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1. Clonal hematopoiesis driven by chromosome 1q/MDM4 trisomy defines a canonical route toward leukemia in Fanconi anemia

Author

Marie Sebert, Stéphanie Gachet, Thierry Leblanc, Alix Rousseau, Olivier Bluteau, Rathana Kim, Raouf Ben Abdelali, Flore Sicre de Fontbrune, Loïc Maillard, Carèle Fedronie, Valentine Murigneux, Léa Bellenger, Naira Naouar, Samuel Quentin, Lucie Hernandez, Nadia Vasquez, Mélanie Da Costa, Pedro H. Prata, Lise Larcher, Marie de Tersant, Matthieu Duchmann, Anna Raimbault, Franck Trimoreau, Odile Fenneteau, Wendy Cuccuini, Nathalie Gachard, Nathalie Auger, Giulia Tueur, Maud Blanluet, Claude Gazin, Michèle Souyri, Francina Langa Vives, Aaron Mendez-Bermudez, Hélène Lapillonne, Etienne Lengline, Emmanuel Raffoux, Pierre Fenaux, Lionel Adès, Edouard Forcade, Charlotte Jubert, Carine Domenech, Marion Strullu, Bénédicte Bruno, Nimrod Buchbinder, Caroline Thomas, Arnaud Petit, Guy Leverger, Gérard Michel, Marina Cavazzana, Eliane Gluckman, Yves Bertrand, Nicolas Boissel, André Baruchel, Jean-Hugues Dalle, Emmanuelle Clappier, Eric Gilson, Ludovic Deriano, Sylvie Chevret, François Sigaux, Gérard Socié, Dominique Stoppa-Lyonnet, Hugues de Thé, Christophe Antoniewski, Dominique Bluteau, Régis Peffault de Latour, Jean Soulier, Institut de Recherche Saint-Louis - Hématologie Immunologie Oncologie (Département de recherche de l’UFR de médecine, ex- Institut Universitaire Hématologie-IUH) (IRSL), Université Paris Cité (UPCité), Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Génomes, biologie cellulaire et thérapeutiques (GenCellDi (U944 / UMR7212)), Collège de France (CdF (institution))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), AP-HP Hôpital universitaire Robert-Debré [Paris], Recherche clinique appliquée à l'hématologie (URP_3518), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), CHU Limoges, Institut Gustave Roussy (IGR), Unité de génétique et biologie des cancers (U830), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de Recherche en Génomique Humaine (CNRGH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Hématopoïèse normale et pathologique : émergence, environnement et recherche translationnelle [Paris] ((UMR_S1131 / U1131)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Centre d'Ingénierie génétique murine - Mouse Genetics Engineering Center (CIGM), Institut Pasteur [Paris] (IP), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), CHU Nice [Cimiez], Hôpital Cimiez [Nice] (CHU), CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Hôpitaux universitaires Est parisien [AP-HP], CHU Bordeaux [Bordeaux], Hospices Civils de Lyon (HCL), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), CHU Rouen, Normandie Université (NU), Centre hospitalier universitaire de Nantes (CHU Nantes), Centre d'études et de recherche sur les services de santé et la qualité de vie (CEReSS), Aix Marseille Université (AMU), Hôpital de la Timone [CHU - APHM] (TIMONE), CIC NECKER BT (CIC 1416), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Collège de France (CdF (institution)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL), This study received support from the European Research Council (ERC) Consolidator Grant to J.S. (CEVAL-311660), the FP7 Eurofancolen program (HEALTH-F5-2012-305421), the ANR program PACRI (Projet alliance parisienne des instituts de recherche en cancérologie), the CONECT-AML (Collaborative Network for Children and Teenagers with Acute Myeloid Leukemia) program supported by a grant from the Institut National du Cancer (INCa), Fondation ARC, Ligue nationale contre le cancer, and Laurette Fugain (INCa-ARC-LIGUE_11905) to J.S. and C.A., and the Association Française pour la Maladie de Fanconi (AFMF) grants 'Histoire naturelle de la maladie de Fanconi' to R.P.L. and J.S., 'Modélisation de la transformation leucémique dans la maladie de Fanconi' to D.B., and 'Cribles fonctionnels à haut débit de gènes modificateurs de la maladie de Fanconi' to C.G. M.S. was supported by the AVIESAN-INCa Program 'Formation à la Recherche Translationnelle,' and A.R. by a grant from the Fondation ARC. The work in E.G.’s lab is supported by Fondation ARC and ANR Telochrom. The work in L.D.’s lab is supported by INCa (PLBIO16-181) and ERC (310917)., ANR-11-PHUC-0002,PACRI,Alliance Parisienne des Instituts de Recherche en Cancérologie(2011), European Project: 311660,EC:FP7:ERC,ERC-2012-StG_20111109,CEVAL(2013), and European Project: 305421,EC:FP7:HEALTH,FP7-HEALTH-2012-INNOVATION-1,EUROFANCOLEN(2013)

Subjects
MDM4, Fanconi anemia, precision medicine, [SDV]Life Sciences [q-bio], Genetics, leukemia, clonal hematopoiesis, Molecular Medicine, Cell Biology, TP53, mutational signature, genomic instability, BRCA2
Abstract

International audience; Fanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects.

Published
2023

2. V(D)J Recombination: Orchestrating Diversity Without Damage

Author

C. Lescale, L. Deriano, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Ralph A. Bradshaw, Gerald W. Hart, and Philip D. Stahl

Subjects
Genome instability, Genetics, Genomic instability, DNA repair, [SDV]Life Sciences [q-bio], V(D)J recombination, Biology, DNA damage response, Antigen receptor diversification, Recombination activating genes (RAGs) and V(D)J recombination, Immune system, Double-strand break, Leukemia and lymphoma, Immunodeficiency, Neoplastic transformation, Epigenetics, Lymphocyte, Gene, Recombination, NHEJ, Cancer
Abstract

International audience; V(D)J recombination assembles immunoglobulin and T-cell receptor genes from the preexisting variable (V), diversity (D), and joining (J) gene segments by a cut and paste mechanism. While this receptor diversification strategy enables efficient immune responses against pathogens, it also poses a constant threat to the genome integrity. Tight regulation of this process at multiple levels is therefore critical to prevent genomic instability and neoplastic transformation. In this article, we discuss the temporal and spatial regulation of V(D)J recombination, in addition to the molecular mechanisms that regulate this process. Finally, we discuss the implication of V(D)J recombination in malignancies of the immune system. “RAGs are the end-point of the strategies developed through evolution to achieve the anticipatory “Promethean” recognition of all universal molecular forms”. – A.A. de Freitas, Tractus Immuno-Logicus.

