40 results on '"Barber LJ"'
Search Results
2. hCLK2 couples FANCD2 to stalled replication forks and functions in the mammalian S-phase checkpoint
- Author
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Collis, SJ, primary, Barber, LJ, additional, Martin, JS, additional, Ward, JD, additional, and Boulton, SJ, additional
- Published
- 2006
- Full Text
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3. AJCN unplugged: easy to use World Wide Web presentation of Journal content
- Author
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Barber, LJ, primary, McGlade, LT, additional, Milot, BA, additional, and Scales, J, additional
- Published
- 1997
- Full Text
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4. Genetic and immune landscape evolution in MMR-deficient colorectal cancer.
- Author
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Challoner BR, Woolston A, Lau D, Buzzetti M, Fong C, Barber LJ, Anandappa G, Crux R, Assiotis I, Fenwick K, Begum R, Begum D, Lund T, Sivamanoharan N, Sansano HB, Domingo-Arada M, Tran A, Pandha H, Church D, Eccles B, Ellis R, Falk S, Hill M, Krell D, Murugaesu N, Nolan L, Potter V, Saunders M, Shiu KK, Guettler S, Alexander JL, Lázare-Iglesias H, Kinross J, Murphy J, von Loga K, Cunningham D, Chau I, Starling N, Ruiz-Bañobre J, Dhillon T, and Gerlinger M
- Subjects
- Humans, B7-H1 Antigen, Phylogeny, Tumor Microenvironment genetics, Colorectal Neoplasms pathology, Colonic Neoplasms
- Abstract
Mismatch repair-deficient (MMRd) colorectal cancers (CRCs) have high mutation burdens, which make these tumours immunogenic and many respond to immune checkpoint inhibitors. The MMRd hypermutator phenotype may also promote intratumour heterogeneity (ITH) and cancer evolution. We applied multiregion sequencing and CD8 and programmed death ligand 1 (PD-L1) immunostaining to systematically investigate ITH and how genetic and immune landscapes coevolve. All cases had high truncal mutation burdens. Despite pervasive ITH, driver aberrations showed a clear hierarchy. Those in WNT/β-catenin, mitogen-activated protein kinase, and TGF-β receptor family genes were almost always truncal. Immune evasion (IE) drivers, such as inactivation of genes involved in antigen presentation or IFN-γ signalling, were predominantly subclonal and showed parallel evolution. These IE drivers have been implicated in immune checkpoint inhibitor resistance or sensitivity. Clonality assessments are therefore important for the development of predictive immunotherapy biomarkers in MMRd CRCs. Phylogenetic analysis identified three distinct patterns of IE driver evolution: pan-tumour evolution, subclonal evolution, and evolutionary stasis. These, but neither mutation burdens nor heterogeneity metrics, significantly correlated with T-cell densities, which were used as a surrogate marker of tumour immunogenicity. Furthermore, this revealed that genetic and T-cell infiltrates coevolve in MMRd CRCs. Low T-cell densities in the subgroup without any known IE drivers may indicate an, as yet unknown, IE mechanism. PD-L1 was expressed in the tumour microenvironment in most samples and correlated with T-cell densities. However, PD-L1 expression in cancer cells was independent of T-cell densities but strongly associated with loss of the intestinal homeobox transcription factor CDX2. This explains infrequent PD-L1 expression by cancer cells and may contribute to a higher recurrence risk of MMRd CRCs with impaired CDX2 expression. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)
- Published
- 2024
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5. Multifactorial Remodeling of the Cancer Immunopeptidome by IFNγ.
- Author
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Newey A, Yu L, Barber LJ, Choudhary JS, Bassani-Sternberg M, and Gerlinger M
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- Humans, Interferon-gamma, Antigens, Peptides chemistry, Proline, Proteomics, Neoplasms genetics
- Abstract
IFNγ alters the immunopeptidome presented on HLA class I (HLA-I), and its activity on cancer cells is known to be important for effective immunotherapy responses. We performed proteomic analyses of untreated and IFNγ-treated colorectal cancer patient-derived organoids and combined this with transcriptomic and HLA-I immunopeptidomics data to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNγ. IFNγ-induced changes in the abundance of source proteins, switching from the constitutive to the immunoproteasome, and differential upregulation of different HLA alleles explained some, but not all, observed peptide abundance changes. By selecting for peptides which increased or decreased the most in abundance, but originated from proteins with limited abundance changes, we discovered that the amino acid composition of presented peptides also influences whether a peptide is upregulated or downregulated on HLA-I through IFNγ. The presence of proline within the peptide core was most strongly associated with peptide downregulation. This was validated in an independent dataset. Proline substitution in relevant core positions did not influence the predicted HLA-I binding affinity or stability, indicating that proline effects on peptide processing may be most relevant. Understanding the multiple factors that influence the abundance of peptides presented on HLA-I in the absence or presence of IFNγ is important to identify the best targets for antigen-specific cancer immunotherapies such as vaccines or T-cell receptor engineered therapeutics., Significance: IFNγ remodels the HLA-I-presented immunopeptidome. We showed that peptide-specific factors influence whether a peptide is upregulated or downregulated and identified a preferential loss or downregulation of those with proline near the peptide center. This will help selecting immunotherapy target antigens which are consistently presented by cancer cells., (© 2023 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2023
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6. Hydrogel Cross-Linking via Thiol-Reactive Pyridazinediones.
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Bahou C, Spears RJ, Ramírez Rosales AM, Rochet LNC, Barber LJ, Stankevich KS, Miranda JF, Butcher TC, Kerrigan AM, Lazarov VK, Grey W, Chudasama V, and Spicer CD
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- Humans, Cross-Linking Reagents chemistry, Hydrogels chemistry, Sulfhydryl Compounds chemistry
- Abstract
Thiol-reactive Michael acceptors are commonly used for the formation of chemically cross-linked hydrogels. In this paper, we address the drawbacks of many Michael acceptors by introducing pyridazinediones as new cross-linking agents. Through the use of pyridazinediones and their mono- or dibrominated analogues, we show that the mechanical strength, swelling ratio, and rate of gelation can all be controlled in a pH-sensitive manner. Moreover, we demonstrate that the degradation of pyridazinedione-gels can be induced by the addition of thiols, thus providing a route to responsive or dynamic gels, and that monobromo-pyridazinedione gels are able to support the proliferation of human cells. We anticipate that our results will provide a valuable and complementary addition to the existing toolkit of cross-linking agents, allowing researchers to tune and rationally design the properties of biomedical hydrogels.
- Published
- 2023
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7. Selectivity and stability of N-terminal targeting protein modification chemistries.
- Author
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Barber LJ, Yates NDJ, Fascione MA, Parkin A, Hemsworth GR, Genever PG, and Spicer CD
- Abstract
Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no 'one size fits all' approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field., Competing Interests: The authors declare no competing financial interests., (This journal is © The Royal Society of Chemistry.)
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- 2022
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8. Catalytic enantioselective synthesis of 1,4-dihydropyridines via the addition of C(1)-ammonium enolates to pyridinium salts.
- Author
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McLaughlin C, Bitai J, Barber LJ, Slawin AMZ, and Smith AD
- Abstract
The regio- and stereoselective addition of C(1)-ammonium enolates - generated in situ from aryl esters and the isothiourea catalyst ( R )-BTM - to pyridinium salts bearing an electron withdrawing substituent in the 3-position allows the synthesis of a range of enantioenriched 1,4-dihydropyridines. This represents the first organocatalytic approach to pyridine dearomatisation using pronucleophiles at the carboxylic acid oxidation level. Optimisation studies revealed a significant solvent dependency upon product enantioselectivity, with only toluene providing significant asymmetric induction. Using DABCO as a base also proved beneficial for product enantioselectivity, while investigations into the nature of the counterion showed that co-ordinating bromide or chloride substrates led to higher product er than the corresponding tetrafluoroborate or hexafluorophosphate. The scope and limitations of this process are developed, with enantioselective addition to 3-cyano- or 3-sulfonylpyridinium salts giving the corresponding 1,4-dihydropyridines (15 examples, up to 95 : 5 dr and 98 : 2 er)., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2021
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9. Mutational signatures impact the evolution of anti-EGFR antibody resistance in colorectal cancer.
