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
The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.
Assuntos
Transformação Celular Neoplásica/genética , Linfoma/genética , Mutação , Complexo Repressor Polycomb 2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Western Blotting , Células da Medula Óssea/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Linfoma/metabolismo , Linfoma/patologia , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Lisina/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Baço/metabolismo , Baço/patologiaRESUMO
NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC. The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.
RESUMO
Tracking the behavior of leukemic samples both in vitro and in vivo plays an increasingly large role in efforts to better understand the leukemogenic processes and the effects of potential new therapies. Such work can be accelerated and made more efficient by methodologies enabling the characterization of leukemia samples in multiplex assays. We recently developed three sets of lentiviral fluorescent genetic barcoding (FGB) vectors that create 26, 14, and 6 unique immunophenotyping-compatible color codes from GFP-, yellow fluorescent protein (YFP)-, and monomeric kusabira orange 2 (mKO2)-derived fluorescent proteins. These vectors allow for labeling and tracking of individual color-coded cell populations in mixed samples by real-time flow cytometry. Using the prototypical Hoxa9/Meis1 murine model of acute myeloid leukemia, we describe the application of the 6xFGB vector system for assessing leukemic cell characteristics in multiplex assays. By transplanting color-coded cell mixes, we investigated the competitive growth behavior of individual color-coded populations, determined leukemia-initiating cell frequencies, and assessed the dose-dependent potential of cells exposed to the histone deacetylase inhibitor Entinostat for bone marrow homing. Thus, FGB provides a useful tool for the multiplex characterization of leukemia samples in a wide variety of applications with a concomitant reduction in workload, processing times, and mouse utilization.
RESUMO
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.
RESUMO
Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Pirimetamina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Antagonistas do Ácido Fólico/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.
Assuntos
Hematopoese/genética , Leucemia/genética , Leucemia/patologia , Fenótipo , Proteínas Supressoras de Tumor/genética , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Leucemia/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Células Progenitoras de Megacariócitos e Eritrócitos/patologia , Camundongos , Mutação , Células Mieloides/metabolismo , Células Mieloides/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Linfócitos T/metabolismo , Linfócitos T/patologia , Transativadores , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismoRESUMO
To determine the contribution of the common N-terminal truncation of NUP98 in NUP98-translocations resulting in acute myeloid leukemia, we have conducted a structure-function analysis of NUP98 in the context of NUP98-HOXA10HD, a novel, canonical NUP98-Hox fusion that significantly enhances the self-renewal capacity of hematopoietic stem cells and collaborates with Meis1 to induce AML in our mouse models. Our results identify that NUP98 functions by transcriptional activation likely by recruitment of CBP/p300 via its FG/GLFG repeats. In contrast, the functional interaction of NUP98 with Rae1 or the anaphase promoting complex appears non-essential for its role in NUP98-leukemogenic fusions.
Assuntos
Proteínas de Homeodomínio/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Doença Aguda , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células da Medula Óssea/metabolismo , Transformação Celular Neoplásica/genética , Células Cultivadas , Feminino , Proteínas Homeobox A10 , Proteínas de Homeodomínio/genética , Estimativa de Kaplan-Meier , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Fusão Oncogênica/genética , Ligação Proteica , Sequências Repetitivas de Aminoácidos , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Pathways defining susceptibility of normal cells to oncogenic transformation may be valuable therapeutic targets. We characterized the cell of origin and its critical pathways in MN1-induced leukemias. Common myeloid (CMP) but not granulocyte-macrophage progenitors (GMP) could be transformed by MN1. Complementation studies of CMP-signature genes in GMPs demonstrated that MN1-leukemogenicity required the MEIS1/AbdB-like HOX-protein complex. ChIP-sequencing identified common target genes of MN1 and MEIS1 and demonstrated identical binding sites for a large proportion of their chromatin targets. Transcriptional repression of MEIS1 targets in established MN1 leukemias demonstrated antileukemic activity. As MN1 relies on but cannot activate expression of MEIS1/AbdB-like HOX proteins, transcriptional activity of these genes determines cellular susceptibility to MN1-induced transformation and may represent a promising therapeutic target.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/patologia , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genes Dominantes/genética , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Células Progenitoras de Granulócitos e Macrófagos/patologia , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteína Meis1 , Regiões Promotoras Genéticas/genética , Ligação ProteicaRESUMO
To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.