RESUMO
There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.
Assuntos
Regulação Leucêmica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/patologia , Regulação para Cima , Quinases Ativadas por p21/análiseRESUMO
Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. Epigenetic deregulation contributes to AML pathogenesis. KDM6 proteins are histone-3-lysine-27-demethylases that play context-dependent roles in AML. We inform that KDM6-demethylase function critically regulates DNA-damage-repair-(DDR) gene expression in AML. Mechanistically, KDM6 expression is regulated by genotoxic stress, with deficiency of KDM6A-(UTX) and KDM6B-(JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations are implicated in chemoresistance, although a significant percentage of relapsed-AML has upregulated KDM6A. Olaparib treatment reduced engraftment of KDM6A-mutant-AML-patient-derived xenografts, highlighting synthetic lethality using Poly-(ADP-ribose)-polymerase-(PARP)-inhibition. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, BCL2A1 associates with venetoclax resistance, and KDM6A loss was accompanied with a downregulated BCL2A1. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support of a novel combination therapy for AML based on subtype-heterogeneity, and establishes KDM6A as a molecular regulator for determining therapeutic efficacy.
Assuntos
Leucemia Mieloide Aguda , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
The nonobese diabetic/severe combined immune deficiency (NOD-scid) xenotransplantation model is the "gold standard" for assaying human hematopoietic stem cell activity. Systematic advancements, such as depletion of natural killer cell activity with anti-CD122 antibody, direct intrafemoral injection, and deletion or truncation of IL2Rgamma, have improved human cell engraftment; however, questions remain whether these mouse models are equivalent or, if not, which model is superior for assaying hematopoietic stem cell activity. To address this, we compared overall engraftment and multilineage differentiation of near-limiting doses of lineage-depleted human umbilical cord blood cells by direct intrafemoral injection into NOD/Lt-scid, NOD/Shi-scid, NOD/Lt-scid/IL2Rgamma(null) (NSG), and NOD/Shi-scid/IL2Rgamma(null) mice. Transplantation into NSG mice generated moderately higher human engraftment levels in bone marrow compared with other strains. At limiting doses, NSG mice of both sexes were 3.6-fold more sensitive in detecting SCID-repopulating cells compared with NOD/Lt-scid mice. However, NSG females exhibited higher engraftment at limiting cell doses, resulting in an overall increase in SCID-repopulating cell detection of 9-fold. Both NSG and NOD/Shi-scid/IL2Rgamma(null) support significantly improved engraftment in peripheral tissues compared with NOD/Lt-scid and NOD/Shi-scid mice, whereas NSG mice provide greater human engraftment in bone marrow than all other strains, especially at limiting doses.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Camundongos Endogâmicos NOD , Modelos Animais , Transplante Heterólogo/imunologia , Transplante Heterólogo/métodos , Animais , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Separação Celular , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Masculino , CamundongosRESUMO
Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells.
Assuntos
Leucemia Mieloide Aguda , MicroRNAs , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , MicroRNAs/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.
Assuntos
Leucemia Mieloide Aguda , Nicotinamida Fosforribosiltransferase , Apoptose , Homeostase , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Lipídeos , Células-Tronco Neoplásicas , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Células-TroncoRESUMO
Children with Down syndrome have a 150-fold increased risk of developing myeloid leukemia, but the mechanism of predisposition is unclear. Because Down syndrome leukemogenesis initiates during fetal development, we characterized the cellular and developmental context of preleukemic initiation and leukemic progression using gene editing in human disomic and trisomic fetal hematopoietic cells and xenotransplantation. GATA binding protein 1 (GATA1) mutations caused transient preleukemia when introduced into trisomy 21 long-term hematopoietic stem cells, where a subset of chromosome 21 microRNAs affected predisposition to preleukemia. By contrast, progression to leukemia was independent of trisomy 21 and originated in various stem and progenitor cells through additional mutations in cohesin genes. CD117+/KIT proto-oncogene (KIT) cells mediated the propagation of preleukemia and leukemia, and KIT inhibition targeted preleukemic stem cells.
