Asunto(s)
Leucemia Megacarioblástica Aguda , Humanos , Leucemia Megacarioblástica Aguda/inmunología , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patología , Ratones , Animales , Niño , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Inmunoterapia Adoptiva , Femenino , Masculino , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/inmunología , Proteínas de Fusión Oncogénica/metabolismoRESUMEN
BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A. METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).
Asunto(s)
Anemia de Células Falciformes , Hemoglobina Fetal , Trasplante de Células Madre Hematopoyéticas , Adolescente , Adulto , Niño , Femenino , Humanos , Masculino , Adulto Joven , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Antígenos CD34 , Busulfano/uso terapéutico , Sistemas CRISPR-Cas , Hemoglobina Fetal/biosíntesis , Hemoglobina Fetal/genética , Edición Génica , Células Madre Hematopoyéticas , Proteínas Represoras , Acondicionamiento Pretrasplante , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Agonistas Mieloablativos/uso terapéutico , Europa (Continente) , América del NorteRESUMEN
The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.
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Receptor 1 de Folato , Inmunoterapia Adoptiva , Leucemia Megacarioblástica Aguda , Proteínas de Fusión Oncogénica , Animales , Niño , Preescolar , Humanos , Lactante , Modelos Animales de Enfermedad , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Leucemia Megacarioblástica Aguda/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Linfocitos T , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.
Asunto(s)
Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/farmacología , Evolución Clonal , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/normas , Sinergismo Farmacológico , Femenino , GTP Fosfohidrolasas/genética , Humanos , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/mortalidad , Leucemia Mielomonocítica Crónica/patología , Proteínas de la Membrana/genética , Ratones , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , ARN Interferente Pequeño/genética , Receptor Notch1/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
PURPOSE: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling. EXPERIMENTAL DESIGN: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N = 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N = 24) and without (N = 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes. RESULTS: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old (P < 0.001), and the presence of this fusion was highly associated with adverse outcome (P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFß, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts. CONCLUSIONS: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.
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Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Leucemia Mieloide Aguda/genética , MicroARNs/genética , Mutación , Proteínas de Fusión Oncogénica/genética , Adulto , Antígeno CD56/genética , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Pronóstico , ARN Mensajero , Receptores de GABA-A/genética , Adulto JovenRESUMEN
The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Tratamiento Basado en Trasplante de Células y Tejidos , Células Madre Hematopoyéticas/citología , Hidrogeles/farmacología , Animales , Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Sangre Fetal/citología , Sangre Fetal/metabolismo , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Biomarcadores , Ciclo Celular/genética , Línea Celular Tumoral , Biología Computacional/métodos , ADN Metiltransferasa 3A , Modelos Animales de Enfermedad , Duplicación de Gen , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Ratones Transgénicos , Mutación , Nucleofosmina , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Secuencias Repetidas en Tándem , Sitio de Iniciación de la Transcripción , TranscriptomaRESUMEN
Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both in vitro and in vivo systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34+ and CD34+CD45RA- hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice. The ability of UM171 to enhance gene transfer to primitive cord blood hematopoietic cells extended to multiple lentiviral pseudotypes, gamma retroviruses, and non-integrating lentiviruses and to adult bone marrow cells. UM171, thus, provides an interesting reagent for improving the ex vivo production of gene-modified cells and for reducing requirements of virus for a broad range of applications.
RESUMEN
Retroviral integration site analysis and barcoding have been instrumental for multiplex clonal fate mapping, although their use imposes an inherent delay between sample acquisition and data analysis. Monitoring of multiple cell populations in real time would be advantageous, but multiplex assays compatible with flow cytometric tracking of competitive growth behavior are currently limited. We here describe the development and initial validation of three generations of lentiviral fluorescent genetic barcoding (FGB) systems that allow the creation of 26, 14, or 6 unique labels. Color-coded populations could be tracked in multiplex in vitro assays for up to 28 days by flow cytometry using all three vector systems. Those involving lower levels of multiplexing eased color-code generation and the reliability of vector expression and enabled functional in vitro and in vivo studies. In proof-of-principle experiments, FGB vectors facilitated in vitro multiplex screening of microRNA (miRNA)-induced growth advantages, as well as the in vivo recovery of color-coded progeny of murine and human hematopoietic stem cells. This novel series of FGB vectors provides new tools for assessing comparative growth properties in in vitro and in vivo multiplexing experiments, while simultaneously allowing for a reduction in sample numbers by up to 26-fold.
Asunto(s)
Rastreo Celular/métodos , Expresión Génica , Genes Reporteros , Vectores Genéticos/genética , Lentivirus/genética , Proteínas Luminiscentes/genética , Diferenciación Celular , Codón , Citometría de Flujo , Orden Génico , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Proteínas Luminiscentes/metabolismo , MicroARNs/genética , Reproducibilidad de los Resultados , Transducción GenéticaRESUMEN
The hematopoietic syndrome of acute radiation syndrome (h-ARS) is characterized by severe bone marrow aplasia, resulting in a significant risk for bleeding, infections, and death. To date, clinical management of h-ARS is limited to supportive care dictated by the level of radiation exposure, with a high incidence of mortality in those exposed to high radiation doses. The ideal therapeutic agent would be an immediately available, easily distributable single-agent therapy capable of rapid in vivo hematopoietic reconstitution until recovery of autologous hematopoiesis occurs. Using a murine model of h-ARS, we herein demonstrate that infusion of ex vivo expanded murine hematopoietic stem and progenitor cells (HSPCs) into major histocompatibility complex mismatched recipient mice exposed to a lethal dose of ionizing radiation (IR) led to rapid myeloid recovery and improved survival. Survival benefit was significant in a dose-dependent manner even when infusion of the expanded cell therapy was delayed 3 days after lethal IR exposure. Most surviving mice (80%) demonstrated long-term in vivo persistence of donor T cells at low levels, and none had evidence of graft versus host disease. Furthermore, survival of donor-derived skin grafts was significantly prolonged in recipients rescued from h-ARS by infusion of the mismatched expanded cell product. These findings provide evidence that ex vivo expanded mismatched HSPCs can provide rapid, high-level hematopoietic reconstitution, mitigate IR-induced mortality, and convey donor-specific immune tolerance in a murine h-ARS model. Stem Cells Translational Medicine 2017;6:566-575.
Asunto(s)
Síndrome de Radiación Aguda/terapia , Proliferación Celular , Supervivencia de Injerto , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de la radiación , Antígenos de Histocompatibilidad/inmunología , Histocompatibilidad , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Trasplante de Piel , Tolerancia al Trasplante , Síndrome de Radiación Aguda/inmunología , Síndrome de Radiación Aguda/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Recuperación de la Función , Transducción de Señal , Factores de TiempoRESUMEN
Cord blood transplantation (CBT) recipients are at increased risk for delayed engraftment and primary graft failure, complications that are often indistinguishable early post-transplantation. Current assays fail to accurately identify recipients with slow hematopoietic recovery and distinguish them from those with pending graft failure. To address this, we prospectively examined the kinetics of immune cell subset recovery in the peripheral blood of 39 patients on days +7 and +14 after double-unit CBT (dCBT) by multiparametric flow cytometry analysis, which we term real-time immunophenotyping (RTIP). RTIP analysis at day +14 revealed distinctive patterns of reconstitution and, importantly, identified patients with slow hematopoietic recovery who went on to engraft. Strikingly, higher absolute numbers of circulating monocytes and natural killer cells at day +14 were predictive of engraftment, but only the absolute number of circulating monocytes was significantly correlated with time to engraftment. This is the first evidence that RTIP on patient peripheral blood mononuclear cells early after dCBT is technically feasible and can be used as a "signature" for predicting the kinetics of hematopoietic recovery. Furthermore, RTIP is a time- and cost-efficient methodology that has the potential to become a clinically feasible diagnostic tool to guide therapeutic interventions in high-risk patients; therefore, its utility should be evaluated in a large cohort of patients.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Supervivencia de Injerto , Inmunofenotipificación , Leucocitos/citología , Adolescente , Adulto , Niño , Trasplante de Células Madre de Sangre del Cordón Umbilical/normas , Funcionamiento Retardado del Injerto , Femenino , Citometría de Flujo , Humanos , Cinética , Leucocitos/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Adulto JovenRESUMEN
Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.
Asunto(s)
Herpesvirus Humano 1/inmunología , Inmunidad Celular , Células Asesinas Naturales/inmunología , Leucemia/inmunología , Rayos Ultravioleta , Inactivación de Virus/efectos de la radiación , Degranulación de la Célula/inmunología , Movimiento Celular/inmunología , Femenino , Humanos , Interleucina-15/inmunología , Interleucina-2/inmunología , Células Jurkat , Masculino , FN-kappa B/inmunología , Proteína Quinasa C/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 2/inmunologíaRESUMEN
Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.