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Woolston A, Barber LJ, Griffiths B, Pich O, Lopez-Bigas N, Matthews N, Rao S, Watkins D, Chau I, Starling N, Cunningham D, and Gerlinger M
- Subjects
- Cetuximab therapeutic use, Humans, Mutation, Antineoplastic Agents, Immunological therapeutic use, Colorectal Neoplasms drug therapy, Colorectal Neoplasms genetics
- Abstract
Anti-EGFR antibodies such as cetuximab are active against KRAS/NRAS wild-type colorectal cancers (CRCs), but acquired resistance invariably evolves. It is unknown which mutational mechanisms enable resistance evolution and whether adaptive mutagenesis (a transient cetuximab-induced increase in mutation generation) contributes in patients. Here, we investigate these questions in exome sequencing data from 42 baseline and progression biopsies from cetuximab-treated CRCs. Mutation loads did not increase from baseline to progression, and evidence for a contribution of adaptive mutagenesis was limited. However, the chemotherapy-induced mutational signature SBS17b was the main contributor of specific KRAS/NRAS and EGFR driver mutations that are enriched at acquired resistance. Detectable SBS17b activity before treatment predicted shorter progression-free survival and the evolution of these specific mutations during subsequent cetuximab treatment. This result suggests that chemotherapy mutagenesis can accelerate resistance evolution. Mutational signatures may be a new class of cancer evolution predictor.
- Published
- 2021
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10. Computational Image Analysis of T-Cell Infiltrates in Resectable Gastric Cancer: Association with Survival and Molecular Subtypes.
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Challoner BR, von Loga K, Woolston A, Griffiths B, Sivamanoharan N, Semiannikova M, Newey A, Barber LJ, Mansfield D, Hewitt LC, Saito Y, Davarzani N, Starling N, Melcher A, Grabsch HI, and Gerlinger M
- Subjects
- Adult, Aged, Aged, 80 and over, CD8 Antigens genetics, CD8 Antigens immunology, Cell Lineage genetics, Cell Lineage immunology, DNA Mismatch Repair genetics, Disease-Free Survival, Female, Forkhead Transcription Factors genetics, Forkhead Transcription Factors immunology, Herpesvirus 4, Human genetics, Herpesvirus 4, Human pathogenicity, Humans, Leukocyte Common Antigens genetics, Leukocyte Common Antigens immunology, Lymphocytes, Tumor-Infiltrating pathology, Male, Middle Aged, Neoplasm Recurrence, Local diagnostic imaging, Neoplasm Recurrence, Local pathology, Neoplasm Recurrence, Local surgery, Stomach Neoplasms diagnostic imaging, Stomach Neoplasms pathology, Stomach Neoplasms surgery, T-Lymphocytes immunology, T-Lymphocytes ultrastructure, T-Lymphocytes, Regulatory pathology, Lymphocytes, Tumor-Infiltrating immunology, Neoplasm Recurrence, Local genetics, Stomach Neoplasms genetics, T-Lymphocytes, Regulatory immunology
- Abstract
Background: Gastric and gastro-esophageal junction cancers (GCs) frequently recur after resection, but markers to predict recurrence risk are missing. T-cell infiltrates have been validated as prognostic markers in other cancer types, but not in GC because of methodological limitations of past studies. We aimed to define and validate the prognostic role of major T-cell subtypes in GC by objective computational quantification., Methods: Surgically resected chemotherapy-naïve GCs were split into discovery (n = 327) and validation (n = 147) cohorts. CD8 (cytotoxic), CD45RO (memory), and FOXP3 (regulatory) T-cell densities were measured through multicolor immunofluorescence and computational image analysis. Cancer-specific survival (CSS) was assessed. All statistical tests were two-sided., Results: CD45RO-cell and FOXP3-cell densities statistically significantly predicted CSS in both cohorts. Stage, CD45RO-cell, and FOXP3-cell densities were independent predictors of CSS in multivariable analysis; mismatch repair (MMR) and Epstein-Barr virus (EBV) status were not statistically significant. Combining CD45RO-cell and FOXP3-cell densities into the Stomach Cancer Immune Score showed highly statistically significant (all P ≤ .002) CSS differences (0.9 years median CSS to not reached). T-cell infiltrates were highest in EBV-positive GCs and similar in MMR-deficient and MMR-proficient GCs., Conclusion: The validation of CD45RO-cell and FOXP3-cell densities as prognostic markers in GC may guide personalized follow-up or (neo)adjuvant treatment strategies. Only those 20% of GCs with the highest T-cell infiltrates showed particularly good CSS, suggesting that a small subgroup of GCs is highly immunogenic. The potential for T-cell densities to predict immunotherapy responses should be assessed. The association of high FOXP3-cell densities with longer CSS warrants studies into the biology of regulatory T cells in GC., (© The Author(s) 2020. Published by Oxford University Press.)
- Published
- 2021
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11. Circulating Tumour DNA Sequencing Identifies a Genetic Resistance-Gap in Colorectal Cancers with Acquired Resistance to EGFR-Antibodies and Chemotherapy.
- Author
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Knebel FH, Barber LJ, Newey A, Kleftogiannis D, Woolston A, Griffiths B, Fenwick K, Bettoni F, Ribeiro MFSA, da Fonseca L, Costa F, Capareli FC, Hoff PM, Sabbaga J, Camargo AA, and Gerlinger M
- Abstract
Epidermal growth factor receptor antibodies (EGFR-Abs) confer a survival benefit in patients with RAS wild-type metastatic colorectal cancer (mCRC), but resistance invariably occurs. Previous data showed that only a minority of cancer cells harboured known genetic resistance drivers when clinical resistance to single-agent EGFR-Abs had evolved, supporting the activity of non-genetic resistance mechanisms. Here, we used error-corrected ctDNA-sequencing (ctDNA-Seq) of 40 cancer genes to identify drivers of resistance and whether a genetic resistance-gap (a lack of detectable genetic resistance mechanisms in a large fraction of the cancer cell population) also occurs in RAS wild-type mCRCs treated with a combination of EGFR-Abs and chemotherapy. We detected one MAP2K1/MEK1 mutation and one ERBB2 amplification in 2/3 patients with primary resistance and KRAS, NRAS, MAP2K1/MEK1 mutations and ERBB2 aberrations in 6/7 patients with acquired resistance. In vitro testing identified MAP2K1/MEK1 P124S as a novel driver of EGFR-Ab resistance. Mutation subclonality analyses confirmed a genetic resistance-gap in mCRCs treated with EGFR-Abs and chemotherapy, with only 13.42% of cancer cells harboring identifiable resistance drivers. Our results support the utility of ctDNA-Seq to guide treatment allocation for patients with resistance and the importance of investigating further non-canonical EGFR-Ab resistance mechanisms, such as microenvironmentally-mediated resistance. The detection of MAP2K1 mutations could inform trials of MEK-inhibitors in these tumours.
- Published
- 2020
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12. Author Correction: Extreme intratumour heterogeneity and driver evolution in mismatch repair deficient gastro-oesophageal cancer.
