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Xie J, Mellado-Lagarde MM, Blankenship K, Ganguly D, Twarog NR, Bianski B, Kieffer M, Atkinson S, Sheppard H, Gartrell J, Cler S, Federico SM, Stewart EA, Tinkle CL, Shelat AA. The Combination of PARP and Topoisomerase 1 Inhibitors Improves Radiation Therapy for Ewing Sarcoma. Cancer Sci 2025. [PMID: 40069935 DOI: 10.1111/cas.70042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2024] [Revised: 02/17/2025] [Accepted: 02/25/2025] [Indexed: 04/02/2025] Open
Abstract
Although primary tumor control rates after surgery and/or radiation therapy (RT) are generally high in patients with Ewing sarcoma (EWS), those with unresectable tumors have failure rates approaching 30% and experience poorer outcomes. Additionally, although metastatic site irradiation is associated with improved survival, dose, and volume effects influence the long-term toxicity risk. Consequently, it is important to identify novel systemic agents to enhance the therapeutic ratio of RT. Given the reported DNA damage response deficits in EWS, we hypothesized that PARP inhibitors (PARPis) would preferentially potentiate radiation relative to standard-of-care (SOC) chemotherapeutics. We investigated primary and recurrent SOC drugs and PARPis with varied trapping potential in combination with radiation in EWS cell lines. At physiologically relevant concentrations, the strong PARP trapper talazoparib (TAL) potentiated radiation to a greater extent than did SOC or other PARPis, although the magnitude of the effect was modest. The radiosensitizing effect of TAL was mediated through the induction of DNA double-strand breaks, rather than through the catalytic inhibition of PARP1. Drug + RT combinations were further tested in vivo by using orthotopic xenograft models of EWS treated with image-guided fractionated radiation. The addition of RT to the combination of TAL plus irinotecan (IRN), a recently evaluated clinical regimen for relapsed pediatric solid tumors, significantly prolonged survival and reduced tumor burden in all EWS-treated mice. This triplet therapy (TAL + IRN + RT) was feasible and yielded responses in several patients with EWS and may represent a useful salvage strategy in recurrent or progressive disease.
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Affiliation(s)
- Jia Xie
- Department of Radiation Oncology, St. Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA
- Department of Chemical Biology and Therapeutics, SJCRH, Memphis, Tennessee, USA
| | | | | | - Debolina Ganguly
- Department of Chemical Biology and Therapeutics, SJCRH, Memphis, Tennessee, USA
| | - Nathaniel R Twarog
- Department of Chemical Biology and Therapeutics, SJCRH, Memphis, Tennessee, USA
| | - Brandon Bianski
- Department of Radiation Oncology, St. Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA
| | | | - Stefan Atkinson
- Department of Developmental Neurobiology, SJCRH, Memphis, Tennessee, USA
| | | | | | - Samuel Cler
- Department of Oncology, SJCRH, Memphis, Tennessee, USA
- Department of Developmental Neurobiology, SJCRH, Memphis, Tennessee, USA
| | | | - Elizabeth A Stewart
- Department of Oncology, SJCRH, Memphis, Tennessee, USA
- Department of Developmental Neurobiology, SJCRH, Memphis, Tennessee, USA
| | - Christopher L Tinkle
- Department of Radiation Oncology, St. Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA
| | - Anang A Shelat
- Department of Chemical Biology and Therapeutics, SJCRH, Memphis, Tennessee, USA
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2
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Her J, Zheng H, Bunting SF. RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication. J Clin Invest 2024; 134:e167419. [PMID: 38530355 PMCID: PMC11093604 DOI: 10.1172/jci167419] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Accepted: 03/20/2024] [Indexed: 03/27/2024] Open
Abstract
The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.
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Affiliation(s)
- Joonyoung Her
- Department of Molecular Biology and Biochemistry and
| | - Haiyan Zheng
- Biological Mass Spectrometry Facility, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA
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3
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Kinzig CG, Zakusilo G, Takai KK, Myler LR, de Lange T. ATR blocks telomerase from converting DNA breaks into telomeres. Science 2024; 383:763-770. [PMID: 38359122 PMCID: PMC11267623 DOI: 10.1126/science.adg3224] [Citation(s) in RCA: 10] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2022] [Accepted: 12/13/2023] [Indexed: 02/17/2024]
Abstract
Telomerase, the enzyme that maintains telomeres at natural chromosome ends, should be repressed at double-strand breaks (DSBs), where neotelomere formation can cause terminal truncations. We developed an assay to detect neotelomere formation at Cas9- or I-SceI-induced DSBs in human cells. Telomerase added telomeric repeats to DSBs, leading to interstitial telomeric repeat insertions or the formation of functional neotelomeres accompanied by terminal deletions. The threat that telomerase poses to genome integrity was minimized by ataxia telangiectasia and Rad3-related (ATR) kinase signaling, which inhibited telomerase at resected DSBs. In addition to acting at resected DSBs, telomerase used the extruded strand in the Cas9 enzyme-product complex as a primer for neotelomere formation. We propose that although neotelomere formation is detrimental in normal human cells, it may allow cancer cells to escape from breakage-fusion-bridge cycles.
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Affiliation(s)
- Charles G. Kinzig
- Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA
- Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD/PhD Program, New York, NY 10065, USA
| | - George Zakusilo
- Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA
- Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD/PhD Program, New York, NY 10065, USA
| | - Kaori K. Takai
- Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA
| | - Logan R. Myler
- Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA
| | - Titia de Lange
- Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA
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4
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Lu M, Billerbeck S. Improving homology-directed repair by small molecule agents for genetic engineering in unconventional yeast?-Learning from the engineering of mammalian systems. Microb Biotechnol 2024; 17:e14398. [PMID: 38376092 PMCID: PMC10878012 DOI: 10.1111/1751-7915.14398] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Revised: 12/08/2023] [Accepted: 12/21/2023] [Indexed: 02/21/2024] Open
Abstract
The ability to precisely edit genomes by deleting or adding genetic information enables the study of biological functions and the building of efficient cell factories. In many unconventional yeasts, such as those promising new hosts for cell factory design but also human pathogenic yeasts and food spoilers, this progress has been limited by the fact that most yeasts favour non-homologous end joining (NHEJ) over homologous recombination (HR) as a DNA repair mechanism, impairing genetic access to these hosts. In mammalian cells, small molecules that either inhibit proteins involved in NHEJ, enhance protein function in HR, or arrest the cell cycle in HR-dominant phases are regarded as promising agents for the simple and transient increase of HR-mediated genome editing without the need for a priori host engineering. Only a few of these chemicals have been applied to the engineering of yeast, although the targeted proteins are mostly conserved, making chemical agents a yet-underexplored area for enhancing yeast engineering. Here, we consolidate knowledge of the available small molecules that have been used to improve HR efficiency in mammalian cells and the few ones that have been used in yeast. We include available high-throughput-compatible NHEJ/HR quantification assays that could be used to screen for and isolate yeast-specific inhibitors.
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Affiliation(s)
- Min Lu
- Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenGroningenThe Netherlands
| | - Sonja Billerbeck
- Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenGroningenThe Netherlands
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5
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Genetta T, Hurwitz J, Clark E, Herold B, Khalil S, Abbas T, Larner J. ZEB1 promotes non-homologous end joining double-strand break repair. Nucleic Acids Res 2023; 51:9863-9879. [PMID: 37665026 PMCID: PMC10570029 DOI: 10.1093/nar/gkad723] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Revised: 07/31/2023] [Accepted: 08/21/2023] [Indexed: 09/05/2023] Open
Abstract
Repair of DSB induced by IR is primarily carried out by Non-Homologous End Joining (NHEJ), a pathway in which 53BP1 plays a key role. We have discovered that the EMT-inducing transcriptional repressor ZEB1 (i) interacts with 53BP1 and that this interaction occurs rapidly and is significantly amplified following exposure of cells to IR; (ii) is required for the localization of 53BP1 to a subset of double-stranded breaks, and for physiological DSB repair; (iii) co-localizes with 53BP1 at IR-induced foci (IRIF); (iv) promotes NHEJ and inhibits Homologous Recombination (HR); (v) depletion increases resection at DSBs and (vi) confers PARP inhibitor (PARPi) sensitivity on BRCA1-deficient cells. Lastly, ZEB1's effects on repair pathway choice, resection, and PARPi sensitivity all rely on its homeodomain. In contrast to the well-characterized therapeutic resistance of high ZEB1-expressing cancer cells, the novel ZEB1-53BP1-shieldin resection axis described here exposes a therapeutic vulnerability: ZEB1 levels in BRCA1-deficient tumors may serve as a predictive biomarker of response to PARPis.
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Affiliation(s)
- Thomas L Genetta
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
| | - Joshua C Hurwitz
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
| | - Evan A Clark
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
| | - Benjamin T Herold
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
| | - Shadi Khalil
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
| | - Tarek Abbas
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
- Dept. of Biochemistry and Molecular Genetics University of Virginia School of Medicine, Charlottesville, VA 22908, USA
| | - James M Larner
- Dept. of Radiation Oncology, University of Virginia School of Medicine, PO Box 800383, Charlottesville, VA 22908, USA
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6
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Xie J, Kuriakose T, Bianski B, Twarog N, Savage E, Xu K, Zhu X, He C, Hansen B, Wang H, High A, Li Y, Rehg JE, Tillman HS, Freeman BB, Rankovic Z, Onar-Thomas A, Fan Y, Wu G, Peng J, Miller S, Baker SJ, Shelat AA, Tinkle CL. ATM inhibition enhances the efficacy of radiation across distinct molecular subgroups of pediatric high-grade glioma. Neuro Oncol 2023; 25:1828-1841. [PMID: 36971093 PMCID: PMC10547515 DOI: 10.1093/neuonc/noad064] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/05/2023] Open
Abstract
BACKGROUND Pediatric high-grade glioma (pHGG) is largely incurable and accounts for most brain tumor-related deaths in children. Radiation is a standard therapy, yet the benefit from this treatment modality is transient, and most children succumb to disease within 2 years. Recent large-scale genomic studies suggest that pHGG has alterations in DNA damage response (DDR) pathways that induce resistance to DNA damaging agents. The aim of this study was to evaluate the therapeutic potential and molecular consequences of combining radiation with selective DDR inhibition in pHGG. METHODS We conducted an unbiased screen in pHGG cells that combined radiation with clinical candidates targeting the DDR and identified the ATM inhibitor AZD1390. Subsequently, we profiled AZD1390 + radiation in an extensive panel of early passage pHGG cell lines, mechanistically characterized response to the combination in vitro in sensitive and resistant cells and evaluated the combination in vivo using TP53 wild-type and TP53 mutant orthotopic xenografts. RESULTS AZD1390 significantly potentiated radiation across molecular subgroups of pHGG by increasing mutagenic nonhomologous end joining and augmenting genomic instability. In contrast to previous reports, ATM inhibition significantly improved the efficacy of radiation in both TP53 wild-type and TP53 mutant isogenic cell lines and distinct orthotopic xenograft models. Furthermore, we identified a novel mechanism of resistance to AZD1390 + radiation that was marked by an attenuated ATM pathway response which dampened sensitivity to ATM inhibition and induced synthetic lethality with ATR inhibition. CONCLUSIONS Our study supports the clinical evaluation of AZD1390 in combination with radiation in pediatric patients with HGG.