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Biomarcadores de Tumor/metabolismo , Proliferación Celular , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animales , Separación Celular , Células Cultivadas , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Células Madre Neoplásicas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Análisis de SupervivenciaRESUMEN
Leukemic transformation of human cells is a complex process. Here we show that forced expression of MN1 in primitive human cord blood cells maintained on stromal cells in vitro induces a transient, but not serially transplantable, myeloproliferation in engrafted mice. However, cotransduction of an activated HOX gene (NUP98HOXD13) with MN1 induces a serially transplantable acute myeloid leukemia (AML). Further characterization of the leukemic cells generated from the dually transduced cells showed the activation of stem cell gene expression signatures also found in primary human AML. These findings show a new forward genetic model of human leukemogenesis and further highlight the relevance of homeobox transcription factors in the transformation process.
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Transformación Celular Neoplásica/metabolismo , Sangre Fetal/metabolismo , Leucemia Mieloide Aguda/metabolismo , Neoplasias Experimentales/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteínas de Fusión Oncogénica/genética , Transactivadores , Proteínas Supresoras de Tumor/genéticaRESUMEN
The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.
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Sangre Fetal/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Indoles/farmacología , Pirimidinas/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Regeneración/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Sangre Fetal/citología , Sangre Fetal/fisiología , Terapia Genética/métodos , Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/fisiología , Humanos , Huésped Inmunocomprometido , Indoles/química , Ratones , Pirimidinas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs), yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast, highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations, one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1), have little intravenous engraftment potential, and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However, varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect.
Asunto(s)
Células Madre Adultas/metabolismo , Aldehído Deshidrogenasa/metabolismo , Antígenos de Diferenciación/metabolismo , Fase G1/fisiología , Supervivencia de Injerto/fisiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/trasplante , Animales , Antígenos de Diferenciación/genética , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Ratones Transgénicos , Trasplante HomólogoRESUMEN
Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34(+) and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34(+) cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.
Asunto(s)
Diferenciación Celular , Células Eritroides/citología , Células Eritroides/metabolismo , Factores de Transcripción/metabolismo , Antígenos CD34/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Factor de Unión a CCCTC , Diferenciación Celular/efectos de los fármacos , Cromatina/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , Proteínas Co-Represoras , Dimetilsulfóxido/farmacología , Células Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HL-60 , Hematopoyesis/efectos de los fármacos , Humanos , Células K562 , Células Mieloides/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacosRESUMEN
High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes.
Asunto(s)
Aldehído Deshidrogenasa/fisiología , Linfocitos B/enzimología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/enzimología , Aldehído Deshidrogenasa/biosíntesis , Aldehído Deshidrogenasa/deficiencia , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Aldehídos/metabolismo , Animales , Animales Congénicos , Linfocitos B/citología , Trasplante de Médula Ósea , Recuento de Células , Ciclo Celular/fisiología , Linaje de la Célula , Células Cultivadas/citología , Células Cultivadas/metabolismo , Ensayo de Unidades Formadoras de Colonias , Daño del ADN , Inducción Enzimática , Regulación de la Expresión Génica/fisiología , Células Madre Hematopoyéticas/citología , Linfopenia/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quimera por Radiación , Especies Reactivas de Oxígeno/metabolismo , Retinal-Deshidrogenasa , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: Leukemia-initiating cells (LICs) have been the subject of considerable investigation because of their ability to self-renew and maintain leukemia. Thus, selective targeting and killing of LICs would provide highly efficient and novel therapeutic strategies. Here we explored whether we could use a canine leukemia cell line (G374) derived from a dog that received HOXB4-transduced repopulating cells to study leukemia in the murine xenograft model and the dog. MATERIALS AND METHODS: G374 cells were infused in dogs intravenously and in nonobese diabetic/severe combined immunodeficient and nonobese diabetic/severe combined immunodeficient/IL2Rγ(null) mice either intravenously or directly into the bone cavity. Animals were bled to track engraftment and proliferation of G374 cells, and were sacrificed when they appeared ill. RESULTS: We found that canine LICs are capable of sustained in vitro self-renewal while maintaining their ability to induce acute myeloid leukemia, which resembles human disease in both dogs and mice. Furthermore, we developed a novel strategy for the quantification of LIC frequency in large animals and showed that this frequency was highly comparable to that determined by limited dilution in mouse xenotransplants. We also demonstrated, using single-cell analysis, that LICs are heterogeneous in their self-renewal capacity and regenerate a leukemic cell population consistent with a hierarchical leukemia model. CONCLUSIONS: The availability of this novel framework should accelerate the characterization of LICs and the translation of animal studies into clinical trials.