- Author
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von Loga K, Woolston A, Punta M, Barber LJ, Griffiths B, Semiannikova M, Spain G, Challoner B, Fenwick K, Simon R, Marx A, Sauter G, Lise S, Matthews N, and Gerlinger M
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2020
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13. Extreme intratumour heterogeneity and driver evolution in mismatch repair deficient gastro-oesophageal cancer.
- Author
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von Loga K, Woolston A, Punta M, Barber LJ, Griffiths B, Semiannikova M, Spain G, Challoner B, Fenwick K, Simon R, Marx A, Sauter G, Lise S, Matthews N, and Gerlinger M
- Subjects
- Adenocarcinoma genetics, DNA-Binding Proteins genetics, Exome, Genes, Neoplasm genetics, Humans, Immune Evasion, Immunotherapy, Mismatch Repair Endonuclease PMS2 genetics, MutL Protein Homolog 1 genetics, MutS Homolog 2 Protein genetics, Mutation, Phenotype, Phylogeny, DNA Mismatch Repair, Esophageal Neoplasms genetics, Genetic Heterogeneity, Stomach Neoplasms genetics
- Abstract
Mismatch repair deficient (dMMR) gastro-oesophageal adenocarcinomas (GOAs) show better outcomes than their MMR-proficient counterparts and high immunotherapy sensitivity. The hypermutator-phenotype of dMMR tumours theoretically enables high evolvability but their evolution has not been investigated. Here we apply multi-region exome sequencing (MSeq) to four treatment-naive dMMR GOAs. This reveals extreme intratumour heterogeneity (ITH), exceeding ITH in other cancer types >20-fold, but also long phylogenetic trunks which may explain the exquisite immunotherapy sensitivity of dMMR tumours. Subclonal driver mutations are common and parallel evolution occurs in RAS, PIK3CA, SWI/SNF-complex genes and in immune evasion regulators. MSeq data and evolution analysis of single region-data from 64 MSI GOAs show that chromosome 8 gains are early genetic events and that the hypermutator-phenotype remains active during progression. MSeq may be necessary for biomarker development in these heterogeneous cancers. Comparison with other MSeq-analysed tumour types reveals mutation rates and their timing to determine phylogenetic tree morphologies.
- Published
- 2020
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14. Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment.
- Author
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Newey A, Griffiths B, Michaux J, Pak HS, Stevenson BJ, Woolston A, Semiannikova M, Spain G, Barber LJ, Matthews N, Rao S, Watkins D, Chau I, Coukos G, Racle J, Gfeller D, Starling N, Cunningham D, Bassani-Sternberg M, and Gerlinger M
- Subjects
- Antigens, Neoplasm genetics, Colorectal Neoplasms genetics, Female, Histocompatibility Antigens Class I genetics, Humans, Interferon-gamma pharmacology, MAP Kinase Kinase Kinases antagonists & inhibitors, Male, Middle Aged, Protein Kinase Inhibitors pharmacology, Proteomics, Antigens, Neoplasm immunology, Colorectal Neoplasms immunology, Histocompatibility Antigens Class I immunology, Organoids immunology, Peptides immunology
- Abstract
Background: Patient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment., Methods: Four microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS., Results: We identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment., Conclusions: Only 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.
- Published
- 2019
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15. Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer.
- Author
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Woolston A, Khan K, Spain G, Barber LJ, Griffiths B, Gonzalez-Exposito R, Hornsteiner L, Punta M, Patil Y, Newey A, Mansukhani S, Davies MN, Furness A, Sclafani F, Peckitt C, Jiménez M, Kouvelakis K, Ranftl R, Begum R, Rana I, Thomas J, Bryant A, Quezada S, Wotherspoon A, Khan N, Fotiadis N, Marafioti T, Powles T, Lise S, Calvo F, Guettler S, von Loga K, Rao S, Watkins D, Starling N, Chau I, Sadanandam A, Cunningham D, and Gerlinger M
- Subjects
- Antineoplastic Agents, Immunological pharmacology, Antineoplastic Agents, Immunological therapeutic use, Biomarkers, Tumor, Biopsy, Colorectal Neoplasms drug therapy, Colorectal Neoplasms mortality, Colorectal Neoplasms pathology, Computational Biology methods, DNA Mutational Analysis, ErbB Receptors antagonists & inhibitors, Gene Expression Profiling, Humans, Kaplan-Meier Estimate, Molecular Targeted Therapy, Mutation, Prognosis, Treatment Outcome, Colorectal Neoplasms etiology, Drug Resistance, Neoplasm genetics, Gene Expression Regulation, Neoplastic drug effects, Immunity, Transcriptome
- Abstract
Despite biomarker stratification, the anti-EGFR antibody cetuximab is only effective against a subgroup of colorectal cancers (CRCs). This genomic and transcriptomic analysis of the cetuximab resistance landscape in 35 RAS wild-type CRCs identified associations of NF1 and non-canonical RAS/RAF aberrations with primary resistance and validated transcriptomic CRC subtypes as non-genetic predictors of benefit. Sixty-four percent of biopsies with acquired resistance harbored no genetic resistance drivers. Most of these had switched from a cetuximab-sensitive transcriptomic subtype at baseline to a fibroblast- and growth factor-rich subtype at progression. Fibroblast-supernatant conferred cetuximab resistance in vitro, confirming a major role for non-genetic resistance through stromal remodeling. Cetuximab treatment increased cytotoxic immune infiltrates and PD-L1 and LAG3 immune checkpoint expression, potentially providing opportunities to treat cetuximab-resistant CRCs with immunotherapy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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16. Detecting and Tracking Circulating Tumour DNA Copy Number Profiles during First Line Chemotherapy in Oesophagogastric Adenocarcinoma.
- Author
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Davidson M, Barber LJ, Woolston A, Cafferkey C, Mansukhani S, Griffiths B, Moorcraft SY, Rana I, Begum R, Assiotis I, Matthews N, Rao S, Watkins D, Chau I, Cunningham D, Starling N, and Gerlinger M
- Abstract
DNA somatic copy number aberrations (SCNAs) are key drivers in oesophagogastric adenocarcinoma (OGA). Whether minimally invasive SCNA analysis of circulating tumour (ct)DNA can predict treatment outcomes and reveal how SCNAs evolve during chemotherapy is unknown. We investigated this by low-coverage whole genome sequencing (lcWGS) of ctDNA from 30 patients with advanced OGA prior to first-line chemotherapy and on progression. SCNA profiles were detectable pretreatment in 23/30 (76.7%) patients. The presence of liver metastases, primary tumour in situ, or of oesophageal or junctional tumour location predicted for a high ctDNA fraction. A low ctDNA concentration associated with significantly longer overall survival. Neither chromosomal instability metrics nor ploidy correlated with chemotherapy outcome. Chromosome 2q and 8p gains before treatment were associated with chemotherapy responses. lcWGS identified all amplifications found by prior targeted tumour tissue sequencing in cases with detectable ctDNA as well as finding additional changes. SCNA profiles changed during chemotherapy, indicating that cancer cell populations evolved during treatment; however, no recurrent SCNA changes were acquired at progression. Tracking the evolution of OGA cancer cell populations in ctDNA is feasible during chemotherapy. The observation of genetic evolution warrants investigation in larger series and with higher resolution techniques to reveal potential genetic predictors of response and drivers of chemotherapy resistance. The presence of liver metastasis is a potential biomarker for the selection of patients with high ctDNA content for such studies.
- Published
- 2019
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17. CEA expression heterogeneity and plasticity confer resistance to the CEA-targeting bispecific immunotherapy antibody cibisatamab (CEA-TCB) in patient-derived colorectal cancer organoids.