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Affiliation(s)
- Jia Xie
- Department of Radiation Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
| | - Teneema Kuriakose
- Department of Radiation Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
| | - Brandon Bianski
- Department of Radiation Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
| | - Nathaniel Twarog
- Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital
| | - Evan Savage
- Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital
| | - Ke Xu
- Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, USA
| | - Xiaoyan Zhu
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital
| | - Chen He
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital
| | - Baranda Hansen
- Center for Advanced Genome Engineering, St. Jude Children’s Research Hospital
| | - Hong Wang
- Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital
| | - Anthony High
- Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital
| | - Yuxin Li
- Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital
| | - Jerold E Rehg
- Department of Pathology, St. Jude Children’s Research Hospital
| | | | - Burgess B Freeman
- Preclinical Pharmacokinetic Shared Resource, St. Jude Children’s Research Hospital
| | - Zoran Rankovic
- Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital
| | - Arzu Onar-Thomas
- Department of Biostatistics, St. Jude Children’s Research Hospital
| | - Yiping Fan
- Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, USA
| | - Gang Wu
- Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, USA
| | - Junmin Peng
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital
- Center for Proteomics and Metabolomics, St. Jude Children’s Research Hospital
- Department of Structural Biology, St. Jude Children’s Research Hospital
| | - Shondra Miller
- Center for Advanced Genome Engineering, St. Jude Children’s Research Hospital
| | - Suzanne J Baker
- Department of Developmental Neurobiology, St. Jude Children’s Research Hospital
| | - Anang A Shelat
- Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital
| | - Christopher L Tinkle
- Department of Radiation Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
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7
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Rasti G, Becker M, Vazquez BN, Espinosa-Alcantud M, Fernández-Duran I, Gámez-García A, Ianni A, Gonzalez J, Bosch-Presegué L, Marazuela-Duque A, Guitart-Solanes A, Segura-Bayona S, Bech-Serra JJ, Scher M, Serrano L, Shankavaram U, Erdjument-Bromage H, Tempst P, Reinberg D, Olivella M, Stracker T, de la Torre C, Vaquero A. SIRT1 regulates DNA damage signaling through the PP4 phosphatase complex. Nucleic Acids Res 2023; 51:6754-6769. [PMID: 37309898 PMCID: PMC10359614 DOI: 10.1093/nar/gkad504] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 05/24/2023] [Accepted: 06/08/2023] [Indexed: 06/14/2023] Open
Abstract
The Sirtuin family of NAD+-dependent enzymes plays an important role in maintaining genome stability upon stress. Several mammalian Sirtuins have been linked directly or indirectly to the regulation of DNA damage during replication through Homologous recombination (HR). The role of one of them, SIRT1, is intriguing as it seems to have a general regulatory role in the DNA damage response (DDR) that has not yet been addressed. SIRT1-deficient cells show impaired DDR reflected in a decrease in repair capacity, increased genome instability and decreased levels of γH2AX. Here we unveil a close functional antagonism between SIRT1 and the PP4 phosphatase multiprotein complex in the regulation of the DDR. Upon DNA damage, SIRT1 interacts specifically with the catalytical subunit PP4c and promotes its inhibition by deacetylating the WH1 domain of the regulatory subunits PP4R3α/β. This in turn regulates γH2AX and RPA2 phosphorylation, two key events in the signaling of DNA damage and repair by HR. We propose a mechanism whereby during stress, SIRT1 signaling ensures a global control of DNA damage signaling through PP4.
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Affiliation(s)
- George Rasti
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
| | - Maximilian Becker
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
| | - Berta N Vazquez
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
| | - Maria Espinosa-Alcantud
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
| | - Irene Fernández-Duran
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
| | - Andrés Gámez-García
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
| | - Alessandro Ianni
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Department of Cardiac Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231Bad Nauheim, Germany
| | - Jessica Gonzalez
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
| | - Laia Bosch-Presegué
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
- Tissue Repair and Regeneration Laboratory (TR2Lab), Institut de Recerca i Innovació en Ciències de la Vida i de la Salut a la Catalunya Central (IrisCC). Experimental Sciences and Methodology Department. Faculty of Health Sciences and Welfare (FCSB), University of Vic - Central University of Catalonia (UVic-UCC), Vic, Spain
| | - Anna Marazuela-Duque
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
| | - Anna Guitart-Solanes
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
| | - Sandra Segura-Bayona
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain
- Current affiliation: The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - Joan-Josep Bech-Serra
- Proteomic Unit, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916, Badalona, Barcelona, Spain
| | - Michael Scher
- Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, NJ08854, USA
| | - Lourdes Serrano
- Department of Science, BMCC, The City University of New York (CUNY), 199 Chambers Street N699P, New Yirk, NY10007, USA
| | - Uma Shankavaram
- Radiation Oncology Branch, National Cancer Institute, Bethesda, MD20892, USA
| | - Hediye Erdjument-Bromage
- Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY10065, USA
- Department of Cell Biology, New York University School of Medicine, New York, NY10016, USA
| | - Paul Tempst
- Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY10065, USA
| | - Danny Reinberg
- Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, NJ08854, USA
- Howard Hughes Medical Institute, Department of Biochemistry, New York University School of Medicine, New York, NY10016, USA
| | - Mireia Olivella
- Bioinfomatics and Medical Statistics Group, Faculty of Science, Technology and Engineering. University of Vic-Central University of Catalonia, Vic, Spain
| | - Travis H Stracker
- Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain
- Radiation Oncology Branch, National Cancer Institute, Bethesda, MD20892, USA
| | - Carolina de la Torre
- Proteomic Unit, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916, Badalona, Barcelona, Spain
| | - Alejandro Vaquero
- Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Ctra de Can Ruti, Camí de les Escoles s/n, 08916 Badalona, Barcelona, Spain
- Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Av. Gran Via de l’Hospitalet, 199-203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain
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8
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Li F, Mladenov E, Sun Y, Soni A, Stuschke M, Timmermann B, Iliakis G. Low CDK Activity and Enhanced Degradation by APC/C CDH1 Abolishes CtIP Activity and Alt-EJ in Quiescent Cells. Cells 2023; 12:1530. [PMID: 37296650 PMCID: PMC10252496 DOI: 10.3390/cells12111530] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Revised: 05/26/2023] [Accepted: 05/27/2023] [Indexed: 06/12/2023] Open
Abstract
Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to benefit from DNA end-resection-a process whereby 3' single-stranded DNA-tails are generated-initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on the cell cycle phase, is at maximum in G2-phase, substantially reduced in G1-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G1- and G0-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G1-phase cells allow modest resection and alt-EJ, as compared to G2-phase cells. Strikingly, CtIP is undetectable in G0-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G0-phase cells. CtIP activation in G0-phase cells also requires CDK-dependent phosphorylation by any available CDK but is restricted to CDK4/6 at the early stages of the normal cell cycle. We suggest that suppression of mutagenic alt-EJ in G0-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms.
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Affiliation(s)
- Fanghua Li
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (F.L.); (E.M.); (Y.S.); (A.S.)
- Department of Particle Therapy, University Hospital Essen, West German Proton Therapy Centre Essen (WPE), West German Cancer Center (WTZ), German Cancer Consortium (DKTK), 45147 Essen, Germany;
| | - Emil Mladenov
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (F.L.); (E.M.); (Y.S.); (A.S.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Yanjie Sun
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (F.L.); (E.M.); (Y.S.); (A.S.)
- Department of Particle Therapy, University Hospital Essen, West German Proton Therapy Centre Essen (WPE), West German Cancer Center (WTZ), German Cancer Consortium (DKTK), 45147 Essen, Germany;
| | - Aashish Soni
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (F.L.); (E.M.); (Y.S.); (A.S.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
| | - Martin Stuschke
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
- German Cancer Consortium (DKTK), Partner Site University Hospital Essen, German Cancer Research Center (DKFZ), 45147 Essen, Germany
| | - Beate Timmermann
- Department of Particle Therapy, University Hospital Essen, West German Proton Therapy Centre Essen (WPE), West German Cancer Center (WTZ), German Cancer Consortium (DKTK), 45147 Essen, Germany;
- German Cancer Consortium (DKTK), Partner Site University Hospital Essen, German Cancer Research Center (DKFZ), 45147 Essen, Germany
| | - George Iliakis
- Institute of Medical Radiation Biology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany; (F.L.); (E.M.); (Y.S.); (A.S.)
- Division of Experimental Radiation Biology, Department of Radiation Therapy, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany;
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9
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Dutta A, Mitra J, Hegde PM, Mitra S, Hegde ML. Characterizing the Repair of DNA Double-Strand Breaks: A Review of Surrogate Plasmid-Based Reporter Methods. Methods Mol Biol 2023; 2701:173-182. [PMID: 37574482 DOI: 10.1007/978-1-0716-3373-1_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
DNA double-strand breaks (DSBs) are the most lethal genomic lesions that are induced endogenously during physiological reactions as well as by external stimuli and genotoxicants. DSBs are repaired in mammalian cells via one of three well-studied pathways depending on the cell cycle status and/or the nature of the break. First, the homologous recombination (HR) pathway utilizes the duplicated sister chromatid as a template in S/G2 cells. Second, the nonhomologous end-joining (NHEJ) is the predominant DSB repair pathway throughout the cell cycle. The third pathway, microhomology-mediated/alternative end-joining (MMEJ/Alt-EJ), is a specialized backup pathway that works not only in the S phase but also in G0/G1 cells that constitute the bulk of human tissues. In vitro experimental methods to recapitulate the repair of physiologically relevant DSBs pose a challenge. Commonly employed plasmid- or oligonucleotide-based substrates contain restriction enzyme-cleaved DSB mimics, which undoubtedly do not mimic DSB ends generated by ionizing radiation (IR), chemotherapeutics, and reactive oxygen species (ROS). DSBs can also be indirectly generated by reactive oxygen species (ROS). All such DSBs invariably contain blocked termini. In this methodology chapter, we describe a method to recapitulate the DSB repair mechanism using in cellulo and in vitro cell-free systems. This methodology enables researchers to assess the contribution of NHEJ vs. Alt-EJ using a reporter plasmid containing DSB lesions with non-ligatable termini. Limitations and challenges of prevailing methods are also addressed.
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Affiliation(s)
- Arijit Dutta
- Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
| | - Joy Mitra
- Division of DNA Repair Research, Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX, USA
| | - Pavana M Hegde
- Division of DNA Repair Research, Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX, USA
| | - Sankar Mitra
- Division of DNA Repair Research, Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX, USA
| | - Muralidhar L Hegde
- Division of DNA Repair Research, Center for Neuroregeneration, Department of Neurosurgery, Houston Methodist Research Institute, Houston, TX, USA.
- Department of Neurosciences, Weill Cornell Medical College, New York, NY, USA.
