RESUMEN
Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.
Asunto(s)
Receptores de Trombopoyetina/metabolismo , Trombopoyetina/metabolismo , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Epítopos/inmunología , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ligandos , Megacariocitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoyetina/inmunología , Receptores de Trombopoyetina/fisiología , Transducción de Señal/fisiología , Trombocitemia Esencial/metabolismo , Trombopoyetina/fisiologíaRESUMEN
Thrombopoietin (Thpo) and its receptor myeloid proliferative leukemia (Mpl) were initially identified as the cytokine signaling that stimulates megakaryopoiesis and platelet production. However, Thpo-Mpl signaling has also been widely characterized as one of the few cytokine systems that directly regulates hematopoietic stem and progenitor cells. The ability of Thpo signaling to stimulate hematopoietic stem cell (HSC) self-renewal has led to the development and utilization of Thpo mimetic drugs to treat hematopoietic diseases with restricted function of HSCs, such as aplastic anemia. This review will cover the mechanisms by which Thpo-Mpl signaling regulates HSCs.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Trombopoyetina/fisiología , Animales , Autorrenovación de las Células/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Receptores de Trombopoyetina/fisiología , Transducción de Señal/efectos de los fármacos , Trombopoyesis/fisiología , Trombopoyetina/farmacologíaRESUMEN
Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL+ polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Receptores de Trombopoyetina/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Xenoinjertos , Humanos , Interferón Tipo I/metabolismo , Ratones , Receptores de Trombopoyetina/agonistas , Receptores de Trombopoyetina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for MPN HSPC expansion is not fully understood, limiting the effectiveness of current treatments. Endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs, suggesting that ECs are involved in the pathogenesis of MPNs. Here we report that JAK2V617F-bearing primary murine ECs have increased cell proliferation and angiogenesis in vitro compared to JAK2WT ECs. While there was no difference between JAK2V617F and JAK2WT HSPC proliferation when co-cultured with JAK2WT EC, the JAK2V617F HSPC displayed a relative growth advantage over the JAK2WT HSPC when co-cultured on JAK2V617F EC. In addition, the thrombopoietin (TPO) receptor MPL is up regulated in JAK2V617F ECs and contributes to the maintenance/expansion of the JAK2V617F clone over JAK2WT clone in vitro. Considering that ECs are an essential component of the hematopoietic niche and most HSPCs reside in the perivascular niche, our studies suggest that the JAK2V617F-bearing ECs form an important component of the MPN vascular niche and contribute to mutant stem/progenitor cell expansion, likely through a critical role of the TPO/MPL signaling axis.
Asunto(s)
Proliferación Celular/genética , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/genética , Receptores de Trombopoyetina/fisiología , Animales , Técnicas de Cocultivo , Células Endoteliales/patología , Humanos , Ratones , Mutación Missense , Trastornos Mieloproliferativos/patología , Neoplasias/genética , Neovascularización Patológica , Receptores de Trombopoyetina/metabolismo , Nicho de Células MadreRESUMEN
BACKGROUND: Rather than a Janus Kinase 2 inhibitor (ruxolitinib), a specific thrombopoietin receptor (TpoR) inhibitor would be more specific for the treatment of myeloproliferative neoplasms due to TpoR mutations. OBJECTIVE: A cell-based phenotypic approach to identify specific TpoR inhibitors was implemented and a library of 505,483 small molecules was screened for inhibitory effects on cells transformed by TpoR mutants. RESULTS: Among the identified hits are two analogs of 3-(4-piperidinyl) indole. The analogs showed about five-fold preferential inhibition of cell viability towards Ba/F3 cells expressing the TpoR W515L mutation compared to the parental cells. There was no significant difference in inhibition of cell viability between the TpoR wild type and the TpoR W515L mutant cells. Preferential inhibition of viability was observed in Ba/F3 cells expressing erythropoietin receptor (EpoR) when stimulated with Epo compared to stimulation with interleukin-3 (IL3). The indole analog inhibited ex vivo colony formations of primary bone marrow cells from heterozygous JAK2 V617F knock-in mice. Drug combination treatment study was performed using ruxolitinib and the indole analog. Drug synergistic effects were observed when cells were stimulated to proliferate through both the IL3 and TpoR pathways. Our compound specifically targets monoamine receptors in the rhodopsin-like receptor family of G protein-coupled receptor. CONCLUSION: This screen has identified a monoamine receptor inhibitor that can inhibit viability of cells with active TpoR or EpoR signalings. Drug synergism with ruxolitib is demonstrated.