- Author
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Gonzalez-Exposito R, Semiannikova M, Griffiths B, Khan K, Barber LJ, Woolston A, Spain G, von Loga K, Challoner B, Patel R, Ranes M, Swain A, Thomas J, Bryant A, Saffery C, Fotiadis N, Guettler S, Mansfield D, Melcher A, Powles T, Rao S, Watkins D, Chau I, Matthews N, Wallberg F, Starling N, Cunningham D, and Gerlinger M
- Subjects
- Antibodies, Bispecific therapeutic use, Antineoplastic Agents, Immunological therapeutic use, CD8-Positive T-Lymphocytes, Coculture Techniques, Colorectal Neoplasms genetics, Colorectal Neoplasms pathology, Drug Screening Assays, Antitumor, GPI-Linked Proteins antagonists & inhibitors, GPI-Linked Proteins genetics, Gene Expression Regulation, Neoplastic, Genetic Heterogeneity, Humans, Tissue Culture Techniques, Antibodies, Bispecific pharmacology, Antineoplastic Agents, Immunological pharmacology, Carcinoembryonic Antigen genetics, Colorectal Neoplasms drug therapy, Drug Resistance, Neoplasm genetics
- Abstract
Background: The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity., Methods: We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells., Results: PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEA
hi (n = 3), CEAlo (n = 1) and CEAmixed PDOs (n = 4), that stably maintained populations of CEAhi and CEAlo cells, which has not previously been described in CRC cell lines. CEAhi PDOs were sensitive whereas CEAlo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEAlo cells maintain cancer cell growth. Culture of FACS-sorted CEAhi and CEAlo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/β-catenin pathway activity in CEAlo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/β-catenin pathway., Conclusions: Based on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/β-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic.- Published
- 2019
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18. Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing.
- Author
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Mansukhani S, Barber LJ, Kleftogiannis D, Moorcraft SY, Davidson M, Woolston A, Proszek PZ, Griffiths B, Fenwick K, Herman B, Matthews N, O'Leary B, Hulkki S, Gonzalez De Castro D, Patel A, Wotherspoon A, Okachi A, Rana I, Begum R, Davies MN, Powles T, von Loga K, Hubank M, Turner N, Watkins D, Chau I, Cunningham D, Lise S, Starling N, and Gerlinger M
- Subjects
- Biomarkers, Tumor genetics, Circulating Tumor DNA genetics, Colorectal Neoplasms blood, Colorectal Neoplasms genetics, Colorectal Neoplasms pathology, Genome-Wide Association Study, Humans, Neoplasm Metastasis, Sensitivity and Specificity, Biomarkers, Tumor blood, Circulating Tumor DNA blood, DNA Copy Number Variations genetics, DNA Mutational Analysis methods, High-Throughput Nucleotide Sequencing methods, Mutation
- Abstract
Background: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies., Methods: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction., Results: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS , parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing., Conclusions: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution., Clinicaltrialsgov Identifier: NCT02112357., (© 2018 American Association for Clinical Chemistry.)
- Published
- 2018
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19. Cancer Evolution and the Limits of Predictability in Precision Cancer Medicine.
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Lipinski KA, Barber LJ, Davies MN, Ashenden M, Sottoriva A, and Gerlinger M
- Abstract
The ability to predict the future behavior of an individual cancer is crucial for precision cancer medicine. The discovery of extensive intratumor heterogeneity and ongoing clonal adaptation in human tumors substantiated the notion of cancer as an evolutionary process. Random events are inherent in evolution and tumor spatial structures hinder the efficacy of selection, which is the only deterministic evolutionary force. This review outlines how the interaction of these stochastic and deterministic processes, which have been extensively studied in evolutionary biology, limits cancer predictability and develops evolutionary strategies to improve predictions. Understanding and advancing the cancer predictability horizon is crucial to improve precision medicine outcomes., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2016
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20. Dissecting cancer evolution at the macro-heterogeneity and micro-heterogeneity scale.
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Barber LJ, Davies MN, and Gerlinger M
- Subjects
- Clonal Evolution, High-Throughput Nucleotide Sequencing methods, Humans, Neoplasms pathology, Sequence Analysis, DNA methods, Single-Cell Analysis methods, Genetic Heterogeneity, Neoplasms genetics
- Abstract
Intratumour heterogeneity complicates biomarker discovery and treatment personalization, and pervasive cancer evolution is a key mechanism leading to therapy failure and patient death. Thus, understanding subclonal heterogeneity architectures and cancer evolution processes is critical for the development of effective therapeutic approaches which can control or thwart cancer evolutionary plasticity. Current insights into heterogeneity are mainly limited to the macroheterogeneity level, established by cancer subclones that have undergone significant clonal expansion. Novel single cell sequencing and blood-based subclonal tracking technologies are enabling detailed insights into microheterogeneity and the dynamics of clonal evolution. We assess how this starts to delineate the rules governing cancer evolution and novel angles for more effective therapeutic intervention., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2015
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21. The genomic landscape of oesophagogastric junctional adenocarcinoma.
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Chong IY, Cunningham D, Barber LJ, Campbell J, Chen L, Kozarewa I, Fenwick K, Assiotis I, Guettler S, Garcia-Murillas I, Awan S, Lambros M, Starling N, Wotherspoon A, Stamp G, Gonzalez-de-Castro D, Benson M, Chau I, Hulkki S, Nohadani M, Eltahir Z, Lemnrau A, Orr N, Rao S, Lord CJ, and Ashworth A
- Subjects
- Adaptor Proteins, Signal Transducing analysis, Adaptor Proteins, Signal Transducing genetics, Adenocarcinoma chemistry, Adenocarcinoma pathology, Adenosine Triphosphatases analysis, Adenosine Triphosphatases genetics, Adult, Aged, DNA Copy Number Variations genetics, DNA Mutational Analysis, DNA Repair Enzymes analysis, DNA Repair Enzymes genetics, DNA-Binding Proteins analysis, DNA-Binding Proteins genetics, Esophageal Neoplasms chemistry, Esophageal Neoplasms pathology, Exome genetics, Female, Genome, Human genetics, Humans, Immunohistochemistry, Loss of Heterozygosity genetics, Male, Microsatellite Instability, Middle Aged, Mismatch Repair Endonuclease PMS2, MutL Protein Homolog 1, MutL Proteins, Neoplasm Proteins analysis, Neoplasm Proteins genetics, Neoplasm Staging, Nuclear Proteins analysis, Nuclear Proteins genetics, Polymerase Chain Reaction methods, Prospective Studies, Stomach Neoplasms chemistry, Stomach Neoplasms pathology, Adenocarcinoma genetics, DNA, Neoplasm genetics, Esophageal Neoplasms genetics, Esophagogastric Junction pathology, Mutation genetics, Stomach Neoplasms genetics
- Abstract
The incidence of oesophagogastric junctional (OGJ) adenocarcinoma is rising rapidly in western countries, in contrast to the declining frequency of distal gastric carcinoma. Treatment options for adenocarcinomas involving the oesophagogastric junction are limited and the overall prognosis is extremely poor. To determine the genomic landscape of OGJ adenocarcinoma, exomes of eight tumours and matched germline DNA were subjected to massively parallel DNA sequencing. Microsatellite instability was observed in three tumours which coincided with an elevated number of somatic mutations. In total, 117 genes were identified that had predicted coding alterations in more than one tumour. Potentially actionable coding mutations were identified in 67 of these genes, including those in CR2, HGF , FGFR4, and ESRRB. Twenty-nine genes harbouring somatic coding mutations and copy number changes in the MSS OGJ dataset are also known to be altered with similar predicted functional consequence in other tumour types. Compared with the published mutational profile of gastric cancers, 49% (57/117) of recurrently mutated genes were unique to OGJ tumours. TP53, SYNE1, and ARID1A were amongst the most frequently mutated genes in a larger OGJ cohort. Our study provides an insight into the mutational landscape of OGJ adenocarcinomas and confirms that this is a highly mutated and heterogeneous disease. Furthermore, we have uncovered somatic mutations in therapeutically relevant genes which may represent candidate drug targets., (2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2013
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22. Efficacy of chemotherapy in BRCA1/2 mutation carrier ovarian cancer in the setting of PARP inhibitor resistance: a multi-institutional study.
