RESUMEN
The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.
Asunto(s)
Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células Progenitoras Mieloides/efectos de los fármacos , Proteolisis/efectos de los fármacos , Triazoles/farmacología , Antineoplásicos/síntesis química , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Línea Celular Tumoral , Proliferación Celular , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Diseño de Fármacos , Descubrimiento de Drogas , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Concentración 50 Inhibidora , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/patología , Piperazinas/farmacología , Cultivo Primario de Células , Piridinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Transducción de Señal , Relación Estructura-Actividad , Transcriptoma , Triazoles/síntesis químicaAsunto(s)
Células de la Médula Ósea , Células Eritroides , Genes Ligados a X , Mutación , Células Mieloides , Células Progenitoras Mieloides , Trastornos Mieloproliferativos , Enzimas Activadoras de Ubiquitina/genética , Vacuolas , Adulto , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/patología , Niño , Células Eritroides/enzimología , Células Eritroides/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Mieloides/enzimología , Células Mieloides/patología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/patología , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Síndrome , Vacuolas/enzimología , Vacuolas/genética , Vacuolas/patologíaRESUMEN
Aged hematopoietic stem cells (HSCs) undergo biased lineage priming and differentiation toward production of myeloid cells. A comprehensive understanding of gene regulatory mechanisms causing HSC aging is needed to devise new strategies to sustainably improve immune function in aged individuals. Here, a focused short hairpin RNA screen of epigenetic factors reveals that the histone acetyltransferase Kat6b regulates myeloid cell production from hematopoietic progenitor cells. Within the stem and progenitor cell compartment, Kat6b is highly expressed in long-term (LT)-HSCs and is significantly decreased with aging at the transcript and protein levels. Knockdown of Kat6b in young LT-HSCs causes skewed production of myeloid cells at the expense of erythroid cells both in vitro and in vivo. Transcriptome analysis identifies enrichment of aging and macrophage-associated gene signatures alongside reduced expression of self-renewal and multilineage priming signatures. Together, our work identifies KAT6B as a novel epigenetic regulator of hematopoietic differentiation and a target to improve aged immune function.
Asunto(s)
Envejecimiento/metabolismo , Diferenciación Celular , Células Eritroides/enzimología , Regulación Enzimológica de la Expresión Génica , Histona Acetiltransferasas/biosíntesis , Células Progenitoras Mieloides/enzimología , Envejecimiento/genética , Envejecimiento/patología , Animales , Epigénesis Genética , Células Eritroides/patología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Histona Acetiltransferasas/genética , Masculino , Ratones , Ratones Transgénicos , Células Progenitoras Mieloides/patología , TranscriptomaRESUMEN
Early damage to transplanted organs initiates excess inflammation that can cause ongoing injury, a leading cause for late graft loss. The endothelial glycocalyx modulates immune reactions and chemokine-mediated haptotaxis, potentially driving graft loss. In prior work, conditional deficiency of the glycocalyx-modifying enzyme N-deacetylase-N-sulfotransferase-1 (Ndst1f/f TekCre+) reduced aortic allograft inflammation. Here we investigated modification of heparan sulfate (HS) and chemokine interactions in whole-organ renal allografts. Conditional donor allograft Ndst1 deficiency (Ndst1-/-; C57Bl/6 background) was compared to systemic treatment with M-T7, a broad-spectrum chemokine-glycosaminoglycan (GAG) inhibitor. Early rejection was significantly reduced in Ndst1-/- kidneys engrafted into wildtype BALB/c mice (Ndst1+/+) and comparable to M-T7 treatment in C57Bl/6 allografts (P < 0.0081). M-T7 lost activity in Ndst1-/- allografts, while M-T7 point mutants with modified GAG-chemokine binding displayed a range of anti-rejection activity. CD3+ T cells (P < 0.0001), HS (P < 0.005) and CXC chemokine staining (P < 0.012), gene expression in NFκB and JAK/STAT pathways, and HS and CS disaccharide content were significantly altered with reduced rejection. Transplant of donor allografts with conditional Ndst1 deficiency exhibit significantly reduced acute rejection, comparable to systemic chemokine-GAG inhibition. Modified disaccharides in engrafted organs correlate with reduced rejection. Altered disaccharides in engrafted organs provide markers for rejection with potential to guide new therapeutic approaches in allograft rejection.
