RESUMO
Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels. Color-coded populations could be tracked in multiplex in vitro assays for up to 28 days by flow cytometry using all three vector systems. Those involving lower levels of multiplexing eased color-code generation and the reliability of vector expression and enabled functional in vitro and in vivo studies. In proof-of-principle experiments, FGB vectors facilitated in vitro multiplex screening of microRNA (miRNA)-induced growth advantages, as well as the in vivo recovery of color-coded progeny of murine and human hematopoietic stem cells. This novel series of FGB vectors provides new tools for assessing comparative growth properties in in vitro and in vivo multiplexing experiments, while simultaneously allowing for a reduction in sample numbers by up to 26-fold.
Assuntos
Rastreamento de Células/métodos , Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Luminescentes/genética , Diferenciação Celular , Códon , Citometria de Fluxo , Ordem dos Genes , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Proteínas Luminescentes/metabolismo , MicroRNAs/genética , Reprodutibilidade dos Testes , Transdução GenéticaRESUMO
Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.
Assuntos
Transformação Celular Neoplásica/metabolismo , Sangue Fetal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas de Fusão Oncogênica/genética , Transativadores , Proteínas Supressoras de Tumor/genéticaRESUMO
The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO-positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO-positive leukemias.
Assuntos
Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição/genética , Translocação Genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Subunidade alfa 2 de Fator de Ligação ao Core , Modelos Animais de Doenças , Feminino , Genes ras/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucopoese/genética , Masculino , Camundongos , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Tirosina Quinase 3 Semelhante a fmsRESUMO
Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of beta-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.
Assuntos
Sangue Fetal/citologia , Vetores Genéticos/metabolismo , Globinas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/metabolismo , Transdução Genética , Animais , Linhagem da Célula , Transplante de Células , Células Cultivadas , Cromossomos Humanos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Globinas/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , TransgenesRESUMO
Myeloid ecotropic viral integration site 1 (MEIS1), a HOX transcription cofactor, is a critical regulator of normal hematopoiesis, and its overexpression is implicated in a wide range of leukemias. Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) gene-editing system, we generated a knock-in transgenic mouse line in which a green fluorescent protein (GFP) reporter and a hemagglutinin (HA) epitope tag are inserted near the translational start site of endogenous Meis1. This novel reporter strain readily enables tracking of MEIS1 expression at single-cell-level resolution via the fluorescence reporter GFP, and facilitates MEIS1 detection and purification via the HA epitope tag. This new Meis1 reporter mouse line provides powerful new approaches to track Meis1-expressing hematopoietic cells and to explore Meis1 function and regulation during normal and leukemic hematopoiesis.