.4% vs. 3.3%, P < .00001.) Median TMB was 5.4 mut/Mb in the MSS/DDR-altered subset versus 3.8 mut/Mb in the MSS/DDR-WT subset (P ≤ .00001), and ATR alterations were enriched in the MSS/TMB-high cases. CONCLUSION: This is the largest study to examine selected DDR defects in tubular GI cancers and confirms that DDR defects are relatively common and that there is an association between the selected DDR defects and a high TMB in more than 20% of cases. Microsatellite stable DDR-defective tumors with elevated TMB warrant further exploration. IMPLICATIONS FOR PRACTICE: Deleterious DNA damage response (DDR) alterations may sensitize tumor cells to poly (ADP-ribose) polymerase inhibition, but also potentially to immune checkpoint inhibitors, owing to accumulation of mutations in DDR-defective tumors. The relevance of DDR defects in gastrointestinal (GI) cancers is understudied. This article characterizes DDR-defective GI malignancies and explores genomic context and tumor mutational burden to provide a platform for future rational investigations.

8.
Cancer ; 125(12): 1963-1972, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30835824

RESUMO

Substantial progress has been made in understanding ovarian cancer at the molecular and cellular level. Significant improvement in 5-year survival has been achieved through cytoreductive surgery, combination platinum-based chemotherapy, and more effective treatment of recurrent cancer, and there are now more than 280,000 ovarian cancer survivors in the United States. Despite these advances, long-term survival in late-stage disease has improved little over the last 4 decades. Poor outcomes relate, in part, to late stage at initial diagnosis, intrinsic drug resistance, and the persistence of dormant drug-resistant cancer cells after primary surgery and chemotherapy. Our ability to accelerate progress in the clinic will depend on the ability to answer several critical questions regarding this disease. To assess current answers, an American Association for Cancer Research Special Conference on "Critical Questions in Ovarian Cancer Research and Treatment" was held in Pittsburgh, Pennsylvania, on October 1-3, 2017. Although clinical, translational, and basic investigators conducted much of the discussion, advocates participated in the meeting, and many presentations were directly relevant to patient care, including treatment with poly adenosine diphosphate ribose polymerase (PARP) inhibitors, attempts to improve immunotherapy by overcoming the immune suppressive effects of the microenvironment, and a better understanding of the heterogeneity of the disease.

10.
Hepatology ; 69(6): 2608-2622, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30693543

RESUMO

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

11.
Cell Rep ; 26(4): 955-968.e3, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673617

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe an assay to visualize ALT-mediated telomeric DNA synthesis in ALT-associated PML bodies (APBs) without DNA-damaging agents or replication inhibitors. Using this assay, we find that ALT occurs through two distinct mechanisms. One of the ALT mechanisms requires RAD52, a protein implicated in break-induced DNA replication (BIR). We demonstrate that RAD52 directly promotes telomeric D-loop formation in vitro and is required for maintaining telomeres in ALT-positive cells. Unexpectedly, however, RAD52 is dispensable for C-circle formation, a hallmark of ALT. In RAD52-knockout ALT cells, C-circle formation and RAD52-independent ALT DNA synthesis gradually increase as telomeres are shortened, and these activities are dependent on BLM and BIR proteins POLD3 and POLD4. These results suggest that ALT occurs through a RAD52-dependent and a RAD52-independent BIR pathway, revealing the bifurcated framework and dynamic nature of this process.

12.
Genes Dev ; 33(1-2): 75-89, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30567999

RESUMO

Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.


Assuntos
Biotinilação/métodos , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Fatores de Transcrição/metabolismo , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Técnicas de Silenciamento de Genes , Humanos , Proteína Homóloga a MRE11/metabolismo , Ligação Proteica , Transporte Proteico/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
13.
Nat Commun ; 9(1): 4115, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30297739

RESUMO

Actively transcribed regions of the genome are protected by transcription-coupled DNA repair mechanisms, including transcription-coupled homologous recombination (TC-HR). Here we used reactive oxygen species (ROS) to induce and characterize TC-HR at a transcribed locus in human cells. As canonical HR, TC-HR requires RAD51. However, the localization of RAD51 to damage sites during TC-HR does not require BRCA1 and BRCA2, but relies on RAD52 and Cockayne Syndrome Protein B (CSB). During TC-HR, RAD52 is recruited by CSB through an acidic domain. CSB in turn is recruited by R loops, which are strongly induced by ROS in transcribed regions. Notably, CSB displays a strong affinity for DNA:RNA hybrids in vitro, suggesting that it is a sensor of ROS-induced R loops. Thus, TC-HR is triggered by R loops, initiated by CSB, and carried out by the CSB-RAD52-RAD51 axis, establishing a BRCA1/2-independent alternative HR pathway protecting the transcribed genome.