Published
2023

3. SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination

Author

Estelle Vincendeau, Wenming Wei, Xuefei Zhang, Cyril Planchais, Wei Yu, Hélène Lenden-Hasse, Thomas Cokelaer, Juliana Pipoli da Fonseca, Hugo Mouquet, David J. Adams, Frederick W. Alt, Stephen P. Jackson, Gabriel Balmus, Chloé Lescale, Ludovic Deriano, Vincendeau, Estelle [0000-0002-0231-1609], Zhang, Xuefei [0000-0002-1873-6679], Yu, Wei [0000-0002-1777-5254], Cokelaer, Thomas [0000-0001-6286-1138], Adams, David J [0000-0001-9490-0306], Jackson, Stephen P [0000-0001-9317-7937], Balmus, Gabriel [0000-0003-2872-4468], Lescale, Chloé [0000-0003-0622-1149], Deriano, Ludovic [0000-0002-9673-9525], Apollo - University of Cambridge Repository, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Howard Hughes Medical Institute (HHMI), Peking University [Beijing], Immunologie humorale - Humoral Immunology, Biomics (plateforme technologique), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, The Wellcome Trust Sanger Institute [Cambridge], University of Cambridge [UK] (CAM), The Deriano laboratory is funded by Institut Pasteur, Institut National de la Santé et de la Recherche Médicale (Inserm), Wordwide Cancer Research (grant # 19-0333), Ligue Nationale Contre le Cancer (Equipe labellisée 2019), and Institut National du Cancer (INCa, grant # PLBIO19-122). E.V. is supported by the French Ministry of Higher Education, Research, and Innovation (Doctoral School Bio Sorbonne Paris Cité) and by the Fondation pour la Recherche Médicale. W.W. is part of the Pasteur–Paris University International Ph.D. program and received funding from the CNBG company, China. The Balmus laboratory at the UK DRI is funded by the Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. Research in the Jackson laboratory is funded by Cancer Research UK (CRUK) program grant DRCPGM\100005 and core grant C6946/A24843, Wellcome Investigator award 206388/Z/17/Z and core grant 203144, and ERC Synergy award 855741. S.P.J. receives a salary from the University of Cambridge. F.W.A is an investigator of the Howard Hughes Medical Institute. H.M. received core grants from the Institut Pasteur, the INSERM and the Milieu Intérieur Program (ANR-10-LABX-69-01)., We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line (Allele: Shld1em1(IMPC)Wtsi), and INFRAFRONTIER/EMMA. We thank the Institut Pasteur animal facility, specifically Quentin Mille, Jeromine Rohard Blanco, and Franck Bourgade for mouse weighing. We thank Jean-Pierre de Villartay for providing the CD21-Cre3a line and Klaus Rajewsky and MGC for permission to use the line. We thank Michela Di Virgilio for providing 53BP1 knockout animals and Junjie Chen for permission to use them. We thank members of the Deriano lab for their discussion and technical help. We thank Bernardo Reina-San-Martin for the discussion and advices. We thank Christopher Carnie for the discussion on the design of SHLD1 mutant sites., ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), and Jackson, Stephen [0000-0001-9317-7937]

Subjects
DNA End-Joining Repair, MESH: V(D)J Recombination, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, General Physics and Astronomy, 13/106, 45/23, 631/250/2152/2497, 631/250/2152/2498, General Biochemistry, Genetics and Molecular Biology, 13/1, 13/21, DNA Breaks, Double-Stranded, 631/337/1427/2122, Multidisciplinary, 45/71, 45, fungi, article, 45/77, General Chemistry, MESH: DNA End-Joining Repair, Immunoglobulin Class Switching, V(D)J Recombination, 49, DNA-Binding Proteins, 13/31, enzymes and coenzymes (carbohydrates), MESH: Immunoglobulin Class Switching, 631/337/149, MESH: DNA-Binding Proteins
Abstract

Funder: Ligue Nationale Contre le Cancer, SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

Published
2022
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4. The Sec61 translocon is a therapeutic vulnerability in multiple myeloma

Author

Antoine Domenger, Caroline Choisy, Ludivine Baron, Véronique Mayau, Emeline Perthame, Ludovic Deriano, Bertrand Arnulf, Jean‐Christophe Bories, Gilles Dadaglio, Caroline Demangel, Immunobiologie de l'Infection - Immunobiology of Infection, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), This work was supported by the Cancéropôle Ile de France (CD and J-CB, Emergence 2019), the Fondation Française pour la Recherche contre le Myélome et les Gammapathies (CD, FFRMG), and the Sanofi iAwards program (CD, 2020). CD also acknowledges core support from Institut Pasteur and INSERM (U1221). LB was recipient of a Roux-Cantarini post-doctoral fellowship (Institut Pasteur). AD is a BioSPC-Université de Paris PhD student, recipient of a doctoral fellowship from the Ministère français de l’Enseignement Supérieur, de la Recherche et de l’Innovation., and Kop, Marie-Luce

Subjects
Sec61 translocon, Proteasome Endopeptidase Complex, [SDV.IMM] Life Sciences [q-bio]/Immunology, proteostatic stress, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Endoplasmic Reticulum Stress, [SDV] Life Sciences [q-bio], Mice, Protein Transport, Cell Line, Tumor, [SDV.IMM]Life Sciences [q-bio]/Immunology, Animals, Humans, Molecular Medicine, Multiple Myeloma, Proteasome Inhibitors, SEC Translocation Channels
Abstract

International audience; Multiple myeloma (MM) is an incurable malignancy characterized by the uncontrolled expansion of plasma cells in the bone marrow. While proteasome inhibitors like bortezomib efficiently halt MM progression, drug resistance inevitably develop, and novel therapeutic approaches are needed. Here, we used a recently discovered Sec61 inhibitor, mycolactone, to assess the interest of disrupting MM proteostasis via protein translocation blockade. In human MM cell lines, mycolactone caused rapid defects in secretion of immunoglobulins and expression of pro-survival interleukin (IL)-6 receptor and CD40, whose activation stimulates IL-6 production. Mycolactone also triggered pro-apoptotic endoplasmic reticulum stress responses synergizing with bortezomib for induction of MM cell death and overriding acquired resistance to the proteasome inhibitor. Notably, the mycolactone-bortezomib combination rapidly killed patient-derived MM cells ex vivo, but not normal mononuclear cells. In immunodeficient mice engrafted with MM cells, it demonstrated superior therapeutic efficacy over single drug treatments, without inducing toxic side effects. Collectively, these findings establish Sec61 blockers as novel anti-MM agents and reveal the interest of targeting both the translocon and the proteasome in proteostasis-addicted tumors.

Published
2022

5. The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

Author

Libri, Alice, Marton, Timea, Deriano, Ludovic, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), and The Deriano laboratory is funded by Institut Pasteur, Institut National de la Santé et de la Recherche Médicale (INSERM), Wordwide Cancer Research (grant # 19–0333), Ligue Nationale Contre le Cancer (Equipe labellis ée 2019) and Institut National du Cancer (INCa, grant # PLBIO19-122). AL received funding from Université de Paris, Sorbonne Paris Cité.

