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1.
Cell ; 176(6): 1265-1281.e24, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30827681

RESUMEN

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.


Asunto(s)
Leucemia Mieloide Aguda/genética , Transcriptoma/genética , Adulto , Secuencia de Bases/genética , Médula Ósea , Células de la Médula Ósea/citología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Genotipo , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/fisiopatología , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , ARN , Transducción de Señal , Análisis de la Célula Individual/métodos , Microambiente Tumoral , Secuenciación del Exoma Completo/métodos
2.
Leukemia ; 33(8): 2061-2077, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30705411

RESUMEN

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/genética
3.
Blood ; 133(20): 2198-2211, 2019 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-30796022

RESUMEN

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/análisis
4.
Cell Rep ; 25(5): 1109-1117.e5, 2018 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-30380403

RESUMEN

Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts.


Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Leucemia/metabolismo , Estrés Fisiológico , Factor de Transcripción Activador 4/metabolismo , Animales , Supervivencia Celular , Citoprotección , Factor 2 Eucariótico de Iniciación/metabolismo , Sangre Fetal/citología , Genes Reporteros , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Células Madre Multipotentes/metabolismo , Fosforilación , Regulación hacia Arriba , Valina/deficiencia
6.
Science ; 360(6386): 331-335, 2018 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-29674595

RESUMEN

Gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) arise primarily in the midline of the central nervous system of young children, suggesting a cooperation between genetics and cellular context in tumorigenesis. Although the genetics of H3K27M-glioma are well characterized, their cellular architecture remains uncharted. We performed single-cell RNA sequencing in 3321 cells from six primary H3K27M-glioma and matched models. We found that H3K27M-glioma primarily contain cells that resemble oligodendrocyte precursor cells (OPC-like), whereas more differentiated malignant cells are a minority. OPC-like cells exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are at least in part sustained by PDGFRA signaling. Our study characterizes oncogenic and developmental programs in H3K27M-glioma at single-cell resolution and across genetic subclones, suggesting potential therapeutic targets in this disease.


Asunto(s)
Neoplasias Encefálicas/patología , Carcinogénesis/genética , Glioma/patología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Oncogenes , Neoplasias Encefálicas/genética , Proliferación Celular , Glioma/genética , Histonas/metabolismo , Humanos , Proteína Quinasa 7 Activada por Mitógenos/genética , Mutación , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos
7.
Cancer Cell ; 33(1): 29-43.e7, 2018 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-29249691

RESUMEN

Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo. Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo.


Asunto(s)
Citocinas/metabolismo , Inflamación/tratamiento farmacológico , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Janus Quinasa 2/genética , Ratones Transgénicos , Mutación/efectos de los fármacos , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos
8.
Cell Stem Cell ; 20(2): 233-246.e7, 2017 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-27989769

RESUMEN

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.


Asunto(s)
Ensamble y Desensamble de Cromatina , Resistencia a Antineoplásicos , Glioblastoma/patología , Células Madre Neoplásicas/patología , Acetilación/efectos de los fármacos , Secuencia de Bases , Biomarcadores de Tumor/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Elementos de Facilitación Genéticos/genética , Glioblastoma/metabolismo , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Metilación/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos
10.
Cancer Cell ; 29(2): 214-28, 2016 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-26832662

RESUMEN

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/metabolismo
11.
Mol Cell ; 61(1): 170-80, 2016 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-26687680

RESUMEN

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.


Asunto(s)
Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina/métodos , Cromatina/metabolismo , Células Madre Hematopoyéticas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/metabolismo , Leucemia/metabolismo , Reacción en Cadena de la Polimerasa Multiplex/métodos , Cromatina/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Código de Barras del ADN Taxonómico , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Histonas/genética , Humanos , Células K562 , Leucemia/genética , Mutación
12.
Blood ; 126(16): 1930-9, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26320100

RESUMEN

Fetal hemoglobin (HbF, α2γ2) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and EHMT2 histone methyltransferases, induces γ-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing γ-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA-mediated knockdown significantly increased γ-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34(+) hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic ß-globin genes Hbb-εy and Hbb-ßh1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the γ-globin gene region. In RNA-sequencing analysis of erythroblasts, γ-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in γ-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in γ-globin repression and represent a novel therapeutic target for SCD.


Asunto(s)
Epigénesis Genética/efectos de los fármacos , Eritroblastos/metabolismo , Hemoglobina Fetal/biosíntesis , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Quinazolinas/farmacología , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Animales , Línea Celular Tumoral , Eritroblastos/citología , Células Eritroides/citología , Células Eritroides/metabolismo , Femenino , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Masculino , Ratones
13.
Exp Hematol ; 43(9): 756-9, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26143581

RESUMEN

Transplantation of hematopoietic stem cells (HSCs) to treat hematologic disorders is routinely used in the clinic. However, HSC therapy is hindered by the requirements of finding human leukocyte antigen (HLA)-matched donors and attaining sufficient numbers of long-term HSCs in the graft. Therefore, ex vivo expansion of transplantable HSCs remains one of the "holy grails" of hematology. Without the ability to maintain and expand human HSCs in vitro, two complementary approaches involving cellular reprogramming to generate transplantable HSCs have emerged. Reprogrammed HSCs represent a potentially inexhaustible supply of autologous tissue. On March 18th, 2015, Dr. George Q. Daley and Dr. Derrick J. Rossi, two pioneers in the field, presented and discussed their most recent research on these topics in a webinar organized by the International Society for Experimental Hematology (ISEH). Here, we summarize these seminars and discuss the possibilities and challenges in the field of hematopoietic specification.


Asunto(s)
Enfermedades Hematológicas , Células Madre Hematopoyéticas , Células Madre Pluripotentes , Animales , Reprogramación Celular , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Enfermedades Hematológicas/patología , Enfermedades Hematológicas/terapia , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología
14.
Phytochemistry ; 110: 166-71, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25482220

RESUMEN

Glucosinolates are secondary plant compounds typically found in members of the Brassicaceae and a few other plant families. Usually each plant species contains a specific subset of the ∼ 130 different glucosinolates identified to date. However, intraspecific variation in glucosinolate profiles is commonly found. Sinalbin (4-hydroxybenzyl glucosinolate) so far has been identified as the main glucosinolate of the heavy metal accumulating plant species Noccaea caerulescens (Brassicaceae). However, a screening of 13 N. caerulescens populations revealed that in 10 populations a structurally related glucosinolate was found as the major component. Based on nuclear magnetic resonance (NMR) and mass spectrometry analyses of the intact glucosinolate as well as of the products formed after enzymatic conversion by sulfatase or myrosinase, this compound was identified as 4-α-rhamnosyloxy benzyl glucosinolate (glucomoringin). So far, glucomoringin had only been reported as the main glucosinolate of Moringa spp. (Moringaceae) which are tropical tree species. There was no apparent relation between the level of soil pollution at the location of origin, and the presence of glucomoringin. The isothiocyanate that is formed after conversion of glucomoringin is a potent antimicrobial and antitumor agent. It has yet to be established whether glucomoringin or its breakdown product have an added benefit to the plant in its natural habitat.


Asunto(s)
Brassicaceae/química , Glucosinolatos/aislamiento & purificación , Isotiocianatos/aislamiento & purificación , Brassicaceae/genética , Europa (Continente) , Glucosinolatos/análisis , Glucosinolatos/química , Glicósido Hidrolasas , Isotiocianatos/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular
15.
Nature ; 510(7504): 268-72, 2014 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-24776803

RESUMEN

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.


Asunto(s)
Estrés del Retículo Endoplásmico , Células Madre Hematopoyéticas/citología , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Xenoinjertos , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteína Fosfatasa 1/metabolismo , Transducción de Señal , Factor de Transcripción CHOP/metabolismo , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/metabolismo
16.
Cell Stem Cell ; 14(1): 94-106, 2014 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-24388174

RESUMEN

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 Heterólogo
17.
Nat Med ; 20(1): 29-36, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24292392

RESUMEN

Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (CICs) in tumors that survive therapy. Together with recent reports showing that CIC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key 'stemness' property unique to CICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation and, more specifically, human colorectal CIC function are dependent on the canonical self-renewal regulator BMI-1. Downregulation of BMI-1 inhibits the ability of colorectal CICs to self-renew, resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with a small-molecule BMI-1 inhibitor resulted in colorectal CIC loss with long-term and irreversible impairment of tumor growth. Targeting the BMI-1-related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer.


Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Compuestos Heterocíclicos con 2 Anillos/farmacología , Recurrencia Local de Neoplasia/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Tiazoles/farmacología , Animales , Western Blotting , Bromodesoxiuridina , Línea Celular Tumoral , Citometría de Flujo , Vectores Genéticos/genética , Compuestos Heterocíclicos con 2 Anillos/uso terapéutico , Humanos , Luciferasas , Ratones Endogámicos NOD , Ratones SCID , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/genética , Tiazoles/uso terapéutico
18.
Antimicrob Agents Chemother ; 57(10): 4794-800, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23877685

RESUMEN

The emergence of resistance against current antibiotics calls for the development of new compounds to treat infectious diseases. Synthetic pantothenamides are pantothenate analogs that possess broad-spectrum antibacterial activity in vitro in minimal media. Pantothenamides were shown to be substrates of the bacterial coenzyme A (CoA) biosynthetic pathway, causing cellular CoA depletion and interference with fatty acid synthesis. In spite of their potential use and selectivity for bacterial metabolic routes, these compounds have never made it to the clinic. In the present study, we show that pantothenamides are not active as antibiotics in the presence of serum, and we found that they were hydrolyzed by ubiquitous pantetheinases of the vanin family. To address this further, we synthesized a series of pantetheinase inhibitors based on a pantothenate scaffold that inhibited serum pantetheinase activity in the nanomolar range. Mass spectrometric analysis showed that addition of these pantetheinase inhibitors prevented hydrolysis of pantothenamides by serum. We found that combinations of these novel pantetheinase inhibitors and prototypic pantothenamides like N5-Pan and N7-Pan exerted antimicrobial activity in vitro, particularly against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes) even in the presence of serum. These results indicate that pantothenamides, when protected against degradation by host pantetheinases, are potentially useful antimicrobial agents.


Asunto(s)
Antibacterianos/farmacología , Bacterias Grampositivas/efectos de los fármacos , Ácido Pantoténico/farmacología , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Ácido Pantoténico/análogos & derivados , Ácido Pantoténico/química , Staphylococcus aureus/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pyogenes/efectos de los fármacos
19.
Science ; 339(6119): 543-8, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23239622

RESUMEN

Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.


Asunto(s)
Evolución Clonal/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/genética , Animales , Linaje de la Célula , Rastreo Celular , Células Clonales , Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN , Humanos , Lentivirus , Ratones , Trasplante de Neoplasias , Transcriptoma , Transducción Genética , Células Tumorales Cultivadas
20.
Cell Stem Cell ; 11(6): 799-811, 2012 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-23142521

RESUMEN

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 Heterólogo
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