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10
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Wilson C, Murnane JP. High-throughput screen to identify compounds that prevent or target telomere loss in human cancer cells. NAR Cancer 2022; 4:zcac029. [PMID: 36196242 PMCID: PMC9527662 DOI: 10.1093/narcan/zcac029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Revised: 09/09/2022] [Accepted: 09/29/2022] [Indexed: 11/14/2022] Open
Abstract
Chromosome instability (CIN) is an early step in carcinogenesis that promotes tumor cell progression and resistance to therapy. Using plasmids integrated adjacent to telomeres, we have previously demonstrated that the sensitivity of subtelomeric regions to DNA double-strand breaks (DSBs) contributes to telomere loss and CIN in cancer. A high-throughput screen was created to identify compounds that affect telomere loss due to subtelomeric DSBs introduced by I-SceI endonuclease, as detected by cells expressing green fluorescent protein (GFP). A screen of a library of 1832 biologically-active compounds identified a variety of compounds that increase or decrease the number of GFP-positive cells following activation of I-SceI. A curated screen done in triplicate at various concentrations found that inhibition of classical nonhomologous end joining (C-NHEJ) increased DSB-induced telomere loss, demonstrating that C-NHEJ is functional in subtelomeric regions. Compounds that decreased DSB-induced telomere loss included inhibitors of mTOR, p38 and tankyrase, consistent with our earlier hypothesis that the sensitivity of subtelomeric regions to DSBs is a result of inappropriate resection during repair. Although this assay was also designed to identify compounds that selectively target cells experiencing telomere loss and/or chromosome instability, no compounds of this type were identified in the current screen.
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Affiliation(s)
- Chris Wilson
- Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, University of California, San Francisco, CA 94143, USA
| | - John P Murnane
- To whom correspondence should be addressed. Tel: +1 415 680 4434;
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11
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Pan-cancer analysis of co-occurring mutations in RAD52 and the BRCA1-BRCA2-PALB2 axis in human cancers. PLoS One 2022; 17:e0273736. [PMID: 36107942 PMCID: PMC9477347 DOI: 10.1371/journal.pone.0273736] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2022] [Accepted: 08/12/2022] [Indexed: 11/19/2022] Open
Abstract
In human cells homologous recombination (HR) is critical for repair of DNA double strand breaks (DSBs) and rescue of stalled or collapsed replication forks. HR is facilitated by RAD51 which is loaded onto DNA by either BRCA2-BRCA1-PALB2 or RAD52. In human culture cells, double-knockdowns of RAD52 and genes in the BRCA1-BRCA2-PALB2 axis are lethal. Mutations in BRCA2, BRCA1 or PALB2 significantly impairs error free HR as RAD51 loading relies on RAD52 which is not as proficient as BRCA2-BRCA1-PALB2. RAD52 also facilitates Single Strand Annealing (SSA) that produces intra-chromosomal deletions. Some RAD52 mutations that affect the SSA function or decrease RAD52 association with DNA can suppress certain BRCA2 associated phenotypes in breast cancers. In this report we did a pan-cancer analysis using data reported on the Catalogue of Somatic Mutations in Cancers (COSMIC) to identify double mutants between RAD52 and BRCA1, BRCA2 or PALB2 that occur in cancer cells. We find that co-occurring mutations are likely in certain cancer tissues but not others. However, all mutations occur in a heterozygous state. Further, using computational and machine learning tools we identified only a handful of pathogenic or driver mutations predicted to significantly affect the function of the proteins. This supports previous findings that co-inactivation of RAD52 with any members of the BRCA2-BRCA1-PALB2 axis is lethal. Molecular modeling also revealed that pathogenic RAD52 mutations co-occurring with mutations in BRCA2-BRCA1-PALB2 axis are either expected to attenuate its SSA function or its interaction with DNA. This study extends previous breast cancer findings to other cancer types and shows that co-occurring mutations likely destabilize HR by similar mechanisms as in breast cancers.
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12
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Liu SC, Feng YL, Sun XN, Chen RD, Liu Q, Xiao JJ, Zhang JN, Huang ZC, Xiang JF, Chen GQ, Yang Y, Lou C, Li HD, Cai Z, Xu SM, Lin H, Xie AY. Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing. Genome Biol 2022; 23:165. [PMID: 35915475 PMCID: PMC9341079 DOI: 10.1186/s13059-022-02736-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 07/22/2022] [Indexed: 12/05/2022] Open
Abstract
BACKGROUND Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.
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Affiliation(s)
- Si-Cheng Liu
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Yi-Li Feng
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
- Department of Biochemistry and Molecular Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, People's Republic of China
| | - Xiu-Na Sun
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Ruo-Dan Chen
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
- Department of Biochemistry and Molecular Biology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, People's Republic of China
| | - Qian Liu
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Jing-Jing Xiao
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Jin-Na Zhang
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
- The First affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
| | - Zhi-Cheng Huang
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Ji-Feng Xiang
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
- Institute of Hepatopancreatobiliary Surgery, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing, 400013, People's Republic of China
| | - Guo-Qiao Chen
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Yi Yang
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Chao Lou
- Shurui Tech Ltd, Hangzhou, Zhejiang, 310005, People's Republic of China
| | - Hao-Dan Li
- Shurui Tech Ltd, Hangzhou, Zhejiang, 310005, People's Republic of China
| | - Zhen Cai
- The First affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
| | - Shi-Ming Xu
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China
| | - Hui Lin
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China
| | - An-Yong Xie
- Innovation Center for Minimally Invasive Technique and Device, Department of General Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310019, People's Republic of China.
- Institute of Translational Medicine, Zhejiang University School of Medicine and Zhejiang University Cancer Center, Hangzhou, Zhejiang, 310029, People's Republic of China.
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13
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van de Kooij B, van Attikum H. Genomic Reporter Constructs to Monitor Pathway-Specific Repair of DNA Double-Strand Breaks. Front Genet 2022; 12:809832. [PMID: 35237296 PMCID: PMC8884240 DOI: 10.3389/fgene.2021.809832] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Accepted: 12/23/2021] [Indexed: 11/29/2022] Open
Abstract
Repair of DNA Double-Strand Breaks (DSBs) can be error-free or highly mutagenic, depending on which of multiple mechanistically distinct pathways repairs the break. Hence, DSB-repair pathway choice directly affects genome integrity, and it is therefore of interest to understand the parameters that direct repair towards a specific pathway. This has been intensively studied using genomic reporter constructs, in which repair of a site-specific DSB by the pathway of interest generates a quantifiable phenotype, generally the expression of a fluorescent protein. The current developments in genome editing with targetable nucleases like Cas9 have increased reporter usage and accelerated the generation of novel reporter constructs. Considering these recent advances, this review will discuss and compare the available DSB-repair pathway reporters, provide essential considerations to guide reporter choice, and give an outlook on potential future developments.
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Affiliation(s)
| | - Haico van Attikum
- Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands
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14
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Lappin KM, Barros EM, Jhujh SS, Irwin GW, McMillan H, Liberante FG, Latimer C, LaBonte MJ, Mills KI, Harkin DP, Stewart GS, Savage KI. CANCER-ASSOCIATED SF3B1 MUTATIONS CONFER A BRCA-LIKE CELLULAR PHENOTYPE AND SYNTHETIC LETHALITY TO PARP INHIBITORS. Cancer Res 2022; 82:819-830. [DOI: 10.1158/0008-5472.can-21-1843] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 11/12/2021] [Accepted: 12/27/2021] [Indexed: 11/16/2022]
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15
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Anand R, Buechelmaier E, Belan O, Newton M, Vancevska A, Kaczmarczyk A, Takaki T, Rueda DS, Powell SN, Boulton SJ. HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51. Nature 2022; 601:268-273. [PMID: 34937945 PMCID: PMC8755542 DOI: 10.1038/s41586-021-04261-0] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Accepted: 11/17/2021] [Indexed: 02/04/2023]
Abstract
DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3' to 5' polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.
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Affiliation(s)
- Roopesh Anand
- DSB Repair Metabolism Laboratory, The Francis Crick Institute, London, UK
| | - Erika Buechelmaier
- Memorial Sloan Kettering Cancer Center, New York, NY, USA
- Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA
| | - Ondrej Belan
- DSB Repair Metabolism Laboratory, The Francis Crick Institute, London, UK
| | - Matthew Newton
- DSB Repair Metabolism Laboratory, The Francis Crick Institute, London, UK
| | | | - Artur Kaczmarczyk
- Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, UK
- Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK
| | - Tohru Takaki
- DSB Repair Metabolism Laboratory, The Francis Crick Institute, London, UK
| | - David S Rueda
- Department of Infectious Disease, Faculty of Medicine, Imperial College London, London, UK.
- Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK.
| | - Simon N Powell
- Memorial Sloan Kettering Cancer Center, New York, NY, USA.
| | - Simon J Boulton
- DSB Repair Metabolism Laboratory, The Francis Crick Institute, London, UK.
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16
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Dow J, Krysztofiak A, Liu Y, Colon-Rios DA, Rogers FA, Glazer PM. Vulnerability of IDH1-Mutant Cancers to Histone Deacetylase Inhibition via Orthogonal Suppression of DNA Repair. Mol Cancer Res 2021; 19:2057-2067. [PMID: 34535560 PMCID: PMC8642278 DOI: 10.1158/1541-7786.mcr-21-0456] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 08/06/2021] [Accepted: 09/10/2021] [Indexed: 11/16/2022]
Abstract
Exploitation of DNA repair defects has enabled major advances in treating specific cancers. Recent work discovered that the oncometabolite 2-hydroxyglutarate (2-HG), produced by neomorphic isocitrate dehydrogenase 1/2 (IDH1/2) mutations, confers a homology-directed repair (HDR) defect through 2-HG-induced histone hypermethylation masking HDR signaling. Here, we report that IDH1-mutant cancer cells are profoundly sensitive to the histone deacetylase inhibitor (HDACi) vorinostat, by further suppressing the residual HDR in 2-HG-producing cells. Vorinostat downregulates repair factors BRCA1 and RAD51 via disrupted E2F-factor regulation, causing increased DNA double-strand breaks, reduced DNA repair factor foci, and functional HDR deficiency even beyond 2-HG's effects. This results in greater cell death of IDH1-mutant cells and confers synergy with radiation and PARPi, both against cells in culture and patient-derived tumor xenografts. Our work identifies HDACi's utility against IDH1-mutant cancers, and presents IDH1/2 mutations as potential biomarkers to guide trials testing HDACi in gliomas and other malignancies. IMPLICATIONS: IDH1-mutant cells show profound vulnerability to HDACi treatment, alone and with PARPi and radiation, via HDR suppression, presenting IDH1/2 mutations as biomarkers for HDACi use in gliomas and other malignancies.
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Affiliation(s)
- Jonathan Dow
- Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut
- Department of Genetics, Yale University School of Medicine. New Haven, Connecticut
| | - Adam Krysztofiak
- Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut
| | - Yanfeng Liu
- Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut
- Department of Genetics, Yale University School of Medicine. New Haven, Connecticut
| | - Daniel A Colon-Rios
- Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut
| | - Faye A Rogers
- Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut
| | - Peter M Glazer
- Department of Therapeutic Radiology, Yale University School of Medicine. New Haven, Connecticut.