- Author
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Ang JE, Gourley C, Powell CB, High H, Shapira-Frommer R, Castonguay V, De Greve J, Atkinson T, Yap TA, Sandhu S, Banerjee S, Chen LM, Friedlander ML, Kaufman B, Oza AM, Matulonis U, Barber LJ, Kozarewa I, Fenwick K, Assiotis I, Campbell J, Chen L, de Bono JS, Gore ME, Lord CJ, Ashworth A, and Kaye SB
- Subjects
- Adult, Aged, Drug Resistance, Neoplasm, Enzyme Inhibitors pharmacology, Enzyme Inhibitors therapeutic use, Female, Humans, Middle Aged, Neoplasm Grading, Neoplasm Staging, Ovarian Neoplasms mortality, Ovarian Neoplasms pathology, Poly(ADP-ribose) Polymerase Inhibitors, Prognosis, Retrospective Studies, Risk Factors, Treatment Outcome, Antineoplastic Combined Chemotherapy Protocols therapeutic use, BRCA1 Protein genetics, BRCA2 Protein genetics, Germ-Line Mutation, Heterozygote, Ovarian Neoplasms drug therapy, Ovarian Neoplasms genetics
- Abstract
Purpose: Preclinical data suggest that exposure to PARP inhibitors (PARPi) may compromise benefit to subsequent chemotherapy, particularly platinum-based regimens, in patients with BRCA1/2 mutation carrier ovarian cancer (PBMCOC), possibly through the acquisition of secondary BRCA1/2 mutations. The efficacy of chemotherapy in the PARPi-resistant setting was therefore investigated., Experimental Design: We conducted a retrospective review of PBMCOC who received chemotherapy following disease progression on olaparib, administered at ≥200 mg twice daily for one month or more. Tumor samples were obtained in the post-olaparib setting where feasible and analyzed by massively parallel sequencing., Results: Data were collected from 89 patients who received a median of 3 (range 1-11) lines of pre-olaparib chemotherapy. The overall objective response rate (ORR) to post-olaparib chemotherapy was 36% (24 of 67 patients) by Response Evaluation Criteria in Solid Tumors (RECIST) and 45% (35 of 78) by RECIST and/or Gynecologic Cancer InterGroup (GCIG) CA125 criteria with median progression-free survival (PFS) and overall survival (OS) of 17 weeks [95% confidence interval (CI), 13-21] and 34 weeks (95% CI, 26-42), respectively. For patients receiving platinum-based chemotherapy, ORRs were 40% (19 of 48) and 49% (26/53), respectively, with a median PFS of 22 weeks (95% CI, 15-29) and OS of 45 weeks (95% CI, 15-75). An increased platinum-to-platinum interval was associated with an increased OS and likelihood of response following post-olaparib platinum. No evidence of secondary BRCA1/2 mutation was detected in tumor samples of six PARPi-resistant patients [estimated frequency of such mutations adjusted for sample size: 0.125 (95%-CI: 0-0.375)]., Conclusions: Heavily pretreated PBMCOC who are PARPi-resistant retain the potential to respond to subsequent chemotherapy, including platinum-based agents. These data support the further development of PARPi in PBMCOC.
- Published
- 2013
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23. Poly (ADP-ribose) polymerase (PARP) inhibitors for the treatment of advanced germline BRCA2 mutant prostate cancer.
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Sandhu SK, Omlin A, Hylands L, Miranda S, Barber LJ, Riisnaes R, Reid AH, Attard G, Chen L, Kozarewa I, Gevensleben H, Campbell J, Fenwick K, Assiotis I, Olmos D, Yap TA, Fong P, Tunariu N, Koh D, Molife LR, Kaye S, Lord CJ, Ashworth A, and de Bono J
- Subjects
- Aged, Androstenes, Androstenols therapeutic use, Bone Neoplasms secondary, Humans, Male, Middle Aged, Mutation, Neoplasm Grading, Phthalazines therapeutic use, Piperazines therapeutic use, Antineoplastic Agents therapeutic use, BRCA2 Protein genetics, Poly(ADP-ribose) Polymerase Inhibitors, Prostatic Neoplasms drug therapy, Prostatic Neoplasms genetics
- Published
- 2013
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24. Secondary mutations in BRCA2 associated with clinical resistance to a PARP inhibitor.
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Barber LJ, Sandhu S, Chen L, Campbell J, Kozarewa I, Fenwick K, Assiotis I, Rodrigues DN, Reis Filho JS, Moreno V, Mateo J, Molife LR, De Bono J, Kaye S, Lord CJ, and Ashworth A
- Subjects
- Adenocarcinoma drug therapy, Adenocarcinoma genetics, Adenocarcinoma secondary, Breast Neoplasms, Male drug therapy, Breast Neoplasms, Male genetics, Breast Neoplasms, Male pathology, Combined Modality Therapy, DNA Mutational Analysis, Female, Gene Expression Regulation, Neoplastic drug effects, Humans, Male, Middle Aged, Ovarian Neoplasms drug therapy, Ovarian Neoplasms genetics, Ovarian Neoplasms pathology, Sequence Analysis, DNA, Antineoplastic Agents pharmacology, BRCA2 Protein genetics, Drug Resistance, Neoplasm, Mutation, Phthalazines pharmacology, Piperazines pharmacology, Poly(ADP-ribose) Polymerase Inhibitors
- Abstract
PARP inhibitors (PARPi) for the treatment of BRCA1 or BRCA2 deficient tumours are currently the focus of seminal clinical trials exploiting the concept of synthetic lethality. Although clinical resistance to PARPi has been described, the mechanism underlying this has not been elucidated. Here, we investigate tumour material from patients who had developed resistance to the PARPi olaparib, subsequent to showing an initial clinical response. Massively parallel DNA sequencing of treatment-naive and post-olaparib treatment biopsies identified tumour-specific BRCA2 secondary mutations in olaparib-resistant metastases. These secondary mutations restored full-length BRCA2 protein, and most likely cause olaparib resistance by re-establishing BRCA2 function in the tumour cells., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2013
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25. Comprehensive genomic analysis of a BRCA2 deficient human pancreatic cancer.
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Barber LJ, Rosa Rosa JM, Kozarewa I, Fenwick K, Assiotis I, Mitsopoulos C, Sims D, Hakas J, Zvelebil M, Lord CJ, and Ashworth A
- Subjects
- Adenocarcinoma genetics, Adenocarcinoma pathology, BRCA2 Protein deficiency, Cell Line, Tumor, Chromosome Mapping, Comparative Genomic Hybridization, Humans, Pancreatic Neoplasms genetics, Pancreatic Neoplasms pathology, BRCA2 Protein genetics, Genome, Human genetics, Genomics methods, Sequence Analysis, DNA methods
- Abstract
Capan-1 is a well-characterised BRCA2-deficient human cell line isolated from a liver metastasis of a pancreatic adenocarcinoma. Here we report a genome-wide assessment of structural variations and high-depth exome characterization of single nucleotide variants and small insertion/deletions in Capan-1. To identify potential somatic and tumour-associated variations in the absence of a matched-normal cell line, we devised a novel method based on the analysis of HapMap samples. We demonstrate that Capan-1 has one of the most rearranged genomes sequenced to date. Furthermore, small insertions and deletions are detected more frequently in the context of short sequence repeats than in other genomes. We also identify a number of novel mutations that may represent genetic changes that have contributed to tumour progression. These data provide insight into the genomic effects of loss of BRCA2 function.
