RESUMO
Hypoxia upregulates the core pluripotency factors NANOG, SOX2, and OCT4, associated with tumor aggressiveness and resistance to conventional anticancer treatments. We have previously reported that hypoxia-induced NANOG contributed in vitro to tumor cell resistance to autologous-specific CTL and in vivo to the in situ recruitment of immune-suppressive cells. In this study, we investigated the mechanisms underlying NANOG-mediated tumor cell resistance to specific lysis under hypoxia. We demonstrated the tumor-promoting effect of hypoxia on tumor initiation into immunodeficient mice using human non-small lung carcinoma cells. We next showed a link between NANOG and autophagy activation under hypoxia because inhibition of NANOG decreased autophagy in tumor cells. Chromatin immunoprecipitation and luciferase reporter assays revealed a direct binding of NANOG to a transcriptionally active site in a BNIP3L enhancer sequence. These data establish a new link between the pluripotency factor NANOG and autophagy involved in resistance to CTL under hypoxia.
Assuntos
Autofagia , Hipóxia Celular , Elementos Facilitadores Genéticos , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Proteína Homeobox Nanog/metabolismo , Regiões Promotoras Genéticas , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/metabolismo , Interferência de RNA , Regulação para CimaAssuntos
Linfo-Histiocitose Hemofagocítica , Linfoma Extranodal de Células T-NK , Linfoma de Células T , Neoplasias Nasais , Humanos , Janus Quinase 3 , Células Matadoras Naturais/patologia , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/etiologia , Linfo-Histiocitose Hemofagocítica/patologia , Linfoma de Células T/complicações , Linfoma de Células T/genética , Linfoma de Células T/patologia , Neoplasias Nasais/patologiaRESUMO
The Wiskott-Aldrich syndrome protein is an essential cytoskeleton regulator found in cells of the hematopoietic lineage and controls the motility of leukocytes. The impact of WAS gene deficiency on the mobilization of hematopoietic progenitor/stem cells in circulation has remained unexplored but information would be pertinent in the context of autologous gene therapy of Wiskott-Aldrich syndrome. The response to granulocyte-colony stimulating factor mobilization was investigated in a murine WAS knock-out model of the disease, by measuring hematologic parameters, circulation and engraftment of hematopoietic progenitor/stem cells. In the steady-state, adult WAS knock-out mice have B-cell lymphopenia, marked neutrophilia, increased counts of circulating hematopoietic progenitor cells and splenomegaly, presumably caused by the retention of hematopoietic progenitor cells due to high levels of splenic CXCL12. In spite of these anomalies, the administration of granulocyte-colony-stimulating factor mobilizes progenitor/stem cells in WAS knock-out mice to the same level and with the same kinetics as in wild-type control mice. Mobilized peripheral blood cells from WAS knock-out mice can be transduced and are able to engraft into lethally-irradiated hosts reconstituting multiple lineages of cells and providing more effective radio-protection than mobilized cells from wild-type control mice. Surprisingly, the homing and the peripheral blood recovery of B lymphocytes was influenced by the background of the host. Thus, in the absence of Wiskott-Aldrich syndrome protein, effective mobilization is achieved but partial correction may occur as a result of an abnormal hematopoietic environment.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/terapia , Animais , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Proteínas Recombinantes/farmacologiaRESUMO
Adipose tissue macrophages (ATMs) play an important role in obesity and inflammation, and they accumulate in adipose tissue (AT) with aging. Furthermore, increased ATM senescence has been shown in obesity-related AT remodeling and dysfunction. However, ATM senescence and its role are unclear in age-related AT dysfunction. Here, we show that ATMs (a) acquire a senescence-like phenotype during chronological aging; (b) display a global decline of basic macrophage functions such as efferocytosis, an essential process to preserve AT homeostasis by clearing dysfunctional or apoptotic cells; and (c) promote AT remodeling and dysfunction. Importantly, we uncover a major role for the age-associated accumulation of osteopontin (OPN) in these processes in visceral AT. Consistently, loss or pharmacologic inhibition of OPN and bone marrow transplantation of OPN-/- mice attenuate the ATM senescence-like phenotype, preserve efferocytosis, and finally restore healthy AT homeostasis in the context of aging. Collectively, our findings implicate pharmacologic OPN inhibition as a viable treatment modality to counter ATM senescence-mediated AT remodeling and dysfunction during aging.
Assuntos
Obesidade , Osteopontina , Camundongos , Animais , Osteopontina/genética , Obesidade/genética , Tecido Adiposo , Macrófagos , FagocitoseRESUMO
Targeting the reprogramming and phagocytic capacities of tumor-associated macrophages (TAMs) has emerged as a therapeutic opportunity for cancer treatment. Here, we demonstrate that tumor cell phagocytosis drives the pro-inflammatory activation of TAMs and identify a key role for the cyclin-dependent kinase inhibitor CDKN1A (p21). Through the transcriptional repression of Signal-Regularity Protein α (SIRPα), p21 promotes leukemia cell phagocytosis and, subsequently, the pro-inflammatory reprogramming of phagocytic macrophages that extends to surrounding macrophages through Interferon γ. In mouse models of human T-cell acute lymphoblastic leukemia (T-ALL), infusion of human monocytes (Mos) engineered to overexpress p21 (p21TD-Mos) leads to Mo differentiation into phagocytosis-proficient TAMs that, after leukemia cell engulfment, undergo pro-inflammatory activation and trigger the reprogramming of bystander TAMs, reducing the leukemic burden and substantially prolonging survival in mice. These results reveal p21 as a trigger of phagocytosis-guided pro-inflammatory TAM reprogramming and highlight the potential for p21TD-Mo-based cellular therapy as a cancer immunotherapy.
Assuntos
Leucemia Mieloide Aguda , Fagocitose , Humanos , Camundongos , Animais , Imunoterapia , Macrófagos/metabolismo , Leucemia Mieloide Aguda/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismoRESUMO
BACKGROUND: hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. METHODS: Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other's (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. RESULTS: A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. CONCLUSION: The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.
Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular/fisiologia , Corpos Embrioides , Células Endoteliais/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , CamundongosRESUMO
The intrinsic properties of self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) play a critical role in the regeneration of mature hematopoietic cells at steady state and in stress conditions including bleeding, inflammation and aging. Common techniques such as flow cytometry and genetic methods have answered many questions about their intrinsic and extrinsic regulation. Using these approaches, it was demonstrated that HSPCs in the bone marrow demonstrate low rates of proliferation and apoptosis. This dormant phenotype is associated with a low production of reactive oxygen species and low mitochondrial activity.Here, we describe the methodology to characterize the physiologic state of HSPCs isolated from their native hematopoietic organ using flow cytometry-based assays. These protocols allow evaluation of their ROS levels and activated signaling pathways under various conditions.
Assuntos
Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Células-Tronco Hematopoéticas/imunologia , Imunofenotipagem , Camundongos Endogâmicos C57BL , Fenótipo , Transdução de SinaisRESUMO
Knowledge about the hematopoietic niche has evolved considerably in recent years, in particular through in vitro analyzes, mouse models and the use of xenografts. Its complexity in the human bone marrow, in particular in a context of hematological malignancy, is more difficult to decipher by these strategies and could benefit from the knowledge acquired on the niches of solid tumors. Indeed, some common features can be suspected, since the bone marrow is a frequent site of solid tumor metastases. Recent research on solid tumors has provided very interesting information on the interactions between tumoral cells and their microenvironment, composed notably of mesenchymal, endothelial and immune cells. This review thus focuses on recent discoveries on tumor niches that could help in understanding hematopoietic niches, with special attention to 4 particular points: i) the heterogeneity of carcinoma/cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs), ii) niche cytokines and chemokines, iii) the energy/oxidative metabolism and communication, especially mitochondrial transfer, and iv) the vascular niche through angiogenesis and endothelial plasticity. This review highlights actors and/or pathways of the microenvironment broadly involved in cancer processes. This opens avenues for innovative therapeutic opportunities targeting not only cancer stem cells but also their regulatory tumor niche(s), in order to improve current antitumor therapies.