- Department of Genetics, Yale University School of Medicine. New Haven, Connecticut
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17
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Chien JCY, Badr CE, Lai CP. Multiplexed bioluminescence-mediated tracking of DNA double-strand break repairs in vitro and in vivo. Nat Protoc 2021; 16:3933-3953. [PMID: 34163064 PMCID: PMC9124064 DOI: 10.1038/s41596-021-00564-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 04/27/2021] [Indexed: 02/06/2023]
Abstract
The dynamics of DNA double-strand break (DSB) repairs including homology-directed repair and nonhomologous end joining play an important role in diseases and therapies. However, investigating DSB repair is typically a low-throughput and cross-sectional process, requiring disruption of cells and organisms for subsequent nuclease-, sequencing- or reporter-based assays. In this protocol, we provide instructions for establishing a bioluminescent repair reporter system using engineered Gaussia and Vargula luciferases for noninvasive tracking of homology-directed repair and nonhomologous end joining, respectively, induced by SceI meganuclease, SpCas9 or SpCas9 D10A nickase-mediated editing. We also describe complementation with orthogonal DSB repair assays and omics analyses to validate the reporter readouts. The bioluminescent repair reporter system provides longitudinal and rapid readout (~seconds per sample) to accurately and efficiently measure the efficacy of genome-editing tools and small-molecule modulators on DSB repair. This protocol takes ~2-4 weeks to establish, and as little as 2 h to complete the assay. The entire bioluminescent repair reporter procedure can be performed by one person with standard molecular biology expertise and equipment. However, orthogonal DNA repair assays would require a specialized facility that performs Sanger sequencing or next-generation sequencing.
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Affiliation(s)
| | - Christian E. Badr
- Department of Neurology, Massachusetts General Hospital, Boston MA, United States,Neuroscience Program, Harvard Medical School, Boston MA, United States,To whom correspondence should be addressed: Christian E. Badr, Tel: 1-617-643-3485; Fax: 1-617-724-1537; ; Charles P. Lai, Tel: 886-2-2366-8204; Fax: 886-2-2362-0200; . C.E.B and C.P.L contributed equally to this work
| | - Charles P. Lai
- Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan,To whom correspondence should be addressed: Christian E. Badr, Tel: 1-617-643-3485; Fax: 1-617-724-1537; ; Charles P. Lai, Tel: 886-2-2366-8204; Fax: 886-2-2362-0200; . C.E.B and C.P.L contributed equally to this work
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18
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Hussain SS, Majumdar R, Moore GM, Narang H, Buechelmaier E, Bazil MJ, Ravindran PT, Leeman J, Li Y, Jalan M, Anderson KS, Farina A, Soni R, Mohibullah N, Hamzic E, Rong-Mullins X, Sifuentes C, Damerla RR, Viale A, Powell SN, Higginson D. Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell. Nucleic Acids Res 2021; 49:e74. [PMID: 33877327 PMCID: PMC8287935 DOI: 10.1093/nar/gkab262] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Revised: 03/23/2021] [Accepted: 04/01/2021] [Indexed: 02/06/2023] Open
Abstract
Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.
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Affiliation(s)
- Suleman S Hussain
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Rahul Majumdar
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Grace M Moore
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Himanshi Narang
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Erika S Buechelmaier
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
- Weill Cornell Medicine, Graduate School of Medical Sciences, New York, NY 10065, USA
| | - Maximilian J Bazil
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | | | - Jonathan E Leeman
- Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02189, USA
| | - Yi Li
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Manisha Jalan
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Kyrie S Anderson
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Andrea Farina
- Integrated Genomics Operations, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Rekha Soni
- Integrated Genomics Operations, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Neeman Mohibullah
- Integrated Genomics Operations, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Edin Hamzic
- Biocomputix, Sarajevo, 71000, Bosnia and Herzegovina
| | - Xiaoqing Rong-Mullins
- Department of Biostatistics, The Ohio State University College of Public Health, Columbus, OH 43210, USA
| | | | - Rama R Damerla
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Agnes Viale
- Integrated Genomics Operations, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Simon N Powell
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Daniel S Higginson
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
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19
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Feng W, Simpson DA, Cho JE, Carvajal-Garcia J, Smith CM, Headley KM, Hathaway N, Ramsden DA, Gupta GP. Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks. Nucleic Acids Res 2021; 49:5095-5105. [PMID: 33963863 PMCID: PMC8136827 DOI: 10.1093/nar/gkab299] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2020] [Revised: 03/24/2021] [Accepted: 05/05/2021] [Indexed: 12/14/2022] Open
Abstract
Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.
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Affiliation(s)
- Wanjuan Feng
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Dennis A Simpson
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Jang-Eun Cho
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Juan Carvajal-Garcia
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.,Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599, USA.,Biological and Biomedical Sciences Program, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Chelsea M Smith
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.,Biological and Biomedical Sciences Program, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kathryn M Headley
- School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Nate Hathaway
- School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Dale A Ramsden
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.,Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Gaorav P Gupta
- Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.,Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.,Department of Radiation Oncology, University of North Carolina, Chapel Hill, NC 27599, USA
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20
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Tatin X, Muggiolu G, Sauvaigo S, Breton J. Evaluation of DNA double-strand break repair capacity in human cells: Critical overview of current functional methods. MUTATION RESEARCH. REVIEWS IN MUTATION RESEARCH 2021; 788:108388. [PMID: 34893153 DOI: 10.1016/j.mrrev.2021.108388] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Revised: 06/17/2021] [Accepted: 06/23/2021] [Indexed: 02/05/2023]
Abstract
DNA double-strand breaks (DSBs) are highly deleterious lesions, responsible for mutagenesis, chromosomal translocation or cell death. DSB repair (DSBR) is therefore a critical part of the DNA damage response (DDR) to restore molecular and genomic integrity. In humans, this process is achieved through different pathways with various outcomes. The balance between DSB repair activities varies depending on cell types, tissues or individuals. Over the years, several methods have been developed to study variations in DSBR capacity. Here, we mainly focus on functional techniques, which provide dynamic information regarding global DSB repair proficiency or the activity of specific pathways. These methods rely on two kinds of approaches. Indirect techniques, such as pulse field gel electrophoresis (PFGE), the comet assay and immunofluorescence (IF), measure DSB repair capacity by quantifying the time-dependent decrease in DSB levels after exposure to a DNA-damaging agent. On the other hand, cell-free assays and reporter-based methods directly track the repair of an artificial DNA substrate. Each approach has intrinsic advantages and limitations and despite considerable efforts, there is currently no ideal method to quantify DSBR capacity. All techniques provide different information and can be regarded as complementary, but some studies report conflicting results. Parameters such as the type of biological material, the required equipment or the cost of analysis may also limit available options. Improving currently available methods measuring DSBR capacity would be a major step forward and we present direct applications in mechanistic studies, drug development, human biomonitoring and personalized medicine, where DSBR analysis may improve the identification of patients eligible for chemo- and radiotherapy.
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Affiliation(s)
- Xavier Tatin
- Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, 38000 Grenoble, France; LXRepair, 5 Avenue du Grand Sablon, 38700 La Tronche, France
| | | | - Sylvie Sauvaigo
- LXRepair, 5 Avenue du Grand Sablon, 38700 La Tronche, France
| | - Jean Breton
- Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, 38000 Grenoble, France.
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21
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Abounader R, Schiff D. A new class of radiosensitizers for glioblastoma. Oncotarget 2021; 12:1199-1200. [PMID: 34194618 PMCID: PMC8238247 DOI: 10.18632/oncotarget.27970] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Indexed: 11/25/2022] Open
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22
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Shkundina IS, Gall AA, Dick A, Cocklin S, Mazin AV. New RAD51 Inhibitors to Target Homologous Recombination in Human Cells. Genes (Basel) 2021; 12:genes12060920. [PMID: 34208492 PMCID: PMC8235719 DOI: 10.3390/genes12060920] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Revised: 06/10/2021] [Accepted: 06/11/2021] [Indexed: 12/31/2022] Open
Abstract
Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.
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Affiliation(s)
- Irina S. Shkundina
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA; (I.S.S.); (A.D.); (S.C.)
| | | | - Alexej Dick
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA; (I.S.S.); (A.D.); (S.C.)
| | - Simon Cocklin
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA; (I.S.S.); (A.D.); (S.C.)
| | - Alexander V. Mazin
- Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA; (I.S.S.); (A.D.); (S.C.)
- Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229, USA
- Correspondence:
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23
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Sule A, Van Doorn J, Sundaram RK, Ganesa S, Vasquez J, Bindra R. Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors. NAR Cancer 2021; 3:zcab018. [PMID: 34027408 PMCID: PMC8127964 DOI: 10.1093/narcan/zcab018] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Revised: 04/17/2021] [Accepted: 05/04/2021] [Indexed: 12/15/2022] Open
Abstract
Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as 'BRCAness', which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.
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Affiliation(s)
- Amrita Sule
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Jinny Van Doorn
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Ranjini K Sundaram
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Sachita Ganesa
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Juan C Vasquez
- Department of Pediatrics, Yale School of Medicine, New Haven, CT 06511, USA
| | - Ranjit S Bindra
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
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24
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Creation of a new class of radiosensitizers for glioblastoma based on the mibefradil pharmacophore. Oncotarget 2021; 12:891-906. [PMID: 33953843 PMCID: PMC8092340 DOI: 10.18632/oncotarget.27933] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Accepted: 03/22/2021] [Indexed: 12/15/2022] Open
Abstract
Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system with a dismal prognosis. Locoregional failure is common despite high doses of radiation therapy, which has prompted great interest in developing novel strategies to radiosensitize these cancers. Our group previously identified a calcium channel blocker (CCB), mibefradil, as a potential GBM radiosensitizer. We discovered that mibefradil selectively inhibits a key DNA repair pathway, alternative non-homologous end joining. We then initiated a phase I clinical trial that revealed promising initial efficacy of mibefradil, but further development was hampered by dose-limiting toxicities, including CCB-related cardiotoxicity, off-target hERG channel and cytochrome P450 enzymes (CYPs) interactions. Here, we show that mibefradil inhibits DNA repair independent of its CCB activity, and report a series of mibefradil analogues which lack CCB activity and demonstrate reduced hERG and CYP activity while retaining potency as DNA repair inhibitors. We present in vivo pharmacokinetic studies of the top analogues with evidence of brain penetration. We also report a targeted siRNA-based screen which suggests a possible role for mTOR and Akt in DNA repair inhibition by this class of drugs. Taken together, these data reveal a new class of mibefradil-based DNA repair inhibitors which can be further advanced into pre-clinical testing and eventually clinical trials, as potential GBM radiosensitizers.