Asunto(s)
Mutación , Trastornos Mieloproliferativos/fisiopatología , Receptores de Trombopoyetina/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas , Médula Ósea/patología , Línea Celular , Sinergismo Farmacológico , Ensayos Analíticos de Alto Rendimiento , Humanos , Nitrilos , Pirazoles/farmacología , Pirimidinas , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
NSCLC accounts for over 80% of all lung cancers and is associated with poor prognosis. Human nuclear distribution C (hNUDC) was predicted to be the target gene of microRNA-194 (miR-194). The present study was designed to demonstrate the mechanism of miR-194 in the regulation of non-small cell lung cancer (NSCLC) via targeting the hNUDC. The hNUDC expression was found to strongly be increased while the miR-194 decreased significantly in the NSCLC cell lines when compared with the healthy controls. Moreover, the luciferase report confirmed the targeting reaction between miR-194 and hNUDC. After transfection with miR-194 mimic into NSCLC cells, we found that the miR-194 overexpression resulted in abnormal nuclear division, decreased cell proliferation and inhibited the expression of hNUDC and Mpl/ERK pathway proteins. Furthermore, the hNUDC overexpression affected the suppression effect of miR-194 in 95D cells, indicating that miR-194 suppresses tumor cell process by inhibiting the hNUDC expression. In brief, the present study suggests that the upregulation of miR-194 affects the hNUDC expression, leading to a downregulated expression of Mpl/ERK pathway proteins, and suppresses the mitosis and proliferation of NSCLC cells. These results offer a potential therapeutic strategy for the treatment of lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular/genética , Neoplasias Pulmonares/patología , MicroARNs/fisiología , Proteínas Nucleares/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/terapia , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/terapia , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Receptores de Trombopoyetina/antagonistas & inhibidores , Receptores de Trombopoyetina/fisiologíaRESUMEN
The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis.
Asunto(s)
Mutación Missense , Receptores de Trombopoyetina/genética , Trombocitopenia/genética , Trombopoyetina/metabolismo , Adulto , Sustitución de Aminoácidos , Células Cultivadas , Niño , Preescolar , Familia , Femenino , Células HeLa , Homeostasis/genética , Humanos , Leucina/genética , Masculino , Linaje , Prolina/genética , Receptores de Trombopoyetina/fisiología , Transducción de Señal/genética , Trombocitopenia/metabolismo , Trombocitosis/genética , Adulto JovenRESUMEN
Thrombopoietin confers immediate protection against injury caused by ischemia/reperfusion in the rat heart. Eltrombopag is a small molecule agonist of the thrombopoietin receptor, the physiologic target of thrombopoietin. However, the ability of eltrombopag and thrombopoietin to protect human cardiac myocytes against injury and the mechanisms underlying myocyte protection are not known. Human cardiac myocytes (n = 6-10/group) were treated with eltrombopag (0.1-30.0 µM) or thrombopoietin (0.1-30.0 ng/ml) and then subjected to 5 hours of hypoxia (95% N2/5% CO2) and 16 hours of reoxygenation to determine their ability to confer resistance to myocardial injury. The thrombopoietin receptor c-Mpl was detected in unstimulated human cardiac myocytes by Western blotting. Eltrombopag and thrombopoietin confer immediate protection to human cardiac myocytes against injury from hypoxia/reoxygenation by decreasing necrotic and apoptotic cell death in a concentration-dependent manner, with an optimal concentration of 3 µM for eltrombopag and 1.0 ng/ml for thrombopoietin. The extent of protection conferred with eltrombopag is equivalent to that of thrombopoietin. Eltrombopag and thrombopoietin activate multiple prosurvival pathways; inhibition of Janus kinase-2, proto-oncogene tyrosine-protein kinase, protein kinase B/phosphatidylinositol-3 kinase, p44/42 mitogen-activated protein kinase (MAPK), and p38 MAPK abolished cardiac myocyte protection by eltrombopag and thrombopoietin. Eltrombopag and thrombopoietin may represent important and potent agents for immediately and substantially increasing protection of human cardiac myocytes, and may offer a long-lasting benefit through activation of prosurvival pathways during ischemia.
Asunto(s)
Benzoatos/farmacología , Cardiotónicos/farmacología , Hidrazinas/farmacología , Miocitos Cardíacos/fisiología , Pirazoles/farmacología , Receptores de Trombopoyetina/agonistas , Receptores de Trombopoyetina/fisiología , Transducción de Señal/fisiología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Miocitos Cardíacos/efectos de los fármacos , Proto-Oncogenes Mas , Transducción de Señal/efectos de los fármacos , Trombopoyetina/farmacologíaRESUMEN
The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F(+) transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F(+) mice, JAK2V617F(+)Mpl(-/-) mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl(+/-) mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F(+)Tpo(-/-) mice, although thrombocytosis was reduced compared with JAK2V617F(+) mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F(+) MPNs, highlighting an entirely novel target for therapeutic intervention.
Asunto(s)
Células Madre Hematopoyéticas/citología , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/metabolismo , Receptores de Trombopoyetina/genética , Animales , Células de la Médula Ósea/citología , Proliferación Celular , Citocinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Hematopoyesis , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Trombopoyetina/fisiología , Células Madre/citologíaRESUMEN
Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC. We show that TBI-induced migration of megakaryocytes to the endosteal niche depends on thrombopoietin signaling through the c-MPL receptor on megakaryocytes, as well as CD41 integrin-mediated adhesion. Moreover, niche osteoblast proliferation post-TBI required megakaryocyte-secreted platelet-derived growth factor-BB. Furthermore, blockade of c-MPL-dependent megakaryocyte migration and function after TBI resulted in a significant decrease in donor HSC engraftment in primary and competitive secondary transplantation assays. Finally, we administered thrombopoietin to mice beginning 5 days before marrow radioablation and ending 24 hours before transplant to enhance megakaryocyte function post-TBI, and found that this strategy significantly enhanced donor HSC engraftment, providing a rationale for improving hematopoietic recovery and perhaps overall outcome after clinical HSC transplantation.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Osteoblastos/citología , Receptores de Trombopoyetina/fisiología , Nicho de Células Madre/fisiología , Irradiación Corporal Total , Animales , Becaplermina , Movimiento Celular/fisiología , Movimiento Celular/efectos de la radiación , Proliferación Celular , Endotelio Vascular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Supervivencia de Injerto , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de la radiación , Megacariocitos/metabolismo , Megacariocitos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoblastos/efectos de la radiación , Proteínas Proto-Oncogénicas c-sis/metabolismo , Transducción de Señal , Trombopoyetina/metabolismoAsunto(s)
Benzoatos/uso terapéutico , Hidrazinas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Pirazoles/uso terapéutico , Receptores de Trombopoyetina/agonistas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Receptores de Trombopoyetina/análisis , Receptores de Trombopoyetina/fisiologíaRESUMEN
Essential thrombocythemia (ET) is a rare type of myeloproliferative neoplasm characterized by clonal expansion of the megakaryocyte and platelet lineage. Here, we describe a novel mutation (Y252H) in the thrombopoietin (TPO) receptor, or MPL, in a JAK2 mutation-negative ET patient. The bone marrow examination revealed increased numbers of dysmorphic megakaryocytes with focal clustering. The x-inactivation pattern suggested clonal expansion of hematopoietic cells in the bone marrow. Furthermore, we found that the patient's bone marrow cells were hypersensitive to TPO in generating megakaryocyte colonies in vitro. More importantly, we demonstrated that this MPL Y252H mutant confers increased TPO/MPL-mediated cell growth and increased cell survival upon cytokine withdrawal in BaF3 cells, indicating it is a disease-driving mutation and may contribute to the development of ET in vivo. In summary, this is the first report describing a mutation in the extracellular domain of MPL underlying ET.