Subjects
DNA end resection, [SDV]Life Sciences [q-bio], fungi, DNA double-strand break repair pathway choice, Review, QH426-470, V(D)J recombination, non-homologous end-joining, enzymes and coenzymes (carbohydrates), homology-directed repair, DNA double-strand break, Genetics, Molecular Medicine, Genetics (clinical)
Abstract

International audience; DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

Published
2022
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6. Fam72a enforces error-prone DNA repair during antibody diversification

Author

Michel Cogné, Brice Laffleur, Ophelie Martin, Bernardo Reina-San-Martin, Mélanie Rogier, Isabelle Robert, Jacques Moritz, Katia Capitani, Arthur Abello, Eric Pinaud, Florence Jouan, Ludovic Deriano, Anne-Sophie Thomas-Claudepierre, Vincent Heyer, Evi Soutoglou, Karin Tarte, Chloé Lescale, Morgane Thomas, Silvestro G. Conticello, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Physiopathologie du système immunitaire (Inserm U1223), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], University of Sussex, Università degli Studi di Siena = University of Siena (UNISI), Contrôle de la Réponse Immune B et des Lymphoproliférations (CRIBL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Génomique, Environnement, Immunité, Santé, Thérapeutique (GEIST), Université de Limoges (UNILIM)-Université de Limoges (UNILIM), Microenvironment, Cell Differentiation, Immunology and Cancer (MICMAC), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), This study was supported by grants from the Fondation Recherche Médicale (Equipe FRM EQU201903007818 to B.R.-S.-M.), Institut National du Cancer (INCa_13852 to L.D. and B.R.-S.-M.), Fondation ARC (ARCPJA32020060002061 to B.R.-S.-M.), the Ligue Nationale contre le Cancer (Equipe labellisée to L.D.) and by the grant ANR-10-LABX-0030-INRT, a French state fund managed by the Agence Nationale de la Recherche under the program Investissements d’Avenir labelled ANR-10-IDEX-0002-02., ANR-10-LABX-0030,INRT,Integrative Biology : Nuclear dynamics- Regenerative medicine - Translational medicine(2010), ANR-10-IDEX-0002,UNISTRA,Par-delà les frontières, l'Université de Strasbourg(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Institut Génomique, Environnement, Immunité, Santé, Thérapeutique (GEIST), Université de Limoges (UNILIM)-Université de Limoges (UNILIM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), and Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )

Subjects
Male, DNA damage, DNA repair, Somatic hypermutation, DNA Mismatch Repair, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, Cytidine deamination, Animals, Uracil, 030304 developmental biology, 0303 health sciences, B-Lymphocytes, Multidisciplinary, Genome, Mutagenesis, Immunoglobulin Class Switching, 3. Good health, Cell biology, Immunoglobulin Switch Region, Up-Regulation, chemistry, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, 030220 oncology & carcinogenesis, Uracil-DNA glycosylase, Mutation, DNA mismatch repair, Female, Somatic Hypermutation, Immunoglobulin, CRISPR-Cas Systems, DNA
Abstract

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1–3. Although uracils arising in DNA are accurately repaired1–4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1–3. Here we performed a genome-wide CRISPR–Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A–UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a−/− B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a−/− B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations. FAM72A interacts with UNG2 to regulate the balance between error-prone and error-free DNA repair.

Published
2021

7. MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair

Author

de Krijger, Inge, Föhr, Bastian, Pérez, Santiago Hernández, Vincendeau, Estelle, Serrat, Judit, Thouin, Alexander Marc, Susvirkar, Vivek, Lescale, Chloé, Paniagua, Inés, Hoekman, Liesbeth, Kaur, Simranjeet, Altelaar, Maarten, Deriano, Ludovic, Faesen, Alex C, Jacobs, Jacqueline J L, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie du système immunitaire (Inserm U1223), Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University [Utrecht], This work was supported by project grant 10999/2017-1 from the Dutch Cancer Society (KWF) to J.J.L.J., by the Institut Pasteur to L.D., by the Institut National Du Cancer (INCA_13852) to L.D., by the Ligue Nationale Contre le Cancer (Equipe labellisée 2019) to L.D. The Proteomics Facility of the Netherlands Cancer Institute was supported by the X-omics Initiative, partially funded by the Dutch Research Council (NWO)(project 184.034.019). E.V. received funding from Université de Paris, Sorbonne Paris Cité. A.C.F. is grateful for generous core funding by the Max Planck Society., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC), Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], and Lescale, Chloé

Subjects
DNA Repair, Chemistry(all), ATPase, MESH: DNA Breaks, Double-Stranded, Gene Expression, General Physics and Astronomy, Cell Cycle Proteins, [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biochemistry, Piperazines, MESH: Recombinant Proteins, Mice, chemistry.chemical_compound, 0302 clinical medicine, DNA Breaks, Double-Stranded, MESH: Animals, 0303 health sciences, Multidisciplinary, biology, Chemistry, MESH: Protein Multimerization, DNA damage and repair, MESH: DNA, Recombinant Proteins, 3. Good health, Cell biology, DNA-Binding Proteins, MESH: ATPases Associated with Diverse Cellular Activities, MESH: HEK293 Cells, Mad2 Proteins, MESH: Phthalazines, MESH: Mad2 Proteins, Protein Binding, MESH: Cell Line, Tumor, MESH: Gene Expression, DNA repair, Science, Physics and Astronomy(all), Article, Chromosomes, General Biochemistry, Genetics and Molecular Biology, Cell Line, 03 medical and health sciences, MESH: Cell Cycle Proteins, Cell Line, Tumor, Animals, Humans, MESH: Protein Binding, Protein Interaction Domains and Motifs, MESH: Mice, 030304 developmental biology, MESH: Protein Interaction Domains and Motifs, [SDV.GEN]Life Sciences [q-bio]/Genetics, Binding Sites, MESH: Humans, TRIP13, Biochemistry, Genetics and Molecular Biology(all), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, General Chemistry, DNA Repair Pathway, Fibroblasts, MESH: Cell Line, HEK293 Cells, MESH: Binding Sites, MESH: Cisplatin, MESH: Fibroblasts, MESH: HeLa Cells, biology.protein, ATPases Associated with Diverse Cellular Activities, Phthalazines, Cisplatin, Protein Multimerization, 030217 neurology & neurosurgery, Function (biology), MESH: DNA-Binding Proteins, HeLa Cells, Genetics and Molecular Biology(all)
Abstract

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity., MAD2L2 — a member of the shieldin complex — is known to play important roles in DNA repair. Here the authors demonstrate how MAD2L2 dimerization mediated through SHLD2 participates in shieldin assembly and function.

Published
2021

8. Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ

Author

Loelia Babin, Caroline Demangel, Hélène Lenden-Hasse, Ludovic Deriano, Ludivine Baron, Erika Brunet, José Yélamos, Marie Bedora-Faure, Wei Yu, Chloé Lescale, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Genome dynamics in the immune system (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunobiologie de l'Infection - Immunobiology of Infection, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), IMIM-Hospital del Mar, Generalitat de Catalunya, This project is funded by the Institut Pasteur (L.D. and C.D. labs), the Institut National du Cancer (INCa Grant # PLBIO16-181 to L.D. and E.B. labs), the Ligue Nationale Contre le Cancer (Équipe Labellisée 2019 L.D. lab and Équipe Labellisée 2017 and 2020 E.B. lab) the Cancéropôle IdF-INCa (Emergence 2016 grant to L.D. and C.D.), the Spanish Ministerio de Economía, Industria y Competitividad (Grant SAF2017-83565-R to J.Y.) as well as by the Fundación Científica de la Asociación Española Contra el Cáncer (AECC) (Grant PROYEI6018YÉLA to J.Y.)., We thank the Institut Pasteur genomics and cytometry platforms for help with sequencing and cell sorting, Frederick Alt and members of his lab for tremendous help with LAM-HTGTS experiments, Barry Sleckman for providing the RAG2-Thy1.1 complementation vector, Joy Bianchi for providing the Rag2−/− p53−/− v-Abl pro-B cell lines, Carine Giovannangeli and Anne de Cian for providing the Cas9 protein and Thomas Mercher for helpful discussions and suggestions., Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Demangel, Caroline, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]

Subjects
0301 basic medicine, Genome instability, DNA End-Joining Repair, MESH: DNA Breaks, Double-Stranded, Poly (ADP-Ribose) Polymerase-1, General Physics and Astronomy, MESH: Cell Cycle, MESH: Poly (ADP-Ribose) Polymerase-1, Genome, Càncer--Tractament, chemistry.chemical_compound, Mice, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, DNA Breaks, Double-Stranded, MESH: Animals, lcsh:Science, Double strand, Multidisciplinary, Cell Cycle, DNA repair protein XRCC4, Cell cycle, 3. Good health, Cell biology, DNA-Binding Proteins, 030220 oncology & carcinogenesis, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Cell division, DNA repair, ADN--Dany, DNA recombination, Science, Double-strand DNA breaks, MESH: G1 Phase, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Targeted therapies, Animals, MESH: Mice, B cells, fungi, G1 Phase, General Chemistry, MESH: DNA End-Joining Repair, enzymes and coenzymes (carbohydrates), 030104 developmental biology, chemistry, lcsh:Q, Homologous recombination, DNA, MESH: DNA-Binding Proteins
Abstract

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies., Depending on the cell cycle stage, cells can repair their genome via different pathways. Here the authors reveal mechanistic insights into repair of double strand breaks induced during G1 in an error-prone manner by Pol θ-dependent and PARP1-independent alt NHEJ during the SG2/M phases of the cell cycle

Published
2020

9. Coordinated signals from the DNA repair enzymes PARP-1 and PARP-2 promotes B-cell development and function

Author

Jordi Farrés, Andrea Cerutti, Coral Ampurdanés, Chloé Lescale, Carlos Martinez, Lucia Moreno-Lama, Ludovic Deriano, Juan Martín-Caballero, Marie Bedora-Faure, José Yélamos, Pedro Aparicio, Françoise Dantzer, Miguel Galindo-Campos, Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS), IMIM-Hospital del Mar, Generalitat de Catalunya, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Universidad de Murcia, Barcelona Biomedical Research Park (PRBB), Departamento de Bioquímica y Biología Molecular [Murcia], Institució Catalana de Recerca i Estudis Avançats (ICREA), We thank P. Moreno, and M. Juan for assistance with mice. The Deriano lab members for help with pro-B-cell lines experiments. O. Fornes for cell sorting and flow cytometry assistance. The Yélamos´s lab is funded by the Spanish Ministerio de Economía, Industria y Competitividad (grant SAF2017-83565-R) and The Fundación Científica de la Asociación Española Contra el Cáncer (AECC). The Deriano´s lab is funded by the Institut Pasteur, the Institut National du Cancer (# PLBIO16-181) and the European Research Council under the ERC (starting grant agreement #310917). The Dantzer’s lab is supported by LABEX ANR-10-LABX-0034_Medalis, Strasbourg University, CNRS. The Cerutti’s lab is supported by the European Research Council (European Advanced Grant ERC-2011-ADG- 20110310) and by the Spanish Ministerio de Economía, Industria y Competitividad (grant SAF2014-52483-R)., ANR-10-IDEX-0002,UNISTRA,Par-delà les frontières, l'Université de Strasbourg(2010), Institut Pasteur [Paris] (IP), and Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, DNA Repair, DNA damage, DNA repair, Poly ADP ribose polymerase, [SDV]Life Sciences [q-bio], Poly (ADP-Ribose) Polymerase-1, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Genetic recombination, Article, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, medicine, Animals, Humans, Molecular Biology, B cell, Polymerase, ComputingMilieux_MISCELLANEOUS, Gene Rearrangement, Mice, Knockout, B-Lymphocytes, biology, Genes, Immunoglobulin, Sciences du Vivant [q-bio]/Biotechnologies, Cell Biology, 3. Good health, Cell biology, 030104 developmental biology, medicine.anatomical_structure, chemistry, Immunoglobulin class switching, 030220 oncology & carcinogenesis, biology.protein, Poly(ADP-ribose) Polymerases, DNA
Abstract

International audience; Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 regulate the function of various DNA-interacting proteins by transferring ADP-ribose emerging from catalytic cleavage of cellular β-NAD+. Hence, mice lacking PARP-1 or PARP-2 show DNA perturbations ranging from altered DNA integrity to impaired DNA repair. These effects stem from the central role that PARP-1 and PARP-2 play on the cellular response to DNA damage. Failure to mount a proper response culminates in cell death. Accordingly, PARP inhibitors are emerging as promising drugs in cancer therapy. However, the full impact of these inhibitors on immunity, including B-cell antibody production, remains elusive. Given that mice carrying dual PARP-1 and PARP-2 deficiency develop early embryonic lethality, we crossed PARP-1-deficient mice with mice carrying a B-cell-conditional PARP-2 gene deletion. We found that the resulting dually PARP-1 and PARP-2 deficient mice had perturbed bone-marrow B-cell development as well as profound peripheral depletion of transitional and follicular but not marginal zone B-cells. Of note, bone-marrow B-cell progenitors and peripheral mature B-cells were conserved in mice carrying either PARP-1 or PARP-2 deficiency. In dually PARP-1 and PARP-2 deficient mice, B-cell lymphopenia was associated with increased DNA damage and accentuated death in actively proliferating B-cells. Moreover, dual PARP-1 and PARP-2 deficiency impaired antibody responses to T-independent carbohydrate but not to T-dependent protein antigens. Notwithstanding the pivotal role of PARP-1 and PARP-2 in DNA repair, combined PARP-1 and PARP-2 deficiency did not perturb the DNA-editing processes required for the generation of a protective antibody repertoire, including Ig V(D)J gene recombination and IgM-to-IgG class switching. These findings provide key information as to the potential impact of PARP inhibitors on humoral immunity, which will facilitate the development of safer PARP-targeting regimens against cancer.

Published
2019

10. NFAT primes the human RORC locus for RORγt expression in CD4+ T cells

Author

Elisabetta Bianchi, Lars Rogge, Tharshana Stephen, Magda Rybczynska, Domenica Lovecchio, Hanane Yahia-Cherbal, Chloé Lescale, Jérôme Larghero, Katarzyna Placek, Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Immunorégulation - Immunoregulation, Institut Pasteur [Paris], Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Département de Génomes et Génétique - Department of Genomes and Genetics, Département d'Immunologie - Department of Immunology, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, CIC - Biotherapie - Saint Louis ((CIC-BT 301)), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Grants from Institut Pasteur, FOREUM Foundation for Research in Rheumatology, the Fondation Arthritis, MSD Avenir (Project iCARE-SpA), and a Bourse Passerelle from Pfizer., Center for Translational Research/Cytometry Biomarkers Unit of Technology and Service (CRT/CB UTechS), Institut Pasteur. Estelle Mottez (CRT/CB UTechS) : drafting of clinical protocols. O. Kaminuma, F. Berberich-Siebelt and G. Natoli: expression constructs (see Methods). Helène Strick-Marchand (Innate Immunity Unit, Institut Pasteur) for human hepatocytes lysates., Institut Pasteur [Paris] (IP), and Kop, Marie-Luce

Subjects
0301 basic medicine, [SDV.IMM] Life Sciences [q-bio]/Immunology, Science, [SDV]Life Sciences [q-bio], Adaptive immunity, General Physics and Astronomy, Biology, Jurkat cells, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, 0302 clinical medicine, RAR-related orphan receptor gamma, Histone code, NF-kappaB, T-helper 17 cells, lcsh:Science, Transcription factor, Regulation of gene expression, Multidisciplinary, NFAT, General Chemistry, NFATC Transcription Factors, Cell biology, [SDV] Life Sciences [q-bio], Thymocyte, Gene regulation in immune cells, 030104 developmental biology, [SDV.IMM]Life Sciences [q-bio]/Immunology, lcsh:Q, 030215 immunology
Abstract

T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4+ T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4+ T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation., The master transcription factor RORγt, encoded by the RORC gene, controls the polarization of CD4+ T cells expressing interleukin-17 (Th17). Here the authors describe several regulatory elements at the RORC locus that are recognized by NFAT and NFkB to induce a permissive epigenetic configuration of the RORC gene for RORγt expression and Th17 differentiation.

Published
2019

11. The immune system profoundly restricts intratumor genetic heterogeneity

Author

Ludovic Deriano, Guy Shakhar, Zacarias Garcia, Philippe Bousso, Leïla Perié, Ronan Thibaut, Idan Milo, Marie Bedora-Faure, Dynamiques des Réponses immunes - Dynamics of Immune Responses, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Physico-Chimie-Curie (PCC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC), Weizmann Institute of Science [Rehovot, Israël], Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, 0301 basic medicine, Lymphoma, B-Cell, Immunology, Computational biology, Biology, Genetic Heterogeneity, Mice, 03 medical and health sciences, Immune system, Antigen, medicine, Animals, B-cell lymphoma, Mice, Knockout, Genetic diversity, Genetic heterogeneity, Neoplasms, Experimental, General Medicine, Intravital Imaging, medicine.disease, 3. Good health, Lymphoma, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, Bone marrow
Abstract

International audience; Tumors develop under the selective pressure of the immune system. However, it remains critical to establish how the immune system affects the clonal heterogeneity of tumors that often display cell-to-cell variation in genetic alterations and antigenic expression. To address these questions, we introduced a multicolor barcoding strategy to study the growth of a MYC-driven B cell lymphoma harboring a large degree of intratumor genetic diversity. Using intravital imaging, we visualized that lymphoma subclones grow as patches of sessile cells in the bone marrow, creating a spatially compartmentalized architecture for tumor diversity. Using multicolor barcoding and whole-exome sequencing, we demonstrated that immune responses strongly restrict intratumor genomic diversity and favor clonal dominance, a process mediated by the selective elimination of more immunogenic cells and amplified by epitope spreading. Anti-PD-1 treatment also narrowed intratumor diversity. Our results provide direct evidence that immune pressure shapes the level of intratumor genetic heterogeneity and have important implications for the design of therapeutic strategies.

Published
2018

12. Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells

Author

Stephen P. Jackson, Mareike Herzog, Alejandra Bruna, Luca Pellegrini, Violeta Serra, Mark J. O'Connor, Zhongwu Lai, Chloé Lescale, Jacqueline J.L. Jacobs, Fengtang Yang, Jonathan Lam, Matylda Sczaniecka-Clift, Abigail Shea, Carlos Caldas, Matthias Ostermaier, Gabriel Balmus, Julia Coates, Wenming Wei, Inge de Krijger, Yaron Galanty, Mukerrem Demir, Ludovic Deriano, Petra Beli, Domenic Pilger, Harveer Dev, Rimma Belotserkovskaya, Alistair Martin, Beiyuan Fu, Ting-Wei Will Chiang, Qian Wu, Dev, Harveer [0000-0003-2874-6894], Yang, Fengtang [0000-0002-3573-2354], Balmus, Gabriel [0000-0003-2872-4468], Serra, Violeta [0000-0001-6620-1065], Beli, Petra [0000-0001-9507-9820], Pellegrini, Luca [0000-0002-9300-497X], Deriano, Ludovic [0000-0002-9673-9525], Jacobs, Jacqueline JL [0000-0002-7704-4795], Jackson, Stephen P [0000-0001-9317-7937], Apollo - University of Cambridge Repository, Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge [UK] (CAM), Department of Biochemistry [Cambridge], Cambridge University Hospitals - NHS (CUH), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP), Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, Cancer Research UK Cambridge Institute [Cambridge, Royaume-Uni] (CRUK), Institute of Molecular Biology (IMB), Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Wellcome Trust Sanger Institute [Hinxton, UK], AstraZeneca US [Waltham, USA], AstraZeneca [Cambridge, UK], The Wellcome Trust Sanger Institute [Cambridge], Vall d'Hebron Institute of Oncology [Barcelone] (VHIO), Vall d'Hebron University Hospital [Barcelona], The SPJ lab is largely funded by a Cancer Research UK (CRUK) Program Grant, C6/A18796, and a Wellcome Trust (WT) Investigator Award, 206388/Z/17/Z. Core infrastructure funding was provided by CRUK grant C6946/A24843 and WT grant WT203144. S.P.J. receives a salary from the University of Cambridge. H.D. is funded by WT Clinical Fellowship 206721/Z/17/Z. TWC was supported by a Cambridge International Scholarship. D.P. is funded by Cancer Research UK studentship C6/A21454. The P.B. lab is supported by the Emmy Noether Program (BE 5342/1-1) from the German Research Foundation and a Marie Curie Career Integration Grant from the European Commission (630763). The L.P. lab is funded by the WT (investigator award 104641/Z/14/Z) and the Medical Research Council (project grant MR/N000161/1). The C.C. lab was supported with funding from CRUK. The J.J. lab was supported by the European Research Council grant ERC-StG 311565, The Dutch Cancer Society (KWF) grant KWF 10999, and the Netherlands Organization for Scientific Research (NWO) as part of the National Roadmap Large-scale Research Facilities of the Netherlands, Proteins@Work (project no. 184.032.201 to the Proteomics Facility of the Netherlands Cancer Institute). The L.D. lab is funded by the Institut Pasteur, the Institut National du Cancer (no. PLBIO16-181) and the European Research Council (starting grant agreement no. 310917). W.W. is part of the Pasteur–Paris University (PPU) International PhD program and this project received funding from the CNBG company, China. Q.W. is funded by the Wellcome Trust (200814/Z/16/Z ). The V.S. lab work was funded by the Instituto de Salud Carlos III (ISCIII), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds (PI17-01080 to VS), the European Research Area-NET, Transcan-2 (AC15/00063), a non-commercial research agreement with AstraZeneca UK, and structural funds from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 540) and the Orozco Family. V.S. received a salary and travel support to C.C.’s lab from ISCIII (CP14/00228, MV15/00041) and the FERO Foundation., The authors thank all S.P.J. laboratory members for support and advice, and Cambridge colleagues N. Lawrence for OMX super-resolution microscopy support and R. Butler for help with computational image analyses and programming. The authors also thank S. Selivanova and S. Hough for help with plasmid amplification, sample preparation and tissue culture maintenance, K. Dry for extensive editorial assistance, F. Muñoz-Martinez for assistance with CRISPR–Cas9 knockout generation, L. Radu for assistance with protein purification, C. Lord (Institute of Cancer Research, London) for SUM149PT cells, D. Durocher (University of Toronto, Canada) for U2OS LacSceIII cells, F. Alt (Harvard University, USA) for CH12F3 cells and 53bp1 knockout CH12F3 cell clones, T. Honjo (Kyoto University, Japan) for permission to use the CH12F3 cell line, and J. Serrat in the Jacobs lab for technical assistance, Institut Pasteur [Paris], and Johannes Gutenberg - Universität Mainz (JGU)

Subjects
MESH: DNA Breaks, Double-Stranded, RAD51, Cell Cycle Proteins, Poly (ADP-Ribose) Polymerase Inhibitor, MESH: Recombinational DNA Repair, Mice, MESH: Animals, DNA Breaks, Double-Stranded, skin and connective tissue diseases, Cancer, Telomere-binding protein, Ovarian Neoplasms, MESH: Breast Neoplasms / metabolism, MESH: Telomere-Binding Proteins / metabolism, 3. Good health, Cell biology, MESH: HEK293 Cells, MESH: Proteins / genetics, MESH: Telomere-Binding Proteins / genetics, MESH: Tumor Suppressor p53-Binding Protein 1 / metabolism, MESH: Xenograft Model Antitumor Assays, Telomere-Binding Proteins, MESH: Ovarian Neoplasms / drug therapy, Bone Neoplasms, MESH: Ovarian Neoplasms / metabolism, Article, 03 medical and health sciences, MESH: Cell Cycle Proteins, MESH: Bone Neoplasms / metabolism, Humans, MESH: Osteosarcoma / metabolism, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, MESH: Tumor Suppressor p53-Binding Protein 1 / genetics, Dose-Response Relationship, Drug, HEK 293 cells, Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, MESH: BRCA1 Protein / deficiency, 030104 developmental biology, Multiprotein Complexes, MESH: Mad2 Proteins / metabolism, MESH: Breast Neoplasms / genetics, MESH: Bone Neoplasms / drug therapy, Cisplatin, Homologous recombination, MESH: Osteosarcoma / genetics, MESH: Female, 0301 basic medicine, DNA End-Joining Repair, MESH: Proteins / metabolism, MESH: Dose-Response Relationship, Drug, chemistry.chemical_compound, MESH: Osteosarcoma / pathology, MESH: Breast Neoplasms / pathology, Homologous Recombination, Polymerase, MESH: Breast Neoplasms / drug therapy, Osteosarcoma, biology, Chemistry, BRCA1 Protein, DNA damage and repair, MESH: Poly(ADP-ribose) Polymerase Inhibitors / pharmacology, MESH: Bone Neoplasms / genetics, DNA-Binding Proteins, MESH: Bone Neoplasms / pathology, Mad2 Proteins, Female, MESH: Ovarian Neoplasms / genetics, Tumor Suppressor p53-Binding Protein 1, MESH: Cisplatin / pharmacology, MESH: Cell Line, Tumor, Lymphocytes, Null, [SDV.CAN]Life Sciences [q-bio]/Cancer, Breast Neoplasms, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: BRCA1 Protein / genetics, Poly(ADP-ribose) Polymerase Inhibitors, Cell Line, Tumor, MESH: Drug Resistance, Neoplasm* / genetics, MESH: Mad2 Proteins / genetics, MESH: Ovarian Neoplasms / pathology, Animals, MESH: Mice, MESH: Osteosarcoma / drug therapy, Oligonucleotide, Protective Devices, Recombinational DNA Repair, Cell Biology, MESH: Multiprotein Complexes, Xenograft Model Antitumor Assays, HEK293 Cells, Drug Resistance, Neoplasm, biology.protein, MESH: DNA End-Joining Repair, MESH: DNA-Binding Proteins
Abstract

International audience; BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

Published
2018

13. The Dynamics of Interleukin-10-Afforded Protection during Dextran Sulfate Sodium-Induced Colitis

Author

Ana Cardoso, Antonio Gil Castro, Ana Catarina Martins, Guilhermina M. Carriche, Valentine Murigneux, Isabel Castro, Ana Cumano, Paulo Vieira, Margarida Saraiva, Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Lymphopoïèse (Lymphopoïèse (UMR_1223 / U1223 / U-Pasteur_4)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Life and Health Sciences Research Institute [Braga] (ICVS), University of Minho [Braga], ICVS/3B's, PT Government Associate Laboratory, Instituto de Biologia Molecular e Celular (IBMC), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], We acknowledge the Portuguese Foundation for Science and Technology (FCT) for providing a PhD grant to AC (SFRH/BD/84704/2012). This article is a result of the project Norte-01-0145-FEDER-000012—Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The MS lab is also financed by a FCT-ANR grant (FCTANR/BIM-MEC/0007/2013). This work was also backed by the COST Action BM1404 European Network of Investigators Triggering Exploratory Research on Myeloid Regulatory Cells (http://www.mye-euniter.eu), which is supported by the Horizon 2020—EU Framework Program Research and Innovation Programme. MS is a FCT Associate Investigator. AGC lab: This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), by the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through FEDER, and by FEDER, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038. PV is funded by ANR, through the project MYELOTEN (ANR-13-ISV1-0003-01)., We are grateful to Dr. Werner Müller (Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom) for providing the IL-10R-deficient mice. We also thank the excellent support given by the animal house workers at IBMC-i3S., ANR-13-ISV1-0003,MYELOTEN,DEREGULATION DE L'HEMATOPOIESE PAR LA SUREXPRESSION DE L'INTERLEUKINE-10 : IMPLICATIONS DANS LE DEVELOPPEMENT DE PATHOLOGIES HEMATOLOGIQUES(2013), Instituto de Investigação e Inovação em Saúde, Vougny, Marie-Christine, Blanc – Accords bilatéraux 2013 - DEREGULATION DE L'HEMATOPOIESE PAR LA SUREXPRESSION DE L'INTERLEUKINE-10 : IMPLICATIONS DANS LE DEVELOPPEMENT DE PATHOLOGIES HEMATOLOGIQUES - - MYELOTEN2013 - ANR-13-ISV1-0003 - Blanc – Accords bilatéraux 2013 - VALID, Universidade do Porto = University of Porto, Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Universidade do Minho, and TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany.

Subjects
0301 basic medicine, colitis, medicine.medical_treatment, Medicina Básica [Ciências Médicas], interleukin-10, Inflammatory bowel disease, Mice, Immunology and Allergy, Receptors, Interleukin-10, Transgenes, Original Research, Mice, Knockout, Dextran Sulfate, Interleukin, Colitis, 3. Good health, Interleukin-10, macrophages, Interleukin 10, Cytokine, Ciências Médicas::Medicina Básica, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, medicine.symptom, Signal Transduction, lcsh:Immunologic diseases. Allergy, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, Inflammation, Mice, Transgenic, 03 medical and health sciences, Immune system, medicine, Animals, Humans, therapy, Science & Technology, business.industry, Macrophages, Chemical colitis, medicine.disease, Inflammatory Bowel Diseases, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, inflammation, Therapy, business, lcsh:RC581-607
Abstract

Inflammatory bowel disease encompasses a group of chronic-inflammatory conditions of the colon and small intestine. These conditions are characterized by exacerbated inflammation of the organ that greatly affects the quality of life of patients. Molecular mechanisms counteracting this hyperinflammatory status of the gut offer strategies for therapeutic intervention. Among these regulatory molecules is the anti-inflammatory cytokine interleukin (IL)-10, as shown in mice and humans. Indeed, IL-10 signaling, particularly in macrophages, is essential for intestinal homeostasis. We sought to investigate the temporal profile of IL-10-mediated protection during chemical colitis and which were the underlying mechanisms. Using a novel mouse model of inducible IL-10 overexpression (pMT-10), described here, we show that mice preconditioned with IL-10 for 8 days before dextran sulfate sodium (DSS) administration developed a milder colitic phenotype. In IL-10-induced colitic mice, Ly6C cells isolated from the lamina propria showed a decreased inflammatory profile. Because our mouse model leads to transcription of the IL-10 transgene in the bone marrow and elevated seric IL-10 concentration, we investigated whether IL-10 could imprint immune cells in a long-lasting way, thus conferring sustained protection to colitis. We show that this was not the case, as IL-10-afforded protection was only observed if IL-10 induction immediately preceded DSS-mediated colitis. Thus, despite the protection afforded by IL-10 in colitis, novel strategies are required, specifically to achieve long-lasting protection., Portuguese Foundation for Science and Technology (FCT) for providing a PhD grant to AC (SFRH/BD/84704/2012). This article is a result of the project Norte-01-0145-FEDER-000012—Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The MS lab is also financed by a FCT-ANR grant (FCTANR/BIM-MEC/0007/2013). This work was also backed by the COST Action BM1404 European Network of Investigators Triggering Exploratory Research on Myeloid Regulatory Cells (http://www.mye-euniter.eu), which is supported by the Horizon 2020—EU Framework Program Research and Innovation Programme. MS is a FCT Associate Investigator. AGC lab: This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER); by the project NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through FEDER; and by FEDER, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-007038. PV is funded by ANR, through the project MYELOTEN (ANR-13-ISV1-0003-01), info:eu-repo/semantics/publishedVersion

Published
2018

14. Chromosomal Translocation Formation Is Sufficient to Produce Fusion Circular RNAs Specific to Patient Tumor Cells

Author

Benjamin Renouf, Khadija Lamribet, Cécile Thirant, Ludovic Deriano, Erika Brunet, Thomas Mercher, Marion Piganeau, Carine Giovannangeli, Loelia Babin, Genome dynamics in the immune system (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Hématopoïèse normale et pathologique (U1170 Inserm), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP), This research work is supported by a joint grant (E.B., T.M., and L.D.) from the Institut National du Cancer and the Cancéropôle IdF (2016-1-PLBIO-07-INSERM11-1). E.B. research was supported by ANR-12-JSV6-0005. E.B.’s group and T.M.’s team belong to two ‘‘La ligue contre le Cancer’’ teams. M.P. was supported by fellowships from Le Cancéropôle IdF and La Ligue Contre le Cancer, and C.T., by a fellowship from la Fondation de France. L.D.’s research is supported by the Institut Pasteur as well as by the European Research Council under the ERC starting grant agreement # 310917, and T.M.’s research, by SIRIC SOCRATE and an Emergence grant from the Canceropole IdF., ANR-12-JSV6-0005,InRevTrans,Induction et réversion de translocations chromosomiques pour comprendre la tumorigenèse: les modèles du sarcome d'Ewing et du lymphome anaplastique à grandes cellules (ALCL)(2012), European Project: 310917,EC:FP7:ERC,ERC-2012-StG_20111109,LYMPHOONCOGENOMICS(2013), Giovannangeli, Carine, Jeunes Chercheuses et Jeunes Chercheurs - Induction et réversion de translocations chromosomiques pour comprendre la tumorigenèse: les modèles du sarcome d'Ewing et du lymphome anaplastique à grandes cellules (ALCL) - - InRevTrans2012 - ANR-12-JSV6-0005 - JC - VALID, Deciphering the Oncogenic Lesions and Pathways of B and T Cell Cancers - LYMPHOONCOGENOMICS - - EC:FP7:ERC2013-02-01 - 2018-01-31 - 310917 - VALID, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR), and Institut Pasteur [Paris]