Assuntos
Microambiente Tumoral , Animais , Fibroblastos Associados a Câncer , Comunicação Celular , Citocinas , Endotélio Vascular , Humanos , Células-Tronco Mesenquimais , Neoplasias , Neovascularização PatológicaRESUMO
Mouse Rhd* and Rhag* genes were targeted using insertional vectors; the resulting knockout mice, and double-knockout descendants, were analysed. Rhag glycoprotein deficiency entailed defective assembly of the erythroid Rh complex with complete loss of Rh and intercellular adhesion molecule 4 (ICAM-4), but not CD47, expression. Absence of the Rh protein induced a loss of ICAM-4, and only a moderate reduction of Rhag expression. Double knockout phenotype was similar to that of Rhag targeted mice. Rhd and Rhag deficient mice exhibited neither the equivalent of human Rh(null) haemolytic anaemia nor any clinical or cellular abnormalities. Rhd-/- and Rhag-/- erythrocytes showed decreased basal adhesion to an endothelial cell line resulting from defective ICAM-4 membrane expression. There was no difference in recovery from phenylhydrazine-induced haematopoietic stress for double knockout mice as compared to controls, suggesting that ICAM-4 might be dispensable during stress erythropoiesis. Ammonia and methylammonia transport in erythrocytes was severely impaired in Rhag-/- but only slightly in Rhd-/- animals that significantly expressed Rhag, supporting the view that RhAG and Rhag, but not Rh, may act as ammonium transporters in human and mouse erythrocytes. These knockout mice should prove useful for further dissecting the physiological roles of Rh and Rhag proteins in the red cell membrane.
Assuntos
Proteínas Sanguíneas/deficiência , Modelos Animais de Doenças , Glicoproteínas de Membrana/deficiência , Sistema do Grupo Sanguíneo Rh-Hr/fisiologia , Animais , Transporte Biológico/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/fisiologia , Adesão Celular/genética , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/fisiologia , Células Cultivadas , Células Endoteliais/fisiologia , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Eritropoese/fisiologia , Feminino , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Metilaminas/sangue , Camundongos , Camundongos Knockout , Fenótipo , Compostos de Amônio Quaternário/sangue , Sistema do Grupo Sanguíneo Rh-Hr/genéticaRESUMO
Previous observations suggested that functional antagonism between FLI-1 and EKLF might be involved in the commitment toward erythrocytic or megakaryocytic differentiation. We show here, using inducible shRNA expression, that EKLF knockdown in mouse erythroleukemia (MEL) cells decreases erythrocytic and increases megakaryocytic as well as Fli-1 gene expression. Chromatin immunoprecipitation analyses revealed that the increase in megakaryocytic gene expression is associated with a marked increase in RNA pol II and FLI-1 occupancy at their promoters, albeit FLI-1 protein levels are only minimally affected. Similarly, we show that human CD34(+) progenitors infected with shRNA lentivirus allowing EKLF knockdown generate an increased number of differentiated megakaryocytic cells associated with increased levels of megakaryocytic and Fli-1 gene transcripts. Single-cell progeny analysis of a cell population enriched in bipotent progenitors revealed that EKLF knockdown increases the number of megakaryocytic at the expense of erythrocytic colonies. Taken together, these data indicate that EKLF restricts megakaryocytic differentiation to the benefit of erythrocytic differentiation and suggest that this might be at least partially mediated by the inhibition of FLI-1 recruitment to megakaryocytic and Fli-1 gene promoters.
Assuntos
Diferenciação Celular , Eritrócitos/citologia , Fatores de Transcrição Kruppel-Like/fisiologia , Megacariócitos/citologia , Animais , Linhagem Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/genética , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologiaRESUMO
Thrombopoietin (TPO), through activation of its cognate receptor Mpl, is the major regulator of platelet production. However, residual platelets observed in TPO- and Mpl-loss-of-function (LOF) mice suggest the existence of an additional factor to TPO in platelet production. As erythropoietin (EPO) exhibited both in vitro megakaryocytic potential, in association with other early-acting cytokines, and in vivo platelet activation activity, we sought to investigate its role in this setting. Here, we used multiple LOF models to decipher the reciprocal role of EPO and TPO in the regulation of platelet production in TPO-LOF and Mpl-LOF mice and of platelet size heterogeneity in wild-type mice. We first identified EPO as the major thrombopoietic factor in the absence of the TPO-Mpl pathway. Based on the study of several mouse models we found that the EPO-EPO receptor pathway acts on late-stage megakaryopoiesis and is responsible for large-sized platelet production, while the TPO-Mpl pathway promotes small-sized platelet production. On the basis of our data, EPO might be used for thrombocytopenia supportive therapy in congenital amegakaryocytopoiesis. Furthermore, as a distribution skewed toward large platelets is an independent risk factor and a poor prognosis indicator in atherothrombosis, the characterization of EPO's role in the production of large-sized platelets, if confirmed in humans, may open new perspectives in the understanding of the role of EPO-induced platelets in atherothrombosis.
Assuntos
Plaquetas/metabolismo , Eritropoetina/metabolismo , Megacariócitos/microbiologia , Trombopoese , Trombopoetina/metabolismo , Animais , Eritropoetina/genética , Feminino , Camundongos , Camundongos Knockout , Trombopoetina/genéticaRESUMO
Bone marrow (BM) niche cells help to keep adult hematopoietic stem cells (HSCs) in a quiescent state via secreted factors and induction of cell-cycle inhibitors. Here, we demonstrate that the adapter protein CABLES1 is a key regulator of long-term hematopoietic homeostasis during stress and aging. Young mice lacking Cables1 displayed hyperproliferation of hematopoietic progenitor cells. This defect was cell intrinsic, since it was reproduced in BM transplantation assays using wild-type animals as recipients. Overexpression and short hairpin RNA-mediated depletion of CABLES1 protein resulted in p21Cip/waf up- and downregulation, respectively. Aged mice lacking Cables1 displayed abnormalities in peripheral blood cell counts accompanied by a significant reduction in HSC compartment, concomitant with an increased mobilization of progenitor cells. In addition, Cables1-/- mice displayed increased sensitivity to the chemotherapeutic agent 5-fluorouracil due to an abnormal microenvironment. Altogether, our findings uncover a key role for CABLES1 in HSC homeostasis and stress hematopoiesis.
Assuntos
Ciclinas/genética , Células-Tronco Hematopoéticas/citologia , Envelhecimento , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclinas/antagonistas & inibidores , Ciclinas/metabolismo , Fluoruracila/farmacologia , Hematopoese/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Antibody-drug conjugates (ADCs) are a new class of therapeutics that use antibodies to deliver potent cytotoxic drugs selectively to cancer cells. CD203c, an ecto-nucleotide pyrophosphatase-phosphodiesterase 3, is overexpressed on neoplastic mast cells (MCs) in systemic mastocytosis (SM), thus representing a promising target for antibody-mediated therapy. In this study, we have found that human neoplastic MC lines (ROSAKIT D816V and ROSAKIT D816V-Gluc), which express high levels of CD203c, are highly and specifically sensitive to the antiproliferative effects of an ADC against CD203c (AGS-16C3F). In these cell lines, AGS-16C3F induced cell apoptosis at very low concentrations. To characterize the effects of AGS-16C3F on leukemia progression in vivo, ROSAKIT D816V-Gluc NOD-SCID γ mouse models of advanced SM (AdvSM) were treated with AGS-16C3F or an ADC control for 2 weeks. Whereas AGS-16C3F had no apparent toxicity in xenotransplanted mice, in vivo neoplastic MC burden significantly decreased in both hematopoietic and nonhematopoietic organs. Furthermore, animals treated with AGS-16C3F had prolonged survival compared with the animals treated with control ADC, and AGS-16C3F efficiently prevented disease relapse. In conclusion, these preclinical studies identified CD203c as a novel therapeutic target on neoplastic MCs, and AGS-16C3F as a promising ADC for the treatment of patients with AdvSM.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Imunoconjugados/uso terapêutico , Mastocitose Sistêmica/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Mastocitose Sistêmica/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Despite advances to engineer transplantable hematopoietic stem and progenitor cells (HSPCs) for research and therapy, an in-depth characterization of the developing human hematopoietic system is still lacking. The human embryonic liver is at the crossroad of several hematopoietic sites and harbors a complex hematopoietic hierarchy, including the first actively dividing HSPCs that will further seed the definitive hematopoietic organs. However, few are known about the phenotypic and functional HSPC organization operating at these stages of development. In this study, using a combination of four endothelial and hematopoietic surface markers, that is, the endothelial-specific marker vascular endothelial-cadherin (Cdh5, CD144), the pan-leukocyte antigen CD45, the hemato-endothelial marker CD34, and the angiotensin-converting enzyme (ACE, CD143), we identified distinct HSPC subsets, and among them, a population co-expressing the four markers that uniquely harbored an outstanding proliferation potential both ex vivo and in vivo. Moreover, we traced back this population to the yolk sac (YS) and aorta-gonad-mesonephros (AGM) sites of hematopoietic emergence. Taken together, our data will help to identify human HSPC self-renewal and amplification mechanisms for future cell therapies.
Assuntos
Antígenos CD/genética , Caderinas/genética , Linhagem da Célula , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Fígado/citologia , Peptidil Dipeptidase A/genética , Antígenos CD/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Fígado/embriologia , Peptidil Dipeptidase A/metabolismoRESUMO
Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs. Nonetheless, whether megakaryocyte-HSC interactions change during pathological/natural aging is unclear. Premature aging in Hutchinson-Gilford progeria syndrome recapitulates physiological aging features, but whether these arise from altered stem or niche cells is unknown. Here, we show that the BM microenvironment promotes myelopoiesis in premature/physiological aging. During physiological aging, HSC-supporting niches decrease near bone but expand further from bone. Increased BM noradrenergic innervation promotes ß2-adrenergic-receptor(AR)-interleukin-6-dependent megakaryopoiesis. Reduced ß3-AR-Nos1 activity correlates with decreased endosteal niches and megakaryocyte apposition to sinusoids. However, chronic treatment of progeroid mice with ß3-AR agonist decreases premature myeloid and HSC expansion and restores the proximal association of HSCs to megakaryocytes. Therefore, normal/premature aging of BM niches promotes myeloid expansion and can be improved by targeting the microenvironment.
Assuntos
Senilidade Prematura/patologia , Envelhecimento/fisiologia , Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Megacariócitos/fisiologia , Células Mieloides/fisiologia , Progéria/patologia , Agonistas Adrenérgicos/administração & dosagem , Envelhecimento/metabolismo , Senilidade Prematura/metabolismo , Animais , Diferenciação Celular , Encapsulamento de Células , Proliferação de Células , Modelos Animais de Doenças , Humanos , Interleucina-6/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo I/metabolismo , Progéria/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais , Nicho de Células-TroncoRESUMO
In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients' bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients' outcome.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
Rituximab is used in the treatment of lymphoma and autoimmune diseases, for which late-onset neutropenia (LON) were reported. LON-related mechanisms remain unclear. To obtain insights into the mechanisms, we assessed serum, peripheral blood and bone marrow (BM) samples of a patient with LON. Factors classically associated with neutropenia such as anti-neutrophil antibodies, T-LGL, soluble Fas Ligand were not detectable. We then evaluated the kinetics of various cytokines involved in B-cell and granulocyte homeostasis. We found that LON is related to a lack of granulopoiesis in the BM that coincides with a very high level of BAFF, a strong stimulator of B-cell recovery, and hypothesized a hematopoietic lineage competition due to an excessive B-cell recovery in the BM by promotion of B-cell lymphopoiesis over granulopoiesis within common developmental niches. Assessment of serum BAFF levels following rituximab could detect patients at risk of developing LON.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Fator Ativador de Células B/fisiologia , Linfócitos B/patologia , Fatores Imunológicos/efeitos adversos , Linfopoese/fisiologia , Mielopoese/fisiologia , Neutropenia/induzido quimicamente , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Fator Ativador de Células B/sangue , Linfócitos B/efeitos dos fármacos , Medula Óssea/patologia , Linhagem da Célula , Células Cultivadas/efeitos dos fármacos , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Citocinas/sangue , Quimioterapia Combinada , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/uso terapêutico , Pessoa de Meia-Idade , Neutropenia/fisiopatologia , Rituximab , Fatores de Tempo , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico , Macroglobulinemia de Waldenstrom/sangueRESUMO
It has been shown that p210(BCR-ABL) significantly impairs CXCR4 signaling. We report here that the migratory response to SDF-1 was profoundly altered in blast crisis, whereas chronic-phase CD34(+) cells migrated normally to this chemokine. This migratory defect was associated with a low CXCR4 membrane expression. In vitro STI-571 treatment of CD34(+) cells from patients in blast crisis markedly increased the CXCR4 transcript and CXCR4 membrane expression. Because p210(BCR-ABL) frequently increases with disease progression, we determined the effects of high and low p210(BCR-ABL) expression on CXCR4 protein in the granulocyte macrophage colony-stimulating factor-dependent human cell line MO7e. p210(BCR-ABL) expression distinctly alters CXCR4 protein through two different mechanisms depending on its expression level. At low expression, a signaling defect was detected with no modification of CXCR4 expression. However, higher p210(BCR-ABL) expression induced a marked down-regulation of CXCR4 that is related to its decreased transcription. The effect of p210(BCR-ABL) required its tyrosine kinase activity. Collectively, these data indicate that p210(BCR-ABL) could affect CXCR4 by more than one mechanism and suggest that down-regulation of CXCR4 may have important implications in chronic myelogenous leukemia pathogenesis.
Assuntos
Quimiocinas CXC/antagonistas & inibidores , Proteínas de Fusão bcr-abl/fisiologia , Receptores CXCR4/fisiologia , Animais , Antígenos CD34/biossíntese , Benzamidas , Crise Blástica , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/fisiologia , Regulação para Baixo , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Células NIH 3T3 , Piperazinas/farmacologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Pirimidinas/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/biossíntese , Transdução de Sinais , Transcrição GênicaRESUMO
JAK2 activation is the driver mechanism in BCR-ABL-negative myeloproliferative neoplasms (MPN). These diseases are characterized by an abnormal retention of hematopoietic stem cells within the bone marrow microenvironment and their increased trafficking to extramedullary sites. The CXCL12/CXCR4 axis plays a central role in hematopoietic stem cell/ progenitor trafficking and retention in hematopoietic sites. The present study explores the crosstalk between JAK2 and CXCL12/CXCR4 signaling pathways in MPN. We show that JAK2, activated by either MPL-W515L expression or cytokine stimulation, cooperates with CXCL12/CXCR4 signaling to increase the chemotactic response of human cell lines and primary CD34+ cells through an increased phosphatidylinositol-3-kinase (PI3K) signaling. Accordingly, primary myelofibrosis (MF) patient cells demonstrate an increased CXCL12-induced chemotaxis when compared to controls. JAK2 inhibition by knock down or chemical inhibitors decreases this effect in MPL-W515L expressing cell lines and reduces the CXCL12/CXCR4 signaling in some patient primary cells. Taken together, these data indicate that CXCL12/CXCR4 pathway is overactivated in MF patients by oncogenic JAK2 that maintains high PI3K signaling over the threshold required for CXCR4 activation. These results suggest that inhibition of this crosstalk may contribute to the therapeutic effects of JAK2 inhibitors.
RESUMO
The administration of ex vivo-expanded Natural Killer (NK) cells in leukemia therapy is still challenging, in part due to the difficulty to generate in sufficient quantities fully mature and functional NK cells and Identification of surface markers indicative of NK maturation and functionality is therefore needed. Here, based on the analysis of surface receptors of ex vivo-expanded NK cells, we identified CD94 as a surface marker correlating with high lytic potential against leukemic cell lines and immunological synapse formation. CD94-positive ex vivo-expanded NK cells displayed higher expression of NKG2 receptors and the adhesion molecule LFA-1, as compared with their CD94-negative counterparts. We also tested the in vivo anti-leukemic capacity of ex vivo-expanded NK cells against patient-derived acute myeloid leukemia cells. Although no anti-leukemic effect was detected, we noticed that only CD94-positive ex vivo-expanded NK cells were detected in leukemic mice at the end of the 2-week treatment. Moreover, flow cytometry analysis showed a subpopulation harboring CD94 (NK) and CD34 (leukemic cells) double staining, indicative of conjugate formation. Therefore surface expression of CD94 on ex vivo-differentiated NK cells emerged as an indicator of in vitro and in vivo killer cell functionality.