Asunto(s)
Mutación Missense , Mutación Puntual , Receptores de Trombopoyetina/genética , Trombocitemia Esencial/genética , Trombopoyetina/farmacología , Sustitución de Aminoácidos , Animales , Médula Ósea/patología , Línea Celular Transformada/efectos de los fármacos , Preescolar , Células Clonales/patología , Análisis Mutacional de ADN , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Janus Quinasa 2/genética , Megacariocitos/patología , Ratones , Receptores de Trombopoyetina/química , Receptores de Trombopoyetina/fisiología , Trombocitemia Esencial/patología , Inactivación del Cromosoma XRESUMEN
To evaluate the grading of fibrosis and immunohistochemical expression of MPL in bone marrow biopsies of ET and PMF. Fibrosis in bone marrow biopsies (BMBs) was evaluated according to the European Consensus for grading of fibrosis, according to reticulin proliferation. Immunohistochemical analysis was performed in samples from 18 ET and 38 PMF patients: 19 were classified as pre-fibrotic and 19 were classified as fibrotic according to the World Health Organization (WHO) criteria, by means of the MPL antibody. Six bone marrow donors' biopsies were used as controls. Average reticulin (p<0.003) and MPL (p<0.008) values differed significantly between the ET group and the pre-fibrotic stage PMF group. The MPL immunohistochemical expression may represent a new marker for differential diagnosis between ET and pre-fibrotic stage PMF.
Asunto(s)
Biomarcadores de Tumor , Mielofibrosis Primaria/diagnóstico , Receptores de Trombopoyetina/metabolismo , Trombocitemia Esencial/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Estudios de Casos y Controles , Diagnóstico Diferencial , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Receptores de Trombopoyetina/análisis , Receptores de Trombopoyetina/fisiología , Trombocitemia Esencial/metabolismo , Trombocitemia Esencial/patología , Adulto JovenAsunto(s)
Membrana Celular/fisiología , Receptores de Trombopoyetina/fisiología , Animales , Membrana Celular/metabolismo , Colesterol/fisiología , Ligandos , Ratones , Microscopía Fluorescente , Imagen Molecular , Fosforilación , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Receptores de Trombopoyetina/química , Receptores de Trombopoyetina/metabolismo , Trombopoyetina/metabolismoRESUMEN
Ligand binding to the thrombopoietin receptor is thought to stabilize an active receptor dimer that regulates megakaryocyte differentiation and platelet formation, as well as haematopoietic stem cell renewal. By fusing a dimeric coiled coil in all seven possible orientations to the thrombopoietin receptor transmembrane (TM)-cytoplasmic domains, we show that specific biological effects and in vivo phenotypes are imparted by distinct dimeric orientations, which can be visualized by cysteine mutagenesis and crosslinking. Using functional assays and computational searches, we identify one orientation that represents the inactive dimeric state and another similar to a physiologically activated receptor. Several other dimeric orientations are identified that induce proliferation and in vivo myeloproliferative and myelodysplastic disorders, indicating the receptor can signal from several dimeric interfaces. The set of dimeric thrombopoietin receptors with different TM orientations may offer new insights into the activation of distinct signalling pathways by a single receptor and suggests that subtle differences in cytokine receptor dimerization provide a new layer of signalling regulation that is relevant for disease.
