RESUMO
V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Switching de Imunoglobulina , Animais , Linfócitos B/imunologia , Proteínas de Ligação a DNA/deficiência , Genes Letais , Lentivirus/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MutaçãoRESUMO
The NOTCH signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment is unclear. We recently identified the NOTCH signaling pathway as a positive regulator of megakaryocyte lineage specification during hematopoiesis, but the developmental pathways that allow hematopoietic stem cell differentiation into the erythro-megakaryocytic lineages remain controversial. Here, we investigated the role of downstream mediators of NOTCH during megakaryopoiesis and report crosstalk between the NOTCH and PI3K/AKT pathways. We demonstrate the inhibitory role of phosphatase with tensin homolog and Forkhead Box class O factors on megakaryopoiesis in vivo. Finally, our data annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of 2 distinct developmental pathways.
Assuntos
Diferenciação Celular , Linhagem da Célula/fisiologia , Megacariócitos/fisiologia , Proteína Oncogênica v-akt/metabolismo , Receptores Notch/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/fisiologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Receptor Cross-Talk/fisiologia , Receptores Notch/genética , Receptores Notch/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Trombopoese/genéticaRESUMO
Nonhomologous end-joining DNA repair factors, including Artemis, are all required for the repair of DNA double-strand breaks, which occur during the assembly of the variable antigen recognition domain of B-cell receptors and T-cell receptors through the V(D)J recombination. Mature B cells further shape their immunoglobulin repertoire on antigen recognition notably through the class switch recombination (CSR) process. To analyze the role of Artemis during CSR, we developed a mature B-cell-specific Artemis conditional knockout mouse to bypass the absence of B cells caused by its early deficit. Although CSR is not overwhelmingly affected in these mice, class switching to certain isotypes is clearly reduced both in vitro on B-cell activation and in vivo after keyhole limpet hemocyanin immunization. The reduced CSR in Artemis-deficient B cells is accompanied by the increase in DNA microhomology usage at CSR junctions, the imprint of an alternative DNA end-joining pathway. Likewise, significant increase in DNA microhomology usage is the signature of CSR junctions obtained from human RS-SCID patients harboring hypomorphic Artemis mutations. Altogether, this indicates that Artemis participates in the repair of a subset of DNA breaks generated during CSR.
Assuntos
Quebras de DNA de Cadeia Dupla , Imunoglobulina A/genética , Switching de Imunoglobulina/genética , Imunoglobulina G/genética , Proteínas Nucleares/fisiologia , Recombinação Genética , Imunodeficiência Combinada Severa/genética , Adulto , Animais , Linfócitos B/metabolismo , Sequência de Bases , Western Blotting , Criança , Endonucleases , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hemocianinas/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Dados de Sequência Molecular , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
JAK3-activating mutations are commonly seen in chronic or acute hematologic malignancies affecting the myeloid, megakaryocytic, lymphoid, and natural killer (NK) cell compartment. Overexpression models of mutant JAK3 or pharmacologic inhibition of its kinase activity have highlighted the role that these constitutively activated mutants play in the T-cell, NK cell, and megakaryocytic lineages, but to date, the functional impact of JAK3 mutations at an endogenous level remains unknown. Here, we report a JAK3A572V knockin mouse model and demonstrate that activated JAK3 leads to a progressive and dose-dependent expansion of CD8+ T cells in the periphery before colonization of the bone marrow. This phenotype is dependent on the γc chain of cytokine receptors and presents several features of the human leukemic form of cutaneous T-cell lymphoma (L-CTCL), including skin involvements. We also showed that the JAK3A572V-positive malignant cells are transplantable and phenotypically heterogeneous in bone marrow transplantation assays. Interestingly, we revealed that activated JAK3 functionally cooperates with partial trisomy 21 in vivo to enhance the L-CTCL phenotype, ultimately leading to a lethal and fully penetrant disorder. Finally, we assessed the efficacy of JAK3 inhibition and showed that CTCL JAK3A572V-positive T cells are sensitive to tofacitinib, which provides additional preclinical insights into the use of JAK3 inhibitors in these disorders. Altogether, this JAK3A572V knockin model is a relevant new tool for testing the efficacy of JAK inhibitors in JAK3-related hematopoietic malignancies.
Assuntos
Cromossomos de Mamíferos/metabolismo , Neoplasias Hematológicas/metabolismo , Janus Quinase 3/metabolismo , Linfoma Cutâneo de Células T/metabolismo , Mutação de Sentido Incorreto , Neoplasias Experimentais/metabolismo , Trissomia , Substituição de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Cromossomos de Mamíferos/genética , Técnicas de Introdução de Genes , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/patologia , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologiaRESUMO
Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.
Assuntos
Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Linfócitos T/citologia , Timócitos/citologia , Animais , Sequência de Bases , Proliferação de Células , Sobrevivência Celular , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Linfócitos T/metabolismo , Timócitos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Recombinação V(D)JRESUMO
Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.
Assuntos
Genômica/métodos , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Ensaios Antitumorais Modelo de Xenoenxerto , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Idoso , Sequência de Aminoácidos , Animais , Aurora Quinase A , Aurora Quinases , Azepinas/farmacologia , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Estimativa de Kaplan-Meier , Fatores de Transcrição Kruppel-Like/genética , Leucemia Megacarioblástica Aguda/patologia , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Proteínas Repressoras/genéticaRESUMO
The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.
Assuntos
Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Sequência de Aminoácidos , DNA Ligase Dependente de ATP , DNA Ligases , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Humanos , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes , Estrutura Terciária de ProteínaRESUMO
B and T lymphocytes are exposed to various genotoxic stresses during their life, which originate from programmed molecular mechanisms during their development and maturation or are secondary to cellular metabolism during acute phases of cell proliferation and activation during immune responses. How lymphocytes handle these multiple genomic assault has become a focus of interest over the years, perhaps beginning with the identification of the murine scid model in the early 80s when it was recognized that DNA repair deficiencies had profound consequences on the immune system. In this respect, the immune system represents an ideal model to study DNA damage responses (DDR) and the survey of immune deficiency conditions in humans or the development of specific animal models provided many major contributions in our understanding of the various biochemical pathways at play during DDR in general. Although the role of DNA repair in the early phases of B and T cell development has been analyzed thoroughly, the role of these functions in various aspects of the mature immune system (homeostasis, immunological memory, ageing) is less well understood. Lastly, the analysis of DNA repair in the immune system has provided many insights in the more general understanding of cancer.