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25
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Liu Q, Palomero L, Moore J, Guix I, Espín R, Aytés A, Mao JH, Paulovich AG, Whiteaker JR, Ivey RG, Iliakis G, Luo D, Chalmers AJ, Murnane J, Pujana MA, Barcellos-Hoff MH. Loss of TGFβ signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types. Sci Transl Med 2021; 13:eabc4465. [PMID: 33568520 PMCID: PMC8208885 DOI: 10.1126/scitranslmed.abc4465] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Accepted: 12/07/2020] [Indexed: 12/17/2022]
Abstract
Among the pleotropic roles of transforming growth factor-β (TGFβ) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFβ signaling increases use of alternative end joining (alt-EJ), an error-prone DNA repair process that typically functions as a "backup" pathway if double-strand break repair by homologous recombination or nonhomologous end joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFβ broadly controls the DNA damage response and suppresses alt-EJ genes that are associated with genomic instability. Mechanistically based TGFβ and alt-EJ gene expression signatures were anticorrelated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFβ and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes were coordinately expressed and anticorrelated with TGFβ competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFβ and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFβ signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anticorrelation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFβ biology.
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Affiliation(s)
- Qi Liu
- Department of Radiation Oncology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA
| | - Luis Palomero
- ProCURE, Catalan Institute of Oncology, Oncobell, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet del Llobregat, Barcelona 08908, Catalonia, Spain
| | - Jade Moore
- Department of Radiation Oncology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA
| | - Ines Guix
- Department of Radiation Oncology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA
| | - Roderic Espín
- ProCURE, Catalan Institute of Oncology, Oncobell, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet del Llobregat, Barcelona 08908, Catalonia, Spain
| | - Alvaro Aytés
- ProCURE, Catalan Institute of Oncology, Oncobell, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet del Llobregat, Barcelona 08908, Catalonia, Spain
| | - Jian-Hua Mao
- Biological Systems and Engineering Division, Berkeley Biomedical Data Science Center, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
| | - Amanda G Paulovich
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Jeffrey R Whiteaker
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Richard G Ivey
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - George Iliakis
- Institute of Medical Radiation Biology, University of Duisburg-Essen, University Hospital Essen, Essen 45147, Germany
| | - Daxian Luo
- Institute of Medical Radiation Biology, University of Duisburg-Essen, University Hospital Essen, Essen 45147, Germany
| | - Anthony J Chalmers
- Institute of Cancer Sciences and Beatson West of Scotland Cancer Centre, University of Glasgow, Glasgow G12 8QQ, Scotland, UK
| | - John Murnane
- Department of Radiation Oncology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA
| | - Miquel Angel Pujana
- ProCURE, Catalan Institute of Oncology, Oncobell, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet del Llobregat, Barcelona 08908, Catalonia, Spain.
| | - Mary Helen Barcellos-Hoff
- Department of Radiation Oncology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94143, USA.
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26
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Zhang P, Brinton LT, Williams K, Sher S, Orwick S, Tzung-Huei L, Mims AS, Coss CC, Kulp SK, Youssef Y, Chan WK, Mitchell S, Mustonen A, Cannon M, Phillips H, Lehman AM, Kauffman T, Beaver L, Canfield D, Grieselhuber NR, Alinari L, Sampath D, Yan P, Byrd JC, Blachly JS, Lapalombella R. Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition. Clin Cancer Res 2021; 27:2352-2366. [PMID: 33542077 DOI: 10.1158/1078-0432.ccr-20-3724] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Revised: 12/24/2020] [Accepted: 02/01/2021] [Indexed: 11/16/2022]
Abstract
PURPOSE Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.
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Affiliation(s)
- Pu Zhang
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.,College of Pharmacy, The Ohio State University, Columbus, Ohio
| | - Lindsey T Brinton
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Katie Williams
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Steven Sher
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Shelley Orwick
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Lai Tzung-Huei
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Alice S Mims
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | | | - Samuel K Kulp
- College of Pharmacy, The Ohio State University, Columbus, Ohio
| | - Youssef Youssef
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Wing Keung Chan
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Shaneice Mitchell
- Department of Pathology, Stanford University School of Medicine, Stanford, California
| | - Allison Mustonen
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Matthew Cannon
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Hannah Phillips
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Amy M Lehman
- Center for Biostatistics, Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio
| | - Tierney Kauffman
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Larry Beaver
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Daniel Canfield
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Nicole R Grieselhuber
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Lapo Alinari
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Deepa Sampath
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Pearlly Yan
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - John C Byrd
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.,College of Pharmacy, The Ohio State University, Columbus, Ohio
| | - James S Blachly
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio
| | - Rosa Lapalombella
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
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27
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Nouws J, Wan F, Finnemore E, Roque W, Kim SJ, Bazan I, Li CX, Skold CM, Dai Q, Yan X, Chioccioli M, Neumeister V, Britto CJ, Sweasy J, Bindra R, Wheelock ÅM, Gomez JL, Kaminski N, Lee PJ, Sauler M. MicroRNA miR-24-3p reduces DNA damage responses, apoptosis, and susceptibility to chronic obstructive pulmonary disease. JCI Insight 2021; 6:134218. [PMID: 33290275 PMCID: PMC7934877 DOI: 10.1172/jci.insight.134218] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Accepted: 12/02/2020] [Indexed: 12/27/2022] Open
Abstract
The pathogenesis of chronic obstructive pulmonary disease (COPD) involves aberrant responses to cellular stress caused by chronic cigarette smoke (CS) exposure. However, not all smokers develop COPD and the critical mechanisms that regulate cellular stress responses to increase COPD susceptibility are not understood. Because microRNAs are well-known regulators of cellular stress responses, we evaluated microRNA expression arrays performed on distal parenchymal lung tissue samples from 172 subjects with and without COPD. We identified miR-24-3p as the microRNA that best correlated with radiographic emphysema and validated this finding in multiple cohorts. In a CS exposure mouse model, inhibition of miR-24-3p increased susceptibility to apoptosis, including alveolar type II epithelial cell apoptosis, and emphysema severity. In lung epithelial cells, miR-24-3p suppressed apoptosis through the BH3-only protein BIM and suppressed homology-directed DNA repair and the DNA repair protein BRCA1. Finally, we found BIM and BRCA1 were increased in COPD lung tissue, and BIM and BRCA1 expression inversely correlated with miR-24-3p. We concluded that miR-24-3p, a regulator of the cellular response to DNA damage, is decreased in COPD, and decreased miR-24-3p increases susceptibility to emphysema through increased BIM and apoptosis.
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Affiliation(s)
- Jessica Nouws
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Feng Wan
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.,Department of Anatomy, Beijing University of Chinese Medicine, Beijing, China
| | - Eric Finnemore
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Willy Roque
- Department of Internal Medicine, Rutgers New Jersey Medical School, Newark, New Jersey, USA
| | - So-Jin Kim
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Isabel Bazan
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Chuan-Xing Li
- Division of Respiratory Medicine and Allergy, Department of Medicine, and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - C Magnus Skold
- Division of Respiratory Medicine and Allergy, Department of Medicine, and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Qile Dai
- Department of Biostatistics, Yale School of Public Health, New Haven, Connecticut, USA
| | - Xiting Yan
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.,Department of Biostatistics, Yale School of Public Health, New Haven, Connecticut, USA
| | - Maurizio Chioccioli
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Veronique Neumeister
- Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Clemente J Britto
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Joann Sweasy
- Department of Radiation Oncology, University of Arizona College of Medicine, Tucson, Arizona, USA.,Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Ranjit Bindra
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Åsa M Wheelock
- Division of Respiratory Medicine and Allergy, Department of Medicine, and Center for Molecular Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Jose L Gomez
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Naftali Kaminski
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Patty J Lee
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.,Section of Pulmonary, Allergy, and Critical Care Medicine, Department of Internal Medicine, Duke University School of Medicine, Durham, North Carolina, USA
| | - Maor Sauler
- Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
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28
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Eki R, She J, Parlak M, Benamar M, Du KP, Kumar P, Abbas T. A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair. Nucleic Acids Res 2020; 48:e126. [PMID: 33068408 PMCID: PMC7708081 DOI: 10.1093/nar/gkaa897] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2020] [Revised: 09/25/2020] [Accepted: 09/30/2020] [Indexed: 12/30/2022] Open
Abstract
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.
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Affiliation(s)
- Rebeka Eki
- Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.,Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.,Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA
| | - Jane She
- Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA
| | - Mahmut Parlak
- Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA
| | - Mouadh Benamar
- Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.,Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.,Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA
| | - Kang-Ping Du
- Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA
| | - Pankaj Kumar
- Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA
| | - Tarek Abbas
- Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.,Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA.,Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA.,Cancer Center, University of Virginia, Charlottesville, VA 22908, USA
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29
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Chen Y, Geng A, Zhang W, Qian Z, Wan X, Jiang Y, Mao Z. Fight to the bitter end: DNA repair and aging. Ageing Res Rev 2020; 64:101154. [PMID: 32977059 DOI: 10.1016/j.arr.2020.101154] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Revised: 08/05/2020] [Accepted: 08/19/2020] [Indexed: 12/11/2022]
Abstract
DNA carries the genetic information that directs complex biological processes; thus, maintaining a stable genome is critical for individual growth and development and for human health. DNA repair is a fundamental and conserved mechanism responsible for mending damaged DNA and restoring genomic stability, while its deficiency is closely related to multiple human disorders. In recent years, remarkable progress has been made in the field of DNA repair and aging. Here, we will extensively discuss the relationship among DNA damage, DNA repair, aging and aging-associated diseases based on the latest research. In addition, the possible role of DNA repair in several potential rejuvenation strategies will be discussed. Finally, we will also review the emerging methods that may facilitate future research on DNA repair.
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30
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Waisertreiger I, Barlow J. Fragile site instability: measuring more than breaks. Oncoscience 2020; 7:60-67. [PMID: 33195735 PMCID: PMC7640903 DOI: 10.18632/oncoscience.513] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Accepted: 05/17/2020] [Indexed: 12/23/2022] Open
Abstract
Genome instability is not only a hallmark of cancer, it is necessary for its initiation and evolution, and naturally accumulates as cells age. Replication stress is a potent source of genome instability found in many tumor types [1]. Chromosomal fragile sites are genomic loci highly prone to DNA damage specifically from replication stress and are frequently mutated in cancer [2-4]2-4]. While tracking the origin of individual mutations has proved challenging, measuring DNA damage and repair at endogenous sites can offer key insights into understanding the etiology of cancer. In the past 15 years, the causal link between replication stress, oncogene activation, and tumor initiation and evolution has become increasingly clear [1, 5-9]. Replication-associated damage accumulates at early stages of tumorigenesis and may promote further transformation. Studying the causes and consequences of fragile site instability can offer a window into the earliest stages of carcinogenesis [10-13]. In particular, fragile site studies will help us understand the molecular underpinnings influencing the frequency of DNA breakage, successful repair processes suppressing genome instability, and unsuccessful repair leading to mutations and chromosome rearrangements. Of these, measuring successful repair is the most challenging as it leaves little evidence behind.