Asunto(s)
Multimerización de Proteína/fisiología , Receptores de Trombopoyetina/química , Receptores de Trombopoyetina/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Mapas de Interacción de Proteínas , Multimerización de Proteína/genética , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/fisiología , Transducción de Señal/fisiología , EstereoisomerismoRESUMEN
Hematopoietic stem cells (HSCs) are rare residents of the bone marrow responsible for the lifelong production of blood cells. Regulation of the balance between HSC self-renewal and differentiation is central to hematopoiesis, allowing precisely regulated generation of mature blood cells at steady state and expanded production at times of rapid need, as well as maintaining ongoing stem cell capacity. Erg, a member of the Ets family of transcription factors, is deregulated in cancers; and although Erg is known to be required for regulation of adult HSCs, its precise role has not been defined. We show here that, although heterozygosity for functional Erg is sufficient for adequate steady-state HSC maintenance, Erg(+/Mld2) mutant mice exhibit impaired HSC self-renewal after bone marrow transplantation or during recovery from myelotoxic stress. Moreover, although mice functionally compromised for either Erg or Mpl, the receptor for thrombopoietin, a key regulator of HSC quiescence, maintained sufficient HSC activity to sustain hematopoiesis, Mpl(-/-) Erg(+/Mld2) compound mutant mice displayed exacerbated stem cell deficiencies and bone marrow failure. Thus, Erg is a critical regulator of adult HSCs, essential for maintaining self-renewal at times of high HSC cycling.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Proteínas Oncogénicas/fisiología , Animales , Trasplante de Médula Ósea , División Celular , Ensayo de Unidades Formadoras de Colonias , Regulación de la Expresión Génica , Genes Reporteros , Hematopoyesis/genética , Heterocigoto , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proteínas Oncogénicas/genética , Quimera por Radiación , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/fisiología , Factores de Transcripción , Regulador Transcripcional ERGRESUMEN
Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet, it is unknown whether this loss of HSCs is an irreversible process. In this study, we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations, namely, HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors, we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression, replenishes the HSC pool, restores stem cell properties, and corrects platelet production. In some mice, megakaryocyte counts were atypically high, accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential, with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal.
Asunto(s)
Anemia Aplásica/genética , Anemia Aplásica/terapia , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/fisiología , Lentivirus/genética , Receptores de Trombopoyetina/genética , Regeneración/genética , Anemia Aplásica/patología , Anemia Aplásica/fisiopatología , Animales , Linaje de la Célula/genética , Células Cultivadas , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras Genéticas , Receptores de Trombopoyetina/metabolismo , Receptores de Trombopoyetina/fisiologíaRESUMEN
In a previous study, we underlined the functional role of the TPO receptor, Mpl, in the establishment of definitive mouse hematopoiesis, by demonstrating that the lack of Mpl led to a delayed production of definitive hematopoietic cells in the aorta-gonad-mesonephros (AGM) region, and resulted in the production of hematopoietic stem cells (HSCs) with an impaired activity at E11.5. In order to more accurately estimate the role of Mpl during generation of HSCs in the aorta, we performed an analysis of these AGMs at the time of the first HSC emergence (E10.5). Our results indicated that while Mpl-/- AGMs were found to contain more hematopoietic cells (HC) than C57Bl6 AGMs at E10.5, a defect in the expansion process of the HC/HSCs was detected in explant cultures of these AGMs, likely due to an increased apoptosis of these cells. To determine the molecular mechanisms by which invalidation of Mpl receptor affects the temporal distribution and expansion of HC/HSCs in the AGM, a study of the transcription level of of Mpl target genes was conducted. Expression of Runx1, a master transcription factor for the formation of hematopoietic progenitor (HP) cells and HSCs from the vasculature, as well as expression of Meis1 and HoxB4, known to play a role in self-renewal and expansion of HSCs, were found to be down regulated in E10.5 Mpl-/- AGMs. Our data indicate that Mpl is an active player during the first steps of definitive hematopoiesis establishment through direct regulation of the expression of transcription factors or genes important for the self-renewal, proliferation and apoptosis of HSCs.
Asunto(s)
Apoptosis/fisiología , Embrión de Mamíferos/irrigación sanguínea , Células Madre Hematopoyéticas/citología , Sistema Hematopoyético/embriología , Receptores de Trombopoyetina/fisiología , Animales , Antígenos CD34/metabolismo , Aorta/citología , Aorta/embriología , Apoptosis/genética , Recuento de Células , Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Embrión de Mamíferos/metabolismo , Femenino , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Gónadas/irrigación sanguínea , Gónadas/citología , Gónadas/embriología , Células Madre Hematopoyéticas/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Masculino , Mesonefro/irrigación sanguínea , Mesonefro/citología , Mesonefro/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Trombopoyetina/deficiencia , Receptores de Trombopoyetina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas de Cultivo de TejidosRESUMEN
OBJECTIVE: To review the pharmacology and pharmacokinetics and evaluate the safety and efficacy of eltrombopag for the treatment of chronic immune (idiopathic) thrombocytopenic purpura (ITP) and thrombocytopenia associated with hepatitis C virus (HCV) cirrhosis. DATA SOURCES: A Cochrane Controlled Trial Register, clinicaltrials.gov, EMBASE, and MEDLINE search was performed (January 1966-March 2010) using the key terms eltrombopag and SB-497115-GR. Searches were limited to published English-language studies in humans and a reference review of the pertinent literature was conducted. STUDY SELECTION AND DATA EXTRACTION: Published pharmacokinetic data and safety and efficacy trials, case reports, and case series on the use of eltrombopag were selected for inclusion. DATA SYNTHESIS: Eltrombopag is a novel second-generation thrombopoietin receptor agonist that was approved by the Food and Drug Administration for the treatment of chronic ITP in patients who had an insufficient response to corticosteroids, intravenous immune globulin, or splenectomy. Eltrombopag has been shown to be superior to placebo in increasing platelet counts, with more patients achieving counts >50 x 10(3)/microL. One study has also shown eltrombopag to be effective in the treatment of thrombocytopenia associated with HCV cirrhosis. Eltrombopag has a boxed warning related to risk of hepatotoxicity, with criteria for discontinuation in patients with elevated liver enzyme levels or clinical signs of liver damage. As such, close monitoring of laboratory parameters is required, and patients must be registered with the PROMACTA CARES program. CONCLUSIONS: Eltrombopag is effective in increasing platelet counts in patients with chronic ITP and in patients with HCV cirrhosis. In the treatment of ITP, eltrombopag has been studied only for short durations and is more expensive than first-line oral corticosteroids; therefore, it should be considered a second-line agent. More studies are needed to identify a place in therapy for eltrombopag in the treatment of thrombocytopenia associated with HCV cirrhosis.
Asunto(s)
Benzoatos/administración & dosificación , Hidrazinas/administración & dosificación , Pirazoles/administración & dosificación , Receptores de Trombopoyetina/agonistas , Trombocitopenia/tratamiento farmacológico , Administración Oral , Animales , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Receptores de Trombopoyetina/fisiología , Trombocitopenia/fisiopatologíaRESUMEN
OBJECTIVE: To review the pharmacology, pharmacokinetics, efficacy, and safety of two new thrombopoietic (TPO) receptor agonists, romiplostim and eltrombopag, in the treatment of chronic idiopathic thrombocytopenic purpura (ITP) in adults. DATA SOURCES: A MEDLINE search was conducted (1966 to March 2009) using the search terms romiplostim, AMG 531, eltrombopag, SB-497115, idiopathic thrombocytopenic purpura. Articles on phases 1-3 clinical trials in patients with ITP were identified and reviewed. References from manufacturer information, and abstracts from recent hematology meetings, were also evaluated. STUDY SELECTION AND DATA EXTRACTION: Controlled clinical trials evaluating romiplostim and eltrombopag for treatment of chronic ITP in adults were selected from the data sources. All published relevant abstracts were also included. DATA SYNTHESIS: Limited randomized controlled trials and open-label ongoing long-term extension studies for romiplostim and eltrombopag, have shown that both TPO agonists are effective in improving the platelet count and reducing the bleeding episodes in adult patients with ITP unresponsive to at least one standard treatment. The most common adverse events associated with the drugs are mild to moderate headaches. The use of these agents has also been associated with rare but serious side-effects including bone marrow reticulin fibrosis, thrombotic events, and myeloid malignancies. CONCLUSIONS: Until more long-term follow-up data regarding the safety, as well as comparative studies that further define the role of TPO agonists versus other agents in the treatment of chronic ITP are available, these agents should be reserved for patients with ITP refractory or intolerant to standard therapy.