Assuntos
Proteínas de Ciclo Celular/genética , Síndrome de Down/genética , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide/genética , Pré-Leucemia/genética , Animais , Antígenos CD34/análise , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células , Transformação Celular Neoplásica , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 21/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Síndrome de Down/complicações , Feminino , Fator de Transcrição GATA1/metabolismo , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Fígado/embriologia , Masculino , Megacariócitos/fisiologia , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Pré-Leucemia/metabolismo , Pré-Leucemia/patologia , Inibidores de Proteínas Quinases/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , CoesinasRESUMO
In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.
Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células/genética , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Animais , Eletroporação/métodos , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante HeterólogoRESUMO
Acute leukemia is an aggressive blood malignancy with low survival rates. A high expression of stem-like programs in leukemias predicts poor prognosis and is assumed to act in an aberrant fashion in the phenotypically heterogeneous leukemia stem cell (LSC) population. A lack of suitable genome engineering tools that can isolate LSCs based on their stemness precludes their comprehensive examination and full characterization. We hypothesized that tagging endogenous stemness-regulatory regions could generate a genome reporter for the putative leukemia stemness-state. Our analysis revealed that the ERG + 85 enhancer region can serve as a marker for stemness-state and a fluorescent lentiviral reporter was developed that can accurately recapitulate the endogenous activity. Using our novel reporter, we revealed cellular heterogeneity in several leukemia cell lines and patient-derived samples. Alterations in reporter activity were associated with transcriptomic and functional changes that were closely related to the hematopoietic stem cell (HSC) identity. Notably, the differentiation potential was skewed towards the erythro-megakaryocytic lineage. Moreover, an ERG + 85High fraction of AML cells could regenerate the original cellular heterogeneity and was enriched for LSCs. RNA-seq analysis coupled with in silico drug-screen analysis identified 4HPR as an effective inhibitor of ERG + 85High leukemia growth. We propose that further utilization of our novel molecular tool will identify crucial determinants of LSCs, thus providing a rationale for their therapeutic targeting.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/fisiologia , Elementos Facilitadores Genéticos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Regulador Transcricional ERG/genéticaRESUMO
Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the ERG+85 enhancer-based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemoresistance genes and demonstrated increased clonogenicity and resistance to chemo- and radiotherapy as compared with their tagBFPNeg counterparts. The tagBFPHigh fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasive ability. Moreover, the tagBFPHigh fraction was enriched for leukemia-initiating cells in a xenograft assay. We identified the ubiquitin hydrolase USP9X as a novel ERG transcriptional target that sustains ERG+85-positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to preferential inhibition of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional heterogeneity and suggest that targeting ERG via USP9X inhibition may be a potential treatment strategy in patients with leukemia. SIGNIFICANCE: This study couples a novel experimental tool with state-of-the-art approaches to delineate molecular mechanisms underlying stem cell-related characteristics in leukemia cells.
Assuntos
Leucemia Mieloide Aguda/genética , Proteínas Oncogênicas/metabolismo , Regulador Transcricional ERG/metabolismo , Transplante Heterólogo/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Humanos , Leucemia Mieloide Aguda/mortalidade , Camundongos , Análise de Sobrevida , TransfecçãoRESUMO
The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.
Assuntos
Elementos de DNA Transponíveis , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Transposases/genética , Animais , Sequência de Bases , Técnicas de Cultura de Células , Células HeLa , Humanos , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Articulação do Joelho/metabolismo , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Coelhos , Transfecção , Transposases/metabolismoRESUMO
A deep proteomics analysis was conducted on a primary acute myeloid leukemia culture system to identify potential protein targets regulated by miR-126. Leukemia cells were transduced either with an empty control lentivirus or one containing the sequence for miR-126, and resulting cells were analyzed using ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD001994. The proteomics data and statistical analysis described in this article is associated with a research article, "miR-126 regulates distinct self-renewal outcomes in normal and malignant hematopoietic stem cells" (Lechman et al., 2016) [1], and serves as a resource for researchers working in the field of microRNAs and their regulation of protein levels.
RESUMO
MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Ciclo Celular , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , MicroRNAs/metabolismo , Neoplasias Experimentais , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.
Assuntos
Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , MicroRNAs/fisiologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos SCID , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.