14.
Mol Cell ; 72(2): 201-203, 2018 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-30340018

RESUMO

DNA replication forks collapse at numerous sites throughout the genome under replication stress. Studies by Shastri et al. (2018) and Tubbs et al. (2018) used different genomics approaches to map the sites of replication fork collapse, revealing the contribution of specific DNA sequences to replication stress.

15.
Cancer Res ; 78(18): 5363-5374, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30054334

RESUMO

Heterozygous somatic mutations in spliceosome genes (U2AF1, SF3B1, ZRSR2, or SRSF2) occur in >50% of patients with myelodysplastic syndrome (MDS). These mutations occur early in disease development, suggesting that they contribute to MDS pathogenesis and may represent a unique genetic vulnerability for targeted therapy. Here, we show that RNA splicing perturbation by expression of the U2AF1(S34F) mutant causes accumulation of R loops, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA, and elicits an ATR response. ATR inhibitors (ATRi) induced DNA damage and cell death in U2AF1(S34F)-expressing cells, and these effects of ATRi were enhanced by splicing modulating compounds. Moreover, ATRi-induced DNA damage was suppressed by overexpression of RNaseH1, an enzyme that specifically removes the RNA in RNA:DNA hybrids, suggesting that the ATRi sensitivity of U2AF1(S34F)-expressing cells arises from R loops. Taken together, our results demonstrate that ATR may represent a novel therapeutic target in patients with MDS carrying the U2AF1(S34F) mutation and potentially other malignancies harboring spliceosome mutations.Significance: This study provides preclinical evidence that patients with MDS or other myeloid malignancies driven by spliceosome mutations may benefit from ATR inhibition to exploit the R loop-associated vulnerability induced by perturbations in splicing. Cancer Res; 78(18); 5363-74. ©2018 AACR.

17.
Science ; 359(6371): 108-114, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29170278

RESUMO

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability.


Assuntos
Centrômero/enzimologia , Segregação de Cromossomos/genética , Mitose/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo
18.
Dev Cell ; 43(6): 689-703.e5, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29233476

RESUMO

To understand the consequences of the complete elimination of E2F regulation, we profiled the proteome of Drosophila dDP mutants that lack functional E2F/DP complexes. The results uncovered changes in the larval fat body, a differentiated tissue that grows via endocycles. We report an unexpected mechanism of E2F/DP action that promotes quiescence in this tissue. In the fat body, dE2F/dDP limits cell-cycle progression by suppressing DNA damage responses. Loss of dDP upregulates dATM, allowing cells to sense and repair DNA damage and increasing replication of loci that are normally under-replicated in wild-type tissues. Genetic experiments show that ectopic dATM is sufficient to promote DNA synthesis in wild-type fat body cells. Strikingly, reducing dATM levels in dDP-deficient fat bodies restores cell-cycle control, improves tissue morphology, and extends animal development. These results show that, in some cellular contexts, dE2F/dDP-dependent suppression of DNA damage signaling is key for cell-cycle control and needed for normal development.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/biossíntese , Proteínas de Drosophila/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Corpo Adiposo/fisiologia , Transativadores/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Drosophila , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Corpo Adiposo/citologia , Transativadores/genética , Transcriptoma
20.
Sci China Life Sci ; 60(10): 1081-1086, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29075944

RESUMO

Repair of DNA double-strand breaks (DSBs) via the homologous recombination (HR) pathway is a highly regulated process. A number of proteins that participate in HR are intricately modulated by the cell cycle and chromatin environments of DSBs. Recent studies have revealed a clear impact of transcription on HR in transcribed regions of the genome. Several models have been put forth to explain how the process of transcription and/or its RNA products may influence HR. Here we discuss the results and models from these studies, presenting an emerging view of transcription-coupled DSB repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga/genética , RNA/genética , Transcrição Genética , Animais , DNA/genética , DNA/metabolismo , Humanos , Modelos Genéticos , RNA/metabolismo
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