RESUMEN
Hematopoietic stem cell (HSC) dormancy is understood as supportive of HSC function and its long-term integrity. Although regulation of stress responses incurred as a result of HSC activation is recognized as important in maintaining stem cell function, little is understood of the preventive machinery present in human HSCs that may serve to resist their activation and promote HSC self-renewal. We demonstrate that the transcription factor PLAG1 is essential for long-term HSC function and, when overexpressed, endows a 15.6-fold enhancement in the frequency of functional HSCs in stimulatory conditions. Genome-wide measures of chromatin occupancy and PLAG1-directed gene expression changes combined with functional measures reveal that PLAG1 dampens protein synthesis, restrains cell growth and division, and enhances survival, with the primitive cell advantages it imparts being attenuated by addition of the potent translation activator, c-MYC. We find PLAG1 capitalizes on multiple regulatory factors to ensure protective diminished protein synthesis including 4EBP1 and translation-targeting miR-127 and does so independently of stress response signaling. Overall, our study identifies PLAG1 as an enforcer of human HSC dormancy and self-renewal through its highly context-specific regulation of protein biosynthesis and classifies PLAG1 among a rare set of bona fide regulators of messenger RNA translation in these cells. Our findings showcase the importance of regulated translation control underlying human HSC physiology, its dysregulation under activating demands, and the potential if its targeting for therapeutic benefit.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas , Factores de Transcripción , Diferenciación Celular/fisiología , Proliferación Celular , Autorrenovación de las Células , Células Madre Hematopoyéticas/metabolismo , Humanos , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide Aguda/genética , Células Madre Neoplásicas/metabolismo , Animales , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Ratones Endogámicos NOD , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/patología , Regulación hacia Arriba , Quinasas p21 Activadas/análisisRESUMEN
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.
Asunto(s)
Leucemia Mieloide Aguda , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
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.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Ratones Endogámicos NOD , Modelos Animales , Trasplante Heterólogo/inmunología , Trasplante Heterólogo/métodos , Animales , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Separación Celular , Femenino , Sangre Fetal/citología , Sangre Fetal/inmunología , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Masculino , RatonesRESUMEN
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.
Asunto(s)
Leucemia Mieloide Aguda , MicroARNs , Línea Celular Tumoral , Autorrenovación de las Células/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , MicroARNs/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.
Asunto(s)
Proteómica/métodos , Análisis de la Célula Individual/métodos , Humanos , Leucemia Mieloide Aguda , Espectrometría de Masas , Células Madre Neoplásicas , Proteoma/metabolismo , ARN , Flujo de TrabajoRESUMEN
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.
Asunto(s)
Leucemia Mieloide Aguda , Nicotinamida Fosforribosiltransferasa , Apoptosis , Homeostasis , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Lípidos , Células Madre Neoplásicas , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Células MadreRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/genética , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide/genética , Preleucemia/genética , Animales , Antígenos CD34/análisis , Proteínas de Ciclo Celular/metabolismo , Linaje de la Célula , Proliferación Celular , Transformación Celular Neoplásica , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 21/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Síndrome de Down/complicaciones , Femenino , Factor de Transcripción GATA1/metabolismo , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Xenoinjertos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Hígado/embriología , Masculino , Megacariocitos/fisiología , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Preleucemia/metabolismo , Preleucemia/patología , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-kit/análisis , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , CohesinasRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Diferenciación Celular/genética , Proliferación Celular/genética , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Electroporación/métodos , Femenino , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/fisiología , Elementos de Facilitación Genéticos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Regulador Transcripcional ERG/genéticaRESUMEN
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.
Asunto(s)
Leucemia Mieloide Aguda/genética , Proteínas Oncogénicas/metabolismo , Regulador Transcripcional ERG/metabolismo , Trasplante Heterólogo/métodos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Leucemia Mieloide Aguda/mortalidad , Ratones , Análisis de Supervivencia , TransfecciónRESUMEN
We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoensayo/métodos , Elementos de la Serie de los Lantanoides/química , Espectrometría de Masas/métodos , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Células K562 , Factor de Crecimiento Derivado de Plaquetas/análisis , Proteína p53 Supresora de Tumor/análisisRESUMEN
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.
Asunto(s)
Elementos Transponibles de ADN , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Transposasas/genética , Animales , Secuencia de Bases , Técnicas de Cultivo de Célula , Células HeLa , Humanos , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Articulación de la Rodilla/metabolismo , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Conejos , Transfección , Transposasas/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis , Ciclo Celular , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , MicroARNs/metabolismo , Neoplasias Experimentales , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas/patología , Leucemia Mieloide Aguda/patología , MicroARNs/fisiología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones SCID , MicroARNs/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Autorrenovación de las Células/genética , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Marcaje Isotópico , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/trasplante , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
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.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Células Madre Hematopoyéticas/citología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Linfocitos/citología , Animales , Biomarcadores/metabolismo , Western Blotting , Proliferación Celular , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Mieloides/citología , Células Mieloides/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción 3/genética , Factor de Transcripción 3/metabolismo , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , MicroARNs/metabolismo , Animales , Línea Celular , Proliferación Celular , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hematopoyesis/genética , Células Madre Hematopoyéticas/enzimología , Humanos , Ratones , MicroARNs/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Trasplante HeterólogoRESUMEN
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.