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Affiliation(s)
- Irina Waisertreiger
- Department of Microbiology and Molecular Genetics, University of California, Davis, CA, USA
| | - Jacqueline Barlow
- Department of Microbiology and Molecular Genetics & Genome Center, University of California, Davis, CA, USA
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31
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Kaplan AR, Gueble SE, Liu Y, Oeck S, Kim H, Yun Z, Glazer PM. Cediranib suppresses homology-directed DNA repair through down-regulation of BRCA1/2 and RAD51. Sci Transl Med 2020; 11:11/492/eaav4508. [PMID: 31092693 DOI: 10.1126/scitranslmed.aav4508] [Citation(s) in RCA: 114] [Impact Index Per Article: 22.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2018] [Revised: 01/30/2019] [Accepted: 04/15/2019] [Indexed: 12/19/2022]
Abstract
Combining the anti-angiogenic agent cediranib with the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib improves progression-free survival compared to olaparib alone in ovarian cancer patients through an unknown mechanism. PARP inhibitors are used primarily in the treatment of patients with DNA repair-associated (BRCA1/2) mutated cancers because these mutations cause a deficit in homology-directed DNA repair (HDR) that confers sensitivity to these agents. However, the combination of cediranib and olaparib was effective in patients without BRCA1/2 mutations. We report here that cediranib confers sensitivity to olaparib by down-regulating HDR in tumor cells. This occurs partially as a result of cediranib inducing hypoxia, which suppresses expression of the HDR factors BRCA1/2 and RAD51 recombinase (RAD51). However, we also observed that cediranib has a direct effect on HDR independent of its ability to induce tumor hypoxia. This direct effect occurs through platelet-derived growth factor receptor (PDGFR) inhibition, activation of protein phosphatase 2A (PP2A), and E2F transcription factor 4 (E2F4)/RB transcriptional corepressor like 2 (RB2/p130)-mediated repression of BRCA1/2 and RAD51 gene expression. This down-regulation was seen in mouse tumor xenografts but not in mouse bone marrow, providing a therapeutic window for combining cediranib and olaparib in cancer therapy. Our work reveals a treatment strategy by which DNA repair can be manipulated in human tumors to induce synthetic lethality, broadening the potential therapeutic scope of cediranib based on its activity as a DNA repair inhibitor.
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Affiliation(s)
- Alanna R Kaplan
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA.,Department of Pathology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Susan E Gueble
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA.,Department of Pathology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Yanfeng Liu
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Sebastian Oeck
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Hoon Kim
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Zhong Yun
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA
| | - Peter M Glazer
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, USA. .,Department of Genetics, Yale University School of Medicine, New Haven, CT 06511, USA
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32
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Adaku N, Park HB, Spakowicz DJ, Tiwari MK, Strobel SA, Crawford JM, Rogers FA. A DNA Repair Inhibitor Isolated from an Ecuadorian Fungal Endophyte Exhibits Synthetic Lethality in PTEN-Deficient Glioblastoma. JOURNAL OF NATURAL PRODUCTS 2020; 83:1899-1908. [PMID: 32407116 DOI: 10.1021/acs.jnatprod.0c00012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
Disruption of the tumor suppressor PTEN, either at the protein or genomic level, plays an important role in human cancer development. The high frequency of PTEN deficiency reported across several cancer subtypes positions therapeutic approaches that exploit PTEN loss-of-function with the ability to significantly impact the treatment strategies of a large patient population. Here, we report that an endophytic fungus isolated from a medicinal plant produces an inhibitor of DNA double-strand-break repair. Furthermore, the novel alkaloid product, which we have named irrepairzepine (1), demonstrated synthetic lethal targeting in PTEN-deficient glioblastoma cells. Our results uncover a new therapeutic lead for PTEN-deficient cancers and an important molecular tool toward enhancing the efficacy of current cancer treatments.
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Affiliation(s)
- Nneoma Adaku
- Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520, United States
| | - Hyun Bong Park
- Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
- Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States
| | - Daniel J Spakowicz
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United States
| | - Meetu Kaushik Tiwari
- Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520, United States
| | - Scott A Strobel
- Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United States
| | - Jason M Crawford
- Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States
- Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States
- Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut 06536, United States
| | - Faye A Rogers
- Department of Therapeutic Radiology, Yale School of Medicine, New Haven, Connecticut 06520, United States
- Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut 06520, United States
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33
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Sulkowski PL, Oeck S, Dow J, Economos NG, Mirfakhraie L, Liu Y, Noronha K, Bao X, Li J, Shuch BM, King MC, Bindra RS, Glazer PM. Oncometabolites suppress DNA repair by disrupting local chromatin signalling. Nature 2020; 582:586-591. [PMID: 32494005 PMCID: PMC7319896 DOI: 10.1038/s41586-020-2363-0] [Citation(s) in RCA: 192] [Impact Index Per Article: 38.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Accepted: 04/28/2020] [Indexed: 01/06/2023]
Abstract
Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.
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Affiliation(s)
- Parker L Sulkowski
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
| | - Sebastian Oeck
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Department of Medical Oncology, University of Duisburg-Essen, Essen, Germany
| | - Jonathan Dow
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
| | - Nicholas G Economos
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
| | - Lily Mirfakhraie
- Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA
| | - Yanfeng Liu
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
| | - Katelyn Noronha
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA
| | - Xun Bao
- Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA
| | - Jing Li
- Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA
| | - Brian M Shuch
- Department of Urology, University of California at Los Angeles, Los Angeles, CA, USA
| | - Megan C King
- Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA
| | - Ranjit S Bindra
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
| | - Peter M Glazer
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
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34
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Soni A, Murmann-Konda T, Siemann-Loekes M, Pantelias GE, Iliakis G. Chromosome breaks generated by low doses of ionizing radiation in G 2-phase are processed exclusively by gene conversion. DNA Repair (Amst) 2020; 89:102828. [PMID: 32143127 DOI: 10.1016/j.dnarep.2020.102828] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Revised: 01/31/2020] [Accepted: 02/21/2020] [Indexed: 02/07/2023]
Abstract
Four repair pathways process DNA double-strand breaks (DSBs). Among these pathways the homologous recombination repair (HRR) subpathway of gene conversion (GC) affords error-free processing, but functions only in S- and G2-phases of the cell cycle. Classical non-homologous end-joining (c-NHEJ) operates throughout the cell cycle, but causes small deletions and translocations. Similar deficiencies in exaggerated form, combined with reduced efficiency, are associated with alternative end-joining (alt-EJ). Finally, single-strand annealing (SSA) causes large deletions and possibly translocations. Thus, processing of a DSB by any pathway, except GC, poses significant risks to the genome, making the mechanisms navigating pathway-engagement critical to genome stability. Logically, the cell ought to attempt engagement of the pathway ensuring preservation of the genome, while accommodating necessities generated by the types of DSBs induced. Thereby, inception of DNA end-resection will be key determinant for GC, SSA and alt-EJ engagement. We reported that during G2-phase, where all pathways are active, GC engages in the processing of almost 50 % of DSBs, at low DSB-loads in the genome, and that this contribution rapidly drops to nearly zero with increasing DSB-loads. At the transition between these two extremes, SSA and alt-EJ compensate, but at extremely high DSB-loads resection-dependent pathways are suppressed and c-NHEJ remains mainly active. We inquired whether in this processing framework all DSBs have similar fates. Here, we analyze in G2-phase the processing of a subset of DSBs defined by their ability to break chromosomes. Our results reveal an absolute requirement for GC in the processing of chromatid breaks at doses in the range of 1 Gy. Defects in c-NHEJ delay significantly the inception of processing by GC, but leave processing kinetics unchanged. These results delineate the essential role of GC in chromatid break repair before mitosis and classify DSBs that underpin this breakage as the exclusive substrate of GC.
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Affiliation(s)
- Aashish Soni
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany
| | - Tamara Murmann-Konda
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany
| | - Maria Siemann-Loekes
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany
| | - Gabriel E Pantelias
- Institute of Nuclear Technology and Radiation Protection, National Centre for Scientific Research "Demokritos,''Aghia Paraskevi Attikis, Athens, Greece
| | - George Iliakis
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.
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35
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Chen Y, Zhang H, Xu Z, Tang H, Geng A, Cai B, Su T, Shi J, Jiang C, Tian X, Seluanov A, Huang J, Wan X, Jiang Y, Gorbunova V, Mao Z. A PARP1-BRG1-SIRT1 axis promotes HR repair by reducing nucleosome density at DNA damage sites. Nucleic Acids Res 2019; 47:8563-8580. [PMID: 31291457 PMCID: PMC7145522 DOI: 10.1093/nar/gkz592] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2019] [Revised: 06/25/2019] [Accepted: 06/27/2019] [Indexed: 12/02/2022] Open
Abstract
Creating access to DNA double-strand break (DSB) sites in the chromatin context is an essential step during the repair process, but much remains to be determined about its regulatory mechanisms. Here, using a novel reporter cassette for simultaneous detection of homologous recombination (HR) and nonhomologous end joining (NHEJ) at the same chromosomal site, we report that the efficiency of HR but not NHEJ negatively correlates with nucleosome density. We demonstrate that PARP1 is required for HR by modulating nucleosome density at damage sites. Mechanistic studies indicate that the ATPase domain of BRG1 and the ZnF domain of SIRT1 interact with poly-ADP ribose (PAR) in response to DNA damage, and are responsible for bringing the two factors to broken DNA ends. At DNA damage sites, BRG1 and SIRT1 physically interact, whereupon SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, stimulating its ATPase activity to remodel chromatin and promote HR.
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Affiliation(s)
- Yu Chen
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Haiping Zhang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Zhu Xu
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Huanyin Tang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Anke Geng
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Bailian Cai
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Tao Su
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Jiejun Shi
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Cizhong Jiang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Xiao Tian
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
| | - Andrei Seluanov
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
| | - Jun Huang
- Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Xiaoping Wan
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Ying Jiang
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
| | - Vera Gorbunova
- Department of Biology, University of Rochester, Rochester, NY 14627, USA
| | - Zhiyong Mao
- Clinical and Translational Research Center of Shanghai First Maternity & Infant Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China
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36
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Iliakis G, Mladenov E, Mladenova V. Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer. Cancers (Basel) 2019; 11:cancers11111671. [PMID: 31661831 PMCID: PMC6896103 DOI: 10.3390/cancers11111671] [Citation(s) in RCA: 76] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Revised: 10/22/2019] [Accepted: 10/24/2019] [Indexed: 12/03/2022] Open
Abstract
Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.
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Affiliation(s)
- George Iliakis
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
| | - Emil Mladenov
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
| | - Veronika Mladenova
- Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
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37
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Ray S, Breuer G, DeVeaux M, Zelterman D, Bindra R, Sweasy JB. DNA polymerase beta participates in DNA End-joining. Nucleic Acids Res 2019; 46:242-255. [PMID: 29161447 PMCID: PMC5758893 DOI: 10.1093/nar/gkx1147] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2017] [Accepted: 10/31/2017] [Indexed: 12/21/2022] Open
Abstract
DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol μ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol β), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol β, fewer small deletions are observed. In addition, depletion of Pol β results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol β in functions in aNHEJ and provide mechanistic insight into its role in this process.
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Affiliation(s)
- Sreerupa Ray
- Department of Therapeutic Radiology, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA
| | - Gregory Breuer
- Department of Therapeutic Radiology, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA.,Department of Pathology, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA
| | - Michelle DeVeaux
- School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA
| | - Daniel Zelterman
- School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA
| | - Ranjit Bindra
- Department of Therapeutic Radiology, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA.,Department of Pathology, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA
| | - Joann B Sweasy
- Department of Therapeutic Radiology, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA.,Department of Genetics, School of Public Health, Yale University School of Medicine, New Haven, CT 06520-8034, USA
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Abstract
Assays that measure DNA damage and repair are critical in evaluating the extent to which therapeutic agents damage DNA and in identifying whether DNA repair can limit the toxicity of chemotherapy. The COMET assays described in this guide should help readers evaluate single and double-strand breaks cause by chemotherapeutic agents and also monitor the ability of the cells to repair such damage. The EJDR assay described is a valuable tool to assess the ability of drugs and DNA repair proteins to modulate DNA repair capacity. Finally, the immunofluorescence assay described should allow accurate assessments of DNA damage and the kinetics of repair as measured by Ɣ-H2AX foci. This procedure can also be used to mechanistically investigate the recruitment of specific DNA damage and repair proteins in CLL cells.
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Affiliation(s)
- Tzung-Huei Lai
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
| | - Deepa Sampath
- Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, USA.
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39
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Quantitative assessment of HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide-mediated DSB repair. DNA Repair (Amst) 2018; 70:67-71. [PMID: 30212742 DOI: 10.1016/j.dnarep.2018.09.002] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2018] [Revised: 09/01/2018] [Accepted: 09/04/2018] [Indexed: 11/22/2022]
Abstract
Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two major mechanisms for the repair of DNA double-strand breaks (DSBs) in eukaryotic cells. Previously, we designed an assay for detecting NHEJ activity by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, however, this approach cannot be used to predict the activity of HR repair. Hence, we developed a novel method that is capable of quantitatively measuring both HR and NHEJ activities via CRISPR/Cas9-induced oligodeoxynucleotide (ODN)-mediated DSB repair. In the present experimental procedures, the CRISPR/Cas9 plasmid was cotransfected with single-stranded ODN (ssODN) or blunt-ended double-stranded ODN (dsODN), both of which harbored a unique marker sequence. After the induction of site-specific DSBs by CRISPR/Cas9 system, the ssODN, functioned as the donor template for HR repair, could insert the marker sequence into the DSB sites, while the dsODN was embedded in the DSB sites through NHEJ pathway. Next, by means of PCR analysis using a specific primer for the marker sequence and the primers that flank the DSB sites, the relative amount of integrated marker sequence in the genomic DNA could be quantitatively determined. The correlation between the marker sequence abundance and the HR and NHEJ activities was confirmed by using the selective HR and NHEJ inhibitors. This accessible and rapid quantitative assay for HR and NHEJ activities might be useful for the future research of the DSB repair mechanisms.
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40
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Sulkowski PL, Sundaram RK, Oeck S, Corso CD, Liu Y, Noorbakhsh S, Niger M, Boeke M, Ueno D, Kalathil AN, Bao X, Li J, Shuch B, Bindra RS, Glazer PM. Krebs-cycle-deficient hereditary cancer syndromes are defined by defects in homologous-recombination DNA repair. Nat Genet 2018; 50:1086-1092. [PMID: 30013182 PMCID: PMC6072579 DOI: 10.1038/s41588-018-0170-4] [Citation(s) in RCA: 136] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Accepted: 06/01/2018] [Indexed: 01/27/2023]
Abstract
The hereditary cancer syndromes hereditary leiomyomatosis and renal cell cancer (HLRCC) and succinate dehydrogenase-related hereditary paraganglioma and pheochromocytoma (SDH PGL/PCC) are linked to germline loss-of-function mutations in genes encoding the Krebs cycle enzymes fumarate hydratase and succinate dehydrogenase, thus leading to elevated levels of fumarate and succinate, respectively1-3. Here, we report that fumarate and succinate both suppress the homologous recombination (HR) DNA-repair pathway required for the resolution of DNA double-strand breaks (DSBs) and for the maintenance of genomic integrity, thus rendering tumor cells vulnerable to synthetic-lethal targeting with poly(ADP)-ribose polymerase (PARP) inhibitors. These results identify HLRCC and SDH PGL/PCC as familial DNA-repair deficiency syndromes, providing a mechanistic basis to explain their cancer predisposition and suggesting a potentially therapeutic approach for advanced HLRCC and SDH PGL/PCC, both of which are incurable when metastatic.
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Affiliation(s)
- Parker L Sulkowski
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
| | - Ranjini K Sundaram
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
| | - Sebastian Oeck
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Essen, Germany
| | - Christopher D Corso
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Southeast Radiation Oncology and Levine Cancer Institute, Atrium Health, Charlotte, NC, USA
| | - Yanfeng Liu
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
| | - Seth Noorbakhsh
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
| | - Monica Niger
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA
- Medical Oncology Department, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
| | - Marta Boeke
- Department of Urology, Yale University School of Medicine, New Haven, CT, USA
| | - Daiki Ueno
- Department of Urology, Yale University School of Medicine, New Haven, CT, USA
| | | | - Xun Bao
- Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA
| | - Jing Li
- Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA
| | - Brian Shuch
- Department of Urology, Yale University School of Medicine, New Haven, CT, USA.
| | - Ranjit S Bindra
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
| | - Peter M Glazer
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA.
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
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41
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Khan AJ, Misenko SM, Thandoni A, Schiff D, Jhawar SR, Bunting SF, Haffty BG. VX-984 is a selective inhibitor of non-homologous end joining, with possible preferential activity in transformed cells. Oncotarget 2018; 9:25833-25841. [PMID: 29899825 PMCID: PMC5995231 DOI: 10.18632/oncotarget.25383] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2017] [Accepted: 04/25/2018] [Indexed: 12/17/2022] Open
Abstract
PURPOSE DNA double-strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). We demonstrate the selectivity of VX-984, a DNA-PK inhibitor, using assays not previously reported. EXPERIMENTAL DESIGN The class switch recombination assay (CSR) in primary B cells was used to measure efficiency of NHEJ. A cellular reporter assay (U2OS EJ-DR) was used to assess the efficiency of HR and NHEJ in cells treated with VX-984. Immunofluorescence assays (IF) evaluated γ-H2AX foci for DSB repair kinetics in human astrocytes and T98G glioma cells. Western blotting was used to evaluate phosphorylation of DNA-PKcs substrates. RESULTS We found a dose-dependent reduction in CSR efficiency with VX-984, and through the EJ-DR assay, dramatic dose-dependent increases in HR and mNHEJ. Immunofluorescence assays showed an inability of malignant cells to resolve γ-H2AX foci in the presence of VX-984. Radiation-induced phosphorylation of DNA-PK substrates was further reduced by treatment with VX-984. CONCLUSIONS VX-984 efficiently inhibits NHEJ, resulting in compensatory increases in alternative repair pathways, increases DSBs, and appears to affect transformed cells preferentially.
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Affiliation(s)
- Atif J. Khan
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY 10011, USA
- Department of Radiation Oncology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
| | - Sarah M. Misenko
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
| | - Aditya Thandoni
- Department of Radiation Oncology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
| | - Devora Schiff
- Department of Radiation Oncology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
| | - Sachin R. Jhawar
- Department of Radiation Oncology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
| | - Samuel F. Bunting
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
| | - Bruce G. Haffty
- Department of Radiation Oncology, Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
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42
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Misenko SM, Patel DS, Her J, Bunting SF. DNA repair and cell cycle checkpoint defects in a mouse model of 'BRCAness' are partially rescued by 53BP1 deletion. Cell Cycle 2018; 17:881-891. [PMID: 29620483 PMCID: PMC6056228 DOI: 10.1080/15384101.2018.1456295] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2017] [Revised: 04/13/2018] [Accepted: 03/17/2018] [Indexed: 10/17/2022] Open
Abstract
'BRCAness' is a term used to describe cancer cells that behave similarly to tumors with BRCA1 or BRCA2 mutations. The BRCAness phenotype is associated with hypersensitivity to chemotherapy agents including PARP inhibitors, which are a promising class of recently-licensed anti-cancer treatments. This hypersensitivity arises because of a deficiency in the homologous recombination (HR) pathway for DNA double-strand break repair. To gain further insight into how genetic modifiers of HR contribute to the BRCAness phenotype, we created a new mouse model of BRCAness by generating mice that are deficient in BLM helicase and the Exo1 exonuclease, which are involved in the early stages of HR. We find that cells lacking BLM and Exo1 exhibit a BRCAness phenotype, with diminished HR, and hypersensitivity to PARP inhibitors. We further tested how 53BP1, an important regulator of HR, affects repair efficiency in our BRCAness model. We find that deletion of 53BP1 can relieve several of the repair deficiencies observed in cells lacking BLM and Exo1, just as it does in cells lacking BRCA1. These results substantiate the importance of BRCAness as a concept for classification of cancer cases, and further clarify the role of 53BP1 in regulation of DNA repair pathway choice in mammalian cells.
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Affiliation(s)
- Sarah M. Misenko
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
| | - Dharm S. Patel
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
| | - Joonyoung Her
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
| | - Samuel F. Bunting
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, USA
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43
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Eccles LJ, Bell AC, Powell SN. Inhibition of non-homologous end joining in Fanconi Anemia cells results in rescue of survival after interstrand crosslinks but sensitization to replication associated double-strand breaks. DNA Repair (Amst) 2018; 64:1-9. [PMID: 29459202 DOI: 10.1016/j.dnarep.2018.02.003] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Revised: 10/16/2017] [Accepted: 02/07/2018] [Indexed: 12/22/2022]
Abstract
When Fanconi Anemia (FA) proteins were depleted in human U2OS cells with integrated DNA repair reporters, we observed decreases in homologous recombination (HR), decreases in mutagenic non-homologous end joining (m-NHEJ) and increases in canonical NHEJ, which was independently confirmed by measuring V(D)J recombination. Furthermore, depletion of FA proteins resulted in reduced HR protein foci and increased NHEJ protein recruitment to replication-associated DSBs, consistent with our observation that the use of canonical NHEJ increases after depletion of FA proteins in cycling cells. FA-depleted cells and FA-mutant cells were exquisitely sensitive to a DNA-PKcs inhibitor (DNA-PKi) after sustaining replication-associated double strand breaks (DSBs). By contrast, after DNA interstrand crosslinks, DNA-PKi resulted in increased survival in FA-deficient cells, implying that NHEJ is contributing to lethality after crosslink repair. Our results suggest FA proteins inhibit NHEJ, since repair intermediates from crosslinks are rendered lethal by NHEJ. The implication is that bone marrow failure in FA could be triggered by naturally occurring DNA crosslinks, and DNA-PK inhibitors would be protective. Since some sporadic cancers have been shown to have deficiencies in the FA-pathway, these tumors should be vulnerable to NHEJ inhibitors with replication stress, but not with crosslinking agents, which could be tested in future clinical trials.
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Affiliation(s)
- Laura J Eccles
- Molecular Biology Program and Radiation Oncology Department, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 USA
| | - Andrew C Bell
- Molecular Biology Program and Radiation Oncology Department, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 USA
| | - Simon N Powell
- Molecular Biology Program and Radiation Oncology Department, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065 USA.