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- 2011
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26. The MRT-1 nuclease is required for DNA crosslink repair and telomerase activity in vivo in Caenorhabditis elegans.
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Meier B, Barber LJ, Liu Y, Shtessel L, Boulton SJ, Gartner A, and Ahmed S
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- Amino Acid Sequence, Animals, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, DNA Damage physiology, DNA, Single-Stranded metabolism, Deoxyribonucleases chemistry, Deoxyribonucleases genetics, Deoxyribonucleases metabolism, Enzyme Activation genetics, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary genetics, Sequence Homology, Amino Acid, Telomere metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins physiology, DNA Repair genetics, Deoxyribonucleases physiology, Telomerase metabolism
- Abstract
The telomerase reverse transcriptase adds de novo DNA repeats to chromosome termini. Here we define Caenorhabditis elegans MRT-1 as a novel factor required for telomerase-mediated telomere replication and the DNA-damage response. MRT-1 is composed of an N-terminal domain homologous to the second OB-fold of POT1 telomere-binding proteins and a C-terminal SNM1 family nuclease domain, which confer single-strand DNA-binding and processive 3'-to-5' exonuclease activity, respectively. Furthermore, telomerase activity in vivo depends on a functional MRT-1 OB-fold. We show that MRT-1 acts in the same telomere replication pathway as telomerase and the 9-1-1 DNA-damage response complex. MRT-1 is dispensable for DNA double-strand break repair, but functions with the 9-1-1 complex to promote DNA interstrand cross-link (ICL) repair. Our data reveal MRT-1 as a dual-domain protein required for telomerase function and ICL repair, which raises the possibility that telomeres and ICL lesions may share a common feature that plays a critical role in de novo telomere repeat addition.
- Published
- 2009
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27. Methotrexate induces oxidative DNA damage and is selectively lethal to tumour cells with defects in the DNA mismatch repair gene MSH2.
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Martin SA, McCarthy A, Barber LJ, Burgess DJ, Parry S, Lord CJ, and Ashworth A
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- Antioxidants pharmacology, Base Sequence, Cell Line, Tumor, Folic Acid pharmacology, Gene Deletion, Gene Expression Regulation, Neoplastic drug effects, Humans, Molecular Sequence Data, Oxidation-Reduction, RNA, Antisense genetics, Antimetabolites, Antineoplastic pharmacology, DNA Damage drug effects, DNA Mismatch Repair drug effects, Methotrexate pharmacology, MutS Homolog 2 Protein genetics, Neoplasms drug therapy
- Abstract
Mutations in the MSH2 gene predispose to a number of tumourigenic conditions, including hereditary non-polyposis colon cancer (HNPCC). MSH2 encodes a protein in the mismatch repair (MMR) pathway which is involved in the removal of mispairs originating during replication or from damaged DNA. To identify new therapeutic strategies for the treatment of cancer arising from MMR deficiency, we screened a small molecule library encompassing previously utilized drugs and drug-like molecules to identify agents selectively lethal to cells lacking functional MSH2. This approach identified the drug methotrexate as being highly selective for cells with MSH2 deficiency. Methotrexate treatment caused the accumulation of potentially lethal 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative DNA lesions in both MSH2 deficient and proficient cells. In MSH2 proficient cells, these lesions were rapidly cleared, while in MSH2 deficient cells 8-OHdG lesions persisted, potentially explaining the selectivity of methotrexate. Short interfering (si)RNA mediated silencing of the target of methotrexate, dihydrofolate reductase (DHFR), was also selective for MSH2 deficiency and also caused an accumulation of 8-OHdG. This suggested that the ability of methotrexate to modulate folate synthesis via inhibition of DHFR, may explain MSH2 selectivity. Consistent with this hypothesis, addition of folic acid to culture media substantially rescued the lethal phenotype caused by methotrexate. While methotrexate has been used for many years as a cancer therapy, our observations suggest that this drug may have particular utility for the treatment of a subset of patients with tumours characterized by MSH2 mutations.
- Published
- 2009
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28. C. elegans: a model of Fanconi anemia and ICL repair.
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Youds JL, Barber LJ, and Boulton SJ
- Subjects
- Animals, Fanconi Anemia Complementation Group Proteins metabolism, Genomic Instability, Caenorhabditis elegans genetics, Cross-Linking Reagents pharmacology, DNA Repair, Fanconi Anemia genetics, Models, Animal
- Abstract
Fanconi anemia (FA) is a severe recessive disorder with a wide range of clinical manifestations [M. Levitus, H. Joenje, J.P. de Winter, The Fanconi anemia pathway of genomic maintenance, Cell Oncol. 28 (2006) 3-29]. In humans, 13 complementation groups have been identified to underlie FA: A, B, C, D1, D2, E, F, G, I, J, L, M, and N [W. Wang, Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins, Nat. Rev. Genet. 8 (2007) 735-748]. Cells defective for any of these genes display chromosomal aberrations and sensitivity to DNA interstrand cross-links (ICLs). It has therefore been suggested that the 13 FA proteins constitute a pathway for the repair of ICLs, and that a deficiency in this repair process causes genomic instability leading to the different clinical phenotypes. However, the exact nature of this repair pathway, or even whether all 13 FA proteins are involved at some stage of a linear repair process, remains to be defined. Undoubtedly, the recent identification and characterisation of FA homologues in model organisms, such as Caenorhabditis elegans, will help facilitate an understanding of the function of the FA proteins by providing new analytical tools. To date, sequence homologues of five FA genes have been identified in C. elegans. Three of these homologues have been confirmed: brc-2 (FANCD1/BRCA2), fcd-2 (FANCD2), and dog-1 (FANCJ/BRIP1); and two remain to be characterised: W02D3.10 (FANCI) and drh-3 (FANCM). Here we review how the nematode can be used to study FA-associated DNA repair, focusing on what is known about the ICL repair genes in C. elegans and which important questions remain for the field.
- Published
- 2009
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29. FANCJ is a structure-specific DNA helicase associated with the maintenance of genomic G/C tracts.
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London TB, Barber LJ, Mosedale G, Kelly GP, Balasubramanian S, Hickson ID, Boulton SJ, and Hiom K
- Subjects
- Binding, Competitive, Cell Line, Cell Line, Tumor, Cross-Linking Reagents pharmacology, DNA Replication, Gene Deletion, Genetic Predisposition to Disease, Genome, Humans, Nucleic Acid Conformation, Nucleic Acid Hybridization, Basic-Leucine Zipper Transcription Factors metabolism, DNA Helicases metabolism, Fanconi Anemia Complementation Group Proteins metabolism, G-Quadruplexes, RecQ Helicases metabolism
- Abstract
Fanconi anemia (FA) is a heritable human cancer-susceptibility disorder, delineating a genetically heterogenous pathway for the repair of replication-blocking lesions such as interstrand DNA cross-links. Here we demonstrate that one component of this pathway, FANCJ, is a structure-specific DNA helicase that dissociates guanine quadruplex DNA (G4 DNA) in vitro. Moreover, in contrast with previously identified G4 DNA helicases, such as the Bloom's helicase (BLM), FANCJ unwinds G4 substrates with 5'-3' polarity. In the FA-J human patient cell line EUFA0030 the loss of FANCJ G4 unwinding function correlates with the accumulation of large genomic deletions in the vicinity of sequences, which match the G4 DNA signature. Together these findings support a role for FANCJ in the maintenance of potentially unstable genomic G/C tracts during replication.
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- 2008
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30. RTEL1 maintains genomic stability by suppressing homologous recombination.
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Barber LJ, Youds JL, Ward JD, McIlwraith MJ, O'Neil NJ, Petalcorin MI, Martin JS, Collis SJ, Cantor SB, Auclair M, Tissenbaum H, West SC, Rose AM, and Boulton SJ
- Subjects
- Animals, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, Caenorhabditis elegans Proteins genetics, DNA metabolism, DNA Helicases genetics, DNA Repair, Humans, Mutation, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism, Caenorhabditis elegans enzymology, Caenorhabditis elegans Proteins metabolism, DNA Helicases metabolism, Genomic Instability, Recombination, Genetic
- Abstract
Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.
- Published
- 2008
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31. DOG-1 is the Caenorhabditis elegans BRIP1/FANCJ homologue and functions in interstrand cross-link repair.
- Author
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Youds JL, Barber LJ, Ward JD, Collis SJ, O'Neil NJ, Boulton SJ, and Rose AM
- Subjects
- Amino Acid Sequence, Animals, Basic-Leucine Zipper Transcription Factors genetics, Basic-Leucine Zipper Transcription Factors metabolism, Caenorhabditis elegans Proteins chemistry, Caenorhabditis elegans Proteins genetics, Cross-Linking Reagents pharmacology, DNA Helicases chemistry, DNA Helicases genetics, DNA Repair drug effects, DNA Repair genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Fanconi Anemia Complementation Group Proteins genetics, Fanconi Anemia Complementation Group Proteins metabolism, Immunohistochemistry, Molecular Sequence Data, Mutation, RNA Helicases genetics, RNA Helicases metabolism, Sequence Homology, Amino Acid, Basic-Leucine Zipper Transcription Factors physiology, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins physiology, DNA Helicases physiology, DNA Repair physiology, DNA-Binding Proteins physiology, Fanconi Anemia Complementation Group Proteins physiology, RNA Helicases physiology
- Abstract
Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by defective DNA interstrand cross-link (ICL) repair. Here, we show that DOG-1 is the Caenorhabditis elegans homologue of FANCJ, a helicase mutated in FA-J patients. DOG-1 performs a conserved role in ICL repair, as dog-1 mutants are hypersensitive to ICL-inducing agents, but not to UVC irradiation or X rays. Genetic analysis indicated that dog-1 is epistatic with fcd-2 (C. elegans FANCD2) but is nonepistatic with brc-1 (C. elegans BRCA1), thus establishing the existence of two distinct pathways of ICL repair in worms. Furthermore, DOG-1 is dispensable for FCD-2 and RAD-51 focus formation, suggesting that DOG-1 operates downstream of FCD-2 and RAD-51 in ICL repair. DOG-1 was previously implicated in poly(G)/poly(C) (G/C) tract maintenance during DNA replication. G/C tracts remain stable in the absence of ATL-1, CLK-2 (FA pathway activators), FCD-2, BRC-2, and MLH-1 (associated FA components), implying that DOG-1 is the sole FA component required for G/C tract maintenance in a wild-type background. However, FCD-2 is required to promote deletion-free repair at G/C tracts in dog-1 mutants, consistent with a role for FA factors at the replication fork. The functional conservation between DOG-1 and FANCJ suggests a possible role for FANCJ in G/C tract maintenance in human cells.
- Published
- 2008
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32. Human SNM1A suppresses the DNA repair defects of yeast pso2 mutants.
- Author
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Hazrati A, Ramis-Castelltort M, Sarkar S, Barber LJ, Schofield CJ, Hartley JA, and McHugh PJ
- Subjects
- Cell Line, Cross-Linking Reagents toxicity, DNA-Binding Proteins metabolism, Electrophoresis, Gel, Pulsed-Field, Endodeoxyribonucleases, Exodeoxyribonucleases, Gene Components, Humans, Mutation genetics, Nuclear Proteins metabolism, Phosphodiesterase I genetics, Phosphodiesterase I metabolism, Protein Structure, Tertiary, Saccharomyces cerevisiae Proteins metabolism, Yeasts, DNA Repair, DNA Repair Enzymes genetics, DNA-Binding Proteins genetics, Nuclear Proteins genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
Pso2/Snm1 plays a key role in the repair of DNA interstrand cross-links in yeast. Human cells possess three orthologues of Pso2; SNM1A, SNM1B/Apollo and SNM1C/Artemis. Studies using mammalian cells disrupted or depleted for these genes have yielded equivocal evidence that any of these is a true functional homologues of the yeast gene. Here we show that ectopic expression of only one of the three human orthologues, hSNM1A, effectively suppresses the sensitivity of yeast pso2 (snm1) disruptants to cross-linking agents. Two other phenotypes of the pso2 mutants are also partially rescued by ectopic expression of hSNM1A, namely the double-strand repair break defect observed during cross-link processing in pso2 cells, as well as the spontaneous intrachromatid recombination defect of pso2 msh2 double mutants. Finally, we show that recombinant hSNM1A is a 5'-exonuclease, as also recently reported for the yeast Pso2 protein. Together our data suggest that hSnm1A is a functional homologue of yeast Pso2/Snm1.
- Published
- 2008
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33. Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.
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Murakawa Y, Sonoda E, Barber LJ, Zeng W, Yokomori K, Kimura H, Niimi A, Lehmann A, Zhao GY, Hochegger H, Boulton SJ, and Takeda S
- Subjects
- Animals, Ataxia Telangiectasia Mutated Proteins, BRCA1 Protein metabolism, BRCA2 Protein metabolism, Cell Cycle Proteins metabolism, DNA-Binding Proteins metabolism, Fibroblasts drug effects, Fibroblasts enzymology, Fibroblasts physiology, Genes, BRCA1, HeLa Cells, Humans, Mice, Proteasome Endopeptidase Complex metabolism, Protein Serine-Threonine Kinases metabolism, Rad51 Recombinase metabolism, Tumor Suppressor Proteins metabolism, Antineoplastic Agents pharmacology, Cysteine Proteinase Inhibitors pharmacology, DNA Breaks, Double-Stranded, DNA Repair drug effects, Leupeptins pharmacology, Proteasome Inhibitors, Recombination, Genetic drug effects
- Abstract
Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.
- Published
- 2007
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34. Replication blocking lesions present a unique substrate for homologous recombination.
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Ward JD, Barber LJ, Petalcorin MI, Yanowitz J, and Boulton SJ
- Subjects
- Animals, Caenorhabditis elegans cytology, Caenorhabditis elegans radiation effects, Caenorhabditis elegans Proteins metabolism, Crossing Over, Genetic radiation effects, DNA Breaks, Double-Stranded radiation effects, DNA Repair radiation effects, Gene Deletion, Meiosis radiation effects, Mitosis radiation effects, Poly C, Poly G, Protein Binding radiation effects, Rad51 Recombinase metabolism, Substrate Specificity radiation effects, Suppression, Genetic radiation effects, Ultraviolet Rays, Caenorhabditis elegans genetics, DNA Damage, DNA Replication radiation effects, Recombination, Genetic radiation effects
- Abstract
Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.
- Published
- 2007
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35. HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability.
- Author
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Collis SJ, Barber LJ, Clark AJ, Martin JS, Ward JD, and Boulton SJ
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Cell Cycle genetics, Cell Cycle physiology, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Line, Tumor, Checkpoint Kinase 1, DNA Damage, DNA Repair, DNA-Binding Proteins, Exodeoxyribonucleases genetics, Exodeoxyribonucleases metabolism, Fanconi Anemia Complementation Group D2 Protein genetics, Fanconi Anemia Complementation Group D2 Protein metabolism, HeLa Cells, Humans, Immunoprecipitation, Models, Biological, Phosphoproteins genetics, Phosphoproteins metabolism, Phosphorylation, Protein Kinases genetics, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases, RNA Interference, RNA, Small Interfering genetics, S Phase genetics, Signal Transduction genetics, Signal Transduction physiology, cdc25 Phosphatases genetics, cdc25 Phosphatases metabolism, Protein Kinases metabolism, Protein Serine-Threonine Kinases physiology, S Phase physiology
- Abstract
Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.
- Published
- 2007
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36. A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination.
- Author
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Zhao GY, Sonoda E, Barber LJ, Oka H, Murakawa Y, Yamada K, Ikura T, Wang X, Kobayashi M, Yamamoto K, Boulton SJ, and Takeda S
- Subjects
- Animals, BRCA1 Protein metabolism, Cell Nucleus drug effects, Cell Nucleus radiation effects, Chickens, Chromosomes drug effects, Chromosomes radiation effects, DNA metabolism, DNA Breaks, Double-Stranded drug effects, DNA Breaks, Double-Stranded radiation effects, DNA Repair drug effects, DNA Repair radiation effects, Enzyme Activation drug effects, Enzyme Activation radiation effects, Exons genetics, Gene Targeting, HeLa Cells, Histones metabolism, Humans, Infrared Rays, Models, Genetic, Mutagens toxicity, Rad51 Recombinase metabolism, Replication Protein A metabolism, Ubiquitin metabolism, Ubiquitin-Conjugating Enzymes deficiency, Ultraviolet Rays, Recombination, Genetic drug effects, Recombination, Genetic radiation effects, Ubiquitin-Conjugating Enzymes metabolism
- Abstract
The ubiquitin (Ub)-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent postreplication repair (PRR) in budding yeast, but its function in vertebrates is not known. We show here that disruption or siRNA depletion of UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13-deficient cells are also compromised for DNA double-strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3 Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13-deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing.
- Published
- 2007
- Full Text
- View/download PDF
37. BRCA1 ubiquitylation of CtIP: Just the tIP of the iceberg?
- Author
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Barber LJ and Boulton SJ
- Subjects
- Animals, Cell Cycle, Ubiquitin-Protein Ligases metabolism, BRCA1 Protein metabolism, Carrier Proteins metabolism, DNA Repair, Nuclear Proteins metabolism, Ubiquitin metabolism
- Abstract
Ubiquitylation is an important regulatory mechanism of many cellular processes. The breast and ovarian cancer-specific tumour suppressor BRCA1 is well acknowledged to be a RING/E3 ubiquitin ligase, however, identification of its physiological substrates has proved elusive. Recently published data have shown that the BRCA1-interacting protein CtIP is in fact ubiquitylated by BRCA1, and opens new avenues for the isolation of other substrate proteins.
- Published
- 2006
- Full Text
- View/download PDF
38. C. elegans FANCD2 responds to replication stress and functions in interstrand cross-link repair.
- Author
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Collis SJ, Barber LJ, Ward JD, Martin JS, and Boulton SJ
- Subjects
- Amino Acid Sequence, Animals, Caenorhabditis elegans drug effects, Caenorhabditis elegans Proteins genetics, Cross-Linking Reagents pharmacology, DNA Repair drug effects, DNA Repair genetics, Fanconi Anemia Complementation Group D2 Protein genetics, Fanconi Anemia Complementation Group D2 Protein metabolism, Models, Genetic, Molecular Sequence Data, Mutation, Prophase physiology, S Phase physiology, Sequence Alignment, Ubiquitin metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans Proteins physiology, DNA Repair physiology, DNA Replication physiology, Fanconi Anemia Complementation Group D2 Protein physiology
- Abstract
One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL. In its mono-ubiquitylated form FANCD2 is recruited to repair foci where it is believed to somehow coordinate ICL repair and restart of impeded replication forks. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2 promotes ICL repair remains unclear. Here we report on a functional homologue of FANCD2 in C. elegans (FCD-2). Although fcd-2 mutants are homozygous viable, they are exquisitely sensitive to ICL-inducing agents, but insensitive to ionizing radiation (IR). fcd-2 is dispensable for meiotic recombination and activation of the S-phase checkpoint, indicating that ICL sensitivity is likely due to a repair rather than a signalling defect. Indeed, we show that FCD-2 is mono-ubiquitylated in response to ICL damage and is recruited to nuclear repair foci. Consistent with the sensitivity of fcd-2 mutants, FCD-2 focus formation is induced in response to ICL damage and replication stress, but not following IR, suggesting that FCD-2 responds to lesions that block DNA replication and not DNA double strand breaks per se. The realization that the FA pathway is conserved in a genetically tractable model system will permit the comprehensive analysis of the interplay between the FA, homologous recombination (HR), translesion synthesis (TLS) and nucleotide excision repair (NER) pathways, critical to the understanding of ICL repair.
- Published
- 2006
- Full Text
- View/download PDF
39. DNA interstrand cross-link repair in the Saccharomyces cerevisiae cell cycle: overlapping roles for PSO2 (SNM1) with MutS factors and EXO1 during S phase.
- Author
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Barber LJ, Ward TA, Hartley JA, and McHugh PJ
- Subjects
- Adenosine Triphosphatases genetics, Adenosine Triphosphatases physiology, DNA Damage, DNA Helicases genetics, DNA Helicases physiology, DNA Repair genetics, DNA-Binding Proteins genetics, Endodeoxyribonucleases, Exodeoxyribonucleases genetics, Mechlorethamine pharmacology, MutS Homolog 2 Protein, Mutation genetics, Nuclear Proteins genetics, Recombination, Genetic physiology, S Phase genetics, S Phase physiology, Saccharomyces cerevisiae drug effects, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins genetics, DNA Repair physiology, DNA-Binding Proteins physiology, Exodeoxyribonucleases physiology, Nuclear Proteins physiology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins physiology
- Abstract
Pso2/Snm1 is a member of the beta-CASP metallo-beta-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5'-3' exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G2, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.
- Published
- 2005
- Full Text
- View/download PDF
40. Schizosaccharomyces pombe checkpoint response to DNA interstrand cross-links.
- Author
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Lambert S, Mason SJ, Barber LJ, Hartley JA, Pearce JA, Carr AM, and McHugh PJ
- Subjects
- Camptothecin pharmacology, Cell Survival, Checkpoint Kinase 2, Cisplatin pharmacology, DNA drug effects, DNA radiation effects, DNA Damage, DNA Repair, Dose-Response Relationship, Drug, Dose-Response Relationship, Radiation, Enzyme Inhibitors pharmacology, G2 Phase, Hydroxyurea pharmacology, Mitomycin pharmacology, Nucleic Acid Synthesis Inhibitors pharmacology, Phosphorylation, Protein Kinases metabolism, Radiation-Sensitizing Agents pharmacology, Recombination, Genetic, S Phase, Schizosaccharomyces pombe Proteins, Temperature, Time Factors, Cross-Linking Reagents pharmacology, Protein Serine-Threonine Kinases, Schizosaccharomyces genetics, Schizosaccharomyces metabolism
- Abstract
Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the "checkpoint Rad" family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G(2) arrest following ICL induction. In cells able to bypass the G(2) block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G(2) arrest-deficient mutants. This likely reflects the fraction of cells escaping the G(2) damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.
- Published
- 2003
- Full Text
- View/download PDF
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