Subjects
0301 basic medicine, medicine.medical_specialty, Multidisciplinary, Cas9, Chromosomal translocation, Biology, medicine.disease_cause, Fusion protein, Cell biology, Fusion gene, Molecular Genetics, 03 medical and health sciences, 030104 developmental biology, Molecular genetics, Cancer cell, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, medicine, CRISPR, lcsh:Q, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, lcsh:Science, Carcinogenesis, Molecular Biology, Cancer
Abstract

Summary: Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process. : Molecular Biology; Molecular Genetics; Cancer Subject Areas: Molecular Biology, Molecular Genetics, Cancer

Published
2018

15. Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination

Author

Lenden Hasse, Hélène, Lescale, Chloé, Bianchi, Joy, Yu, Wei, Bedora-Faure, Marie, Deriano, Ludovic, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, This work was supported by the Institut Pasteur, the European Research Council under the ERC starting grant agreement # 310917 and the Institut National du Cancer (# PLBIO16-181). J.J.B. is a BioSPC/Université Paris 5 doctoral Fellow., We thank Bernardo Reina San Martin, Erika Brunet and Jean-Pierre de Villartay for initial discussion and reagents and the Institut Pasteur Cytometry Platform and Center for Human Immunology for their help., European Project: 310917,EC:FP7:ERC,ERC-2012-StG_20111109,LYMPHOONCOGENOMICS(2013), and Institut Pasteur [Paris] (IP)

Subjects
MESH: Gene Editing, MESH: G1 Phase Cell Cycle Checkpoints, MESH: CRISPR-Cas Systems, DNA End-Joining Repair, CRISPR-Associated Proteins, MESH: DNA Breaks, Double-Stranded, Apoptosis, MESH: Genotype, hemic and lymphatic diseases, MESH: Protein Kinase Inhibitors, Clustered Regularly Interspaced Short Palindromic Repeats, DNA Breaks, Double-Stranded, MESH: Animals, Cell Line, Transformed, Gene Editing, v-Abl transformed pro-B cells, DNA-Binding Proteins, MESH: Recombinational DNA Repair, Phenotype, Proto-Oncogene Proteins c-bcl-2, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Imatinib Mesylate, Genotype, MESH: Imatinib Mesylate, Recombination-activating gene (RAG) endonuclease, [SDV.CAN]Life Sciences [q-bio]/Cancer, CRISPR/Cas9-mediated gene knock-out, MESH: Phenotype, Article, MESH: Mice, Inbred C57BL, MESH: Homeodomain Proteins, Animals, MESH: Oncogene Proteins v-abl, MESH: Cell Line, Transformed, Oncogene Proteins v-abl, Protein Kinase Inhibitors, neoplasms, Homeodomain Proteins, Precursor Cells, B-Lymphoid, Nonhomologous end joining (NHEJ), MESH: Apoptosis, MESH: CRISPR-Associated Proteins, Recombinational DNA Repair, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: DNA End-Joining Repair, G1 Phase Cell Cycle Checkpoints, V(D)J recombination, Mice, Inbred C57BL, MESH: Proto-Oncogene Proteins c-bcl-2, MESH: Precursor Cells, B-Lymphoid, MESH: Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, MESH: DNA-Binding Proteins
Abstract

International audience; Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.

Published
2017

16. Breakage-Fusion-Bridge Events Trigger Complex Genome Rearrangements and Amplifications in Developmentally Arrested T Cell Lymphomas

Author

Valentine Murigneux, Marie Bedora-Faure, Ludovic Deriano, Joy J. Bianchi, Chloé Lescale, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP), Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, The Deriano Lab is funded by the Institut Pasteur, the Institut National Du Cancer (PLBIO16-181) as well as by the European Research Council under the ERC starting grant agreement 310917. J.J.B. is a BioSPC/Université Paris 5 and Fondation pour la Recherche Médicale (FDT20170739035) doctoral fellow., We thank the Institut Pasteur genomics platform and the New York Genome Center team for help with whole-genome sequencing, Philippe Bousso for providing Rag2−/− OTII animals, Ana Cumano for providing the anti-CD28 antibody, and Erika Brunet and Thomas Mercher for helpful discussions., and Institut Pasteur [Paris]

Subjects
0301 basic medicine, T-Lymphocytes, Genome, Translocation, Genetic, T cell lymphoma, Malignant transformation, Mice, 0302 clinical medicine, hemic and lymphatic diseases, RNA-Seq, lcsh:QH301-705.5, Mice, Knockout, cancer genome landscape, Amplicon, 3. Good health, DNA-Binding Proteins, Gene Expression Regulation, Neoplastic, KMT2A, RAG1/2 nuclease, Myeloid-Lymphoid Leukemia Protein, DNA damage, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Lymphoma, T-Cell, Genomic Instability, Article, General Biochemistry, Genetics and Molecular Biology, Recombination-activating gene, 03 medical and health sciences, RAG2, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, Humans, Mitosis, [SDV.GEN]Life Sciences [q-bio]/Genetics, T cell development, Gene Amplification, structural variation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Histone-Lysine N-Methyltransferase, Molecular biology, Mice, Inbred C57BL, 030104 developmental biology, lcsh:Biology (General), breakage-fusion-bridge, biology.protein, Tumor Suppressor Protein p53, 030217 neurology & neurosurgery
Abstract

Summary To reveal the relative contribution of the recombination activating gene (RAG)1/2 nuclease to lymphomagenesis, we conducted a genome-wide analysis of T cell lymphomas from p53-deficient mice expressing or lacking RAG2. We found that while p53−/− lymphoblastic T cells harbor primarily ectopic DNA deletions, Rag2−/−p53−/− T cell lymphomas display complex genomic rearrangements associated with amplification of the chromosomal location 9qA4-5.3. We show that this amplicon is generated by breakage-fusion-bridge during mitosis and arises distinctly in T cell lymphomas originating from an early progenitor stage. Notably, we report amplification of the corresponding syntenic region (11q23) in a subset of human leukemia leading to the overexpression of several cancer genes, including MLL/KMT2A. Our findings provide direct evidence that lymphocytes undergo malignant transformation through distinct genome architectural routes that are determined by both RAG-dependent and RAG-independent DNA damage and a block in cell development., Graphical Abstract, Highlights • Lymphomas from RAG2/p53- and p53-deficient mice bear distinct genome architectures • Block in T cell development leads to 9qA4-5.3 rearrangements and amplifications • Breakage-fusion-bridge events trigger 9qA4-5.3 aberrations in early T cell lymphomas • The syntenic region 11q23 is amplified in some human hematological cancers, Using p53-deficient T cell lymphoma mouse models, Bianchi et al. show that breakage-fusion-bridge events trigger complex focal genome rearrangements and amplifications in developmentally arrested lymphoblastic cells, leading to unique genome and transcriptome signatures found in some human hematological cancers.

Published
2019

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