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44
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Sulkowski PL, Corso CD, Robinson ND, Scanlon SE, Purshouse KR, Bai H, Liu Y, Sundaram RK, Hegan DC, Fons NR, Breuer GA, Song Y, Mishra-Gorur K, De Feyter HM, de Graaf RA, Surovtseva YV, Kachman M, Halene S, Günel M, Glazer PM, Bindra RS. 2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity. Sci Transl Med 2018; 9:9/375/eaal2463. [PMID: 28148839 DOI: 10.1126/scitranslmed.aal2463] [Citation(s) in RCA: 408] [Impact Index Per Article: 58.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2016] [Revised: 12/08/2016] [Accepted: 12/23/2016] [Indexed: 12/12/2022]
Abstract
2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.
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Affiliation(s)
- Parker L Sulkowski
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA.,Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Christopher D Corso
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Nathaniel D Robinson
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Susan E Scanlon
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA.,Department of Experimental Pathology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Karin R Purshouse
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Hanwen Bai
- Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Yanfeng Liu
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Ranjini K Sundaram
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Denise C Hegan
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Nathan R Fons
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA.,Department of Experimental Pathology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Gregory A Breuer
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA.,Department of Experimental Pathology, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Yuanbin Song
- Section of Hematology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Ketu Mishra-Gorur
- Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Henk M De Feyter
- Department of Radiology and Biomedical Imaging, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Robin A de Graaf
- Department of Radiology and Biomedical Imaging, Yale University School of Medicine, New Haven, CT 06520, USA
| | | | - Maureen Kachman
- Michigan Regional Comprehensive Metabolomics Resource Core, National Institute of Environmental Health Sciences (NIEHS) Children's Health Exposure Analysis Resource for Metabolomics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Stephanie Halene
- Section of Hematology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Murat Günel
- Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA.,Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Peter M Glazer
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA. .,Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
| | - Ranjit S Bindra
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06520, USA. .,Department of Experimental Pathology, Yale University School of Medicine, New Haven, CT 06520, USA
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45
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Processing-Challenges Generated by Clusters of DNA Double-Strand Breaks Underpin Increased Effectiveness of High-LET Radiation and Chromothripsis. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2018; 1044:149-168. [DOI: 10.1007/978-981-13-0593-1_10] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
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46
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Patel DS, Misenko SM, Her J, Bunting SF. BLM helicase regulates DNA repair by counteracting RAD51 loading at DNA double-strand break sites. J Cell Biol 2017; 216:3521-3534. [PMID: 28912125 PMCID: PMC5674892 DOI: 10.1083/jcb.201703144] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Revised: 06/28/2017] [Accepted: 08/04/2017] [Indexed: 11/22/2022] Open
Abstract
The BLM gene product, BLM, is a RECQ helicase that is involved in DNA replication and repair of DNA double-strand breaks by the homologous recombination (HR) pathway. During HR, BLM has both pro- and anti-recombinogenic activities, either of which may contribute to maintenance of genomic integrity. We find that in cells expressing a mutant version of BRCA1, an essential HR factor, ablation of BLM rescues genomic integrity and cell survival in the presence of DNA double-strand breaks. Improved genomic integrity in these cells is linked to a substantial increase in the stability of RAD51 at DNA double-strand break sites and in the overall efficiency of HR. Ablation of BLM also rescues RAD51 foci and HR in cells lacking BRCA2 or XRCC2. These results indicate that the anti-recombinase activity of BLM is of general importance for normal retention of RAD51 at DNA break sites and regulation of HR.
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Affiliation(s)
- Dharm S Patel
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ
| | - Sarah M Misenko
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ
| | - Joonyoung Her
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ
| | - Samuel F Bunting
- Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ
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47
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Chand SN, Zarei M, Schiewer MJ, Kamath AR, Romeo C, Lal S, Cozzitorto JA, Nevler A, Scolaro L, Londin E, Jiang W, Meisner-Kober N, Pishvaian MJ, Knudsen KE, Yeo CJ, Pascal JM, Winter JM, Brody JR. Posttranscriptional Regulation of PARG mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors. Cancer Res 2017; 77:5011-5025. [PMID: 28687616 PMCID: PMC5663502 DOI: 10.1158/0008-5472.can-16-2704] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2016] [Revised: 12/23/2016] [Accepted: 06/29/2017] [Indexed: 01/08/2023]
Abstract
The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9-mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 3' untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR-PARG axis as an opportunity to enhance PARPi-based therapies. Cancer Res; 77(18); 5011-25. ©2017 AACR.
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MESH Headings
- Animals
- Apoptosis
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Carcinoma, Pancreatic Ductal/genetics
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/pathology
- Cell Nucleus/drug effects
- Cell Nucleus/genetics
- Cell Proliferation
- DNA Damage/drug effects
- DNA Damage/genetics
- DNA Repair/drug effects
- DNA Repair/genetics
- Drug Resistance, Neoplasm/genetics
- ELAV-Like Protein 1/antagonists & inhibitors
- ELAV-Like Protein 1/genetics
- ELAV-Like Protein 1/metabolism
- Female
- Glycoside Hydrolases/genetics
- Humans
- Mice
- Mice, Nude
- Pancreatic Neoplasms/genetics
- Pancreatic Neoplasms/metabolism
- Pancreatic Neoplasms/pathology
- Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
- Poly(ADP-ribose) Polymerases/chemistry
- RNA Processing, Post-Transcriptional
- RNA, Messenger/genetics
- Tumor Cells, Cultured
- Up-Regulation
- Xenograft Model Antitumor Assays
- Pancreatic Neoplasms
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Affiliation(s)
- Saswati N Chand
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Mahsa Zarei
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Matthew J Schiewer
- Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
- Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Akshay R Kamath
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Carmella Romeo
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Shruti Lal
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Joseph A Cozzitorto
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Avinoam Nevler
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Laura Scolaro
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Eric Londin
- Computational Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania
- Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Wei Jiang
- Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania
| | | | - Michael J Pishvaian
- Division of Hematology and Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC
| | - Karen E Knudsen
- Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania
- Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Charles J Yeo
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - John M Pascal
- Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada
| | - Jordan M Winter
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Jonathan R Brody
- Department of Surgery, The Jefferson Pancreas, Biliary and Related Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania.
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Fujii N. Potential Strategies to Target Protein-Protein Interactions in the DNA Damage Response and Repair Pathways. J Med Chem 2017; 60:9932-9959. [PMID: 28654754 DOI: 10.1021/acs.jmedchem.7b00358] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This review article discusses some insights about generating novel mechanistic inhibitors of the DNA damage response and repair (DDR) pathways by focusing on protein-protein interactions (PPIs) of the key DDR components. General requirements for PPI strategies, such as selecting the target PPI site on the basis of its functionality, are discussed first. Next, on the basis of functional rationale and biochemical feasibility to identify a PPI inhibitor, 26 PPIs in DDR pathways (BER, MMR, NER, NHEJ, HR, TLS, and ICL repair) are specifically discussed for inhibitor discovery to benefit cancer therapies using a DNA-damaging agent.
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Affiliation(s)
- Naoaki Fujii
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital , 262 Danny Thomas Place, MS1000, Memphis, Tennessee 38105, United States
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49
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King AR, Corso CD, Chen EM, Song E, Bongiorni P, Chen Z, Sundaram RK, Bindra RS, Saltzman WM. Local DNA Repair Inhibition for Sustained Radiosensitization of High-Grade Gliomas. Mol Cancer Ther 2017; 16:1456-1469. [PMID: 28566437 DOI: 10.1158/1535-7163.mct-16-0788] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2016] [Revised: 04/14/2017] [Accepted: 05/16/2017] [Indexed: 11/16/2022]
Abstract
High-grade gliomas, such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), are characterized by an aggressive phenotype with nearly universal local disease progression despite multimodal treatment, which typically includes chemotherapy, radiotherapy, and possibly surgery. Radiosensitizers that have improved the effects of radiotherapy for extracranial tumors have been ineffective for the treatment of GBM and DIPG, in part due to poor blood-brain barrier penetration and rapid intracranial clearance of small molecules. Here, we demonstrate that nanoparticles can provide sustained drug release and minimal toxicity. When administered locally, these nanoparticles conferred radiosensitization in vitro and improved survival in rats with intracranial gliomas when delivered concurrently with a 5-day course of fractionated radiotherapy. Compared with previous work using locally delivered radiosensitizers and cranial radiation, our approach, based on the rational selection of agents and a clinically relevant radiation dosing schedule, produces the strongest synergistic effects between chemo- and radiotherapy approaches to the treatment of high-grade gliomas. Mol Cancer Ther; 16(8); 1456-69. ©2017 AACR.
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Affiliation(s)
- Amanda R King
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Christopher D Corso
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut
| | - Evan M Chen
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Eric Song
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut
| | - Paul Bongiorni
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut
| | - Zhe Chen
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut
| | - Ranjini K Sundaram
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut
| | - Ranjit S Bindra
- Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut. .,Department of Experimental Pathology, Yale University School of Medicine, New Haven, Connecticut
| | - W Mark Saltzman
- Department of Biomedical Engineering, Yale University, New Haven, Connecticut.
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50
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Differences in the recruitment of DNA repair proteins at subtelomeric and interstitial I-SceI endonuclease-induced DNA double-strand breaks. DNA Repair (Amst) 2016; 49:1-8. [PMID: 27842255 DOI: 10.1016/j.dnarep.2016.10.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Revised: 09/21/2016] [Accepted: 10/26/2016] [Indexed: 12/28/2022]
Abstract
Telomeres are nucleoprotein structures that are required to protect chromosome ends. Dysfunctional telomeres are recognized as DNA double-strand breaks (DSBs), and elicit the activation of a DNA damage response (DDR). We have previously reported that DSBs near telomeres are poorly repaired, resulting in a high frequency of large deletions and gross chromosome rearrangements (GCRs). Our previous genetic studies have demonstrated that this sensitivity of telomeric regions to DSBs is a result of excessive processing. In the current study, we have further investigated the sensitivity of telomeric regions to DSBs through the analysis of repair proteins associated with DSBs at interstitial and telomeric sites. Following the inducible expression of I-SceI endonuclease, chromatin immunoprecipitation (ChIP) and real-time quantitative PCR were used to compare the recruitment of repair proteins at I-SceI-induced DSBs at interstitial and subtelomeric sites. We observed that proteins that are specifically associated with processing of DSBs during homologous recombination repair, RAD51, BRCA1, and CtIP, are present at a much greater abundance at subtelomeric DSBs. In contrast, Ku70, which is specifically involved in classical nonhomologous end joining, showed no difference at interstitial and subtelomeric DSBs. Importantly, ATM was lower in abundance at subtelomeric DSBs, while ATR was in greater abundance at subtelomeric DSBs, consistent with the accumulation of processed DSBs near telomeres, since processing is accompanied by a transition from ATM to ATR binding. Combined, our results suggest that excessive processing is responsible for the increased frequency of large deletions and GCRs at DSBs near telomeres.
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