Assuntos
Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Autorrenovação Celular/genética , Redes Reguladoras de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Marcação por Isótopo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Modelos Biológicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/transplante , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.
Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Linfócitos/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Mieloides/citologia , Células Mieloides/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 3 de Transcrição/genética , Fator 3 de Transcrição/metabolismo , Transplante HeterólogoRESUMO
Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3ß pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hematopoese/genética , Células-Tronco Hematopoéticas/enzimologia , Humanos , Camundongos , MicroRNAs/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transplante HeterólogoRESUMO
Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/fisiopatologia , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Animais , Ensaio de Unidades Formadoras de Colônias , Biologia Computacional , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IL-10 is a Th2 cytokine important for inhibiting cell-mediated immunity while promoting humoral responses. Human IL-10 (hIL-10) has anti-inflammatory, immunosuppressive as well as immunostimulatory characteristics, whereas viral IL-10 (vIL-10), a homologue of hIL-10 encoded by Epstein Barr virus (EBV), lacks several immunostimulatory functions. The immunostimulatory characteristic of hIL-10 has been attributed to a single amino acid, isoleucine at position 87, which in vIL-10 is alanine. A mutant hIL-10 in which isoleucine has been substituted (mut.hIL-10) is biologically active with only immunosuppressive, but not immunostimulatory, functions, making it a potentially superior therapeutic for inflammatory diseases. To compare the efficacy of mut.hIL-10 with hIL-10 and vIL-10 in blocking the progression of rheumatoid arthritis, we used replication defective adenoviral vectors to deliver intra-articularly the gene encoding hIL-10, vIL-10 or mut.hIL-10 to antigen-induced arthritic (AIA) knee joints in rabbits. Intra-articular expression of hIL-10, vIL-10, and mut.hIL-10 resulted in significant improvement of the pathology in the treated joints to similar levels. These observed changes included a significant reduction in intra-articular leukocytosis and the degree of synovitis, as well as normalization of cartilage matrix metabolism. Our results suggest that hIL-10, vIL-10, and mut.hIL-10 are all equally therapeutic in the rabbit AIA model for treating disease pathology.
Assuntos
Artrite Experimental/patologia , Herpesvirus Humano 4/metabolismo , Interleucina-10/farmacologia , Articulação do Joelho/patologia , Mutação , Adenoviridae/genética , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Cartilagem Articular/metabolismo , Matriz Extracelular/metabolismo , Feminino , Técnicas de Transferência de Genes , Humanos , Injeções Intra-Articulares , Interleucina-10/genética , Interleucina-10/metabolismo , Ovalbumina/imunologia , CoelhosRESUMO
We previously have demonstrated the ability of primary murine bone marrow-derived DC (BM-DC), genetically modified by adenoviral infection to express FasL, to inhibit progression of established collagen-induced arthritis (CIA) following systemic delivery. Here we demonstrate that exosomes derived from genetically modified BM-DC expressing FasL are able to inhibit inflammation in a murine footpad model of delayed-type hypersensitivity (DTH). Local administration of exosomes derived from DC expressing FasL (Exo/FasL) as well as the parental DC/FasL resulted in a significant reduction in swelling in both the treated and the untreated distal paw. However, both the DC/FasL and the Exo/FasL were unable to suppress the DTH response in lpr (Fas-deficient) mice. Gene transfer of FasL to BM-DC from gld (FasL-deficient) mice resulted in restoration of the ability of DC as well as DC-derived exosomes to suppress DTH. The ability of DC-derived exosomes and DC to suppress DTH responses was antigen specific and MHC class II dependent, but class I independent. The injected exosomes were found to be internalized into CD11c(+) cells at the site of injection and in the draining popliteal lymph node. Systemic injection of exosome/FasL into mice with established CIA resulted in significant disease amelioration. These results demonstrate that both systemic and local administration of exosomes derived from FasL-expressing DC are able to suppress antigen-specific immune responses through an MHC class II-dependent pathway, resulting in effective and sustained treatment of established collagen-induced arthritis and suppression of the DTH inflammatory response. These results suggest that DC/FasL-derived exosomes could be used clinically for the treatment of inflammatory and autoimmune diseases.