Asunto(s)
Células Alogénicas/enzimología , Aorta/trasplante , Células Progenitoras Endoteliales/enzimología , Rechazo de Injerto/enzimología , Células Progenitoras Mieloides/enzimología , Sulfotransferasas , Células Alogénicas/patología , Animales , Aorta/patología , Células Progenitoras Endoteliales/patología , Eliminación de Gen , Rechazo de Injerto/genética , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Ratones , Ratones Endogámicos BALB C , Células Progenitoras Mieloides/patología , Sulfotransferasas/genética , Sulfotransferasas/metabolismoRESUMEN
Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKI) targeted against BCR-ABL. We previously reported the investigation of residual CML diseases during TKI treatment using FACS-sorting and quantitative RT-PCR of BCR-ABL among each population; total mononuclear cells, hematopoietic stem cells, and myeloid progenitors. The observations also implied that the second-generation of ABL-tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib therapy can be more promising approach for efficient reduction of the CML stem cells. Moreover, we need to develop the evaluation method of the residual CML diseases to establish rational therapy-cessation strategies in CML.
Asunto(s)
Dasatinib/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Células Progenitoras Mieloides , Células Madre Neoplásicas , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/patología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/patología , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patologíaRESUMEN
OBJECTIVE: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery. APPROACH AND RESULTS: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF-macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density. CONCLUSIONS: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.
Asunto(s)
Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Hiperlipoproteinemia Tipo I/enzimología , Lipoproteína Lipasa/deficiencia , Macrófagos/enzimología , Monocitos/enzimología , Células Progenitoras Mieloides/enzimología , Mielopoyesis , Animales , Aorta/patología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/sangre , Aterosclerosis/genética , Aterosclerosis/patología , Proliferación Celular , Citocinas/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hiperlipoproteinemia Tipo I/sangre , Hiperlipoproteinemia Tipo I/genética , Hiperlipoproteinemia Tipo I/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lipoproteína Lipasa/genética , Macrófagos/patología , Ratones Noqueados , Monocitos/patología , Células Progenitoras Mieloides/patología , Transducción de Señal , Triglicéridos/metabolismoRESUMEN
The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear. Late-onset neurodegenerative disease observed in patients with histiocytoses, which are clonal myeloid diseases associated with somatic mutations in the RAS-MEK-ERK pathway such as BRAF(V600E), suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases.
Asunto(s)
Células Precursoras Eritroides/patología , Sistema de Señalización de MAP Quinasas , Mutación , Células Progenitoras Mieloides/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Células Clonales/enzimología , Células Clonales/metabolismo , Células Clonales/patología , Modelos Animales de Enfermedad , Células Precursoras Eritroides/enzimología , Células Precursoras Eritroides/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Histiocitosis/enzimología , Histiocitosis/genética , Histiocitosis/metabolismo , Histiocitosis/patología , Humanos , Macrófagos/enzimología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Microglía/enzimología , Microglía/metabolismo , Microglía/patología , Mosaicismo , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/metabolismo , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1(flfl)), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.
Asunto(s)
Monóxido de Carbono/farmacología , Diferenciación Celular/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Macrófagos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Células Progenitoras Mieloides/efectos de los fármacos , Animales , Trasplante de Médula Ósea , Monóxido de Carbono/metabolismo , Linaje de la Célula , Proliferación Celular , Quimiocina CCL2/sangre , Gases , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Hemo-Oxigenasa 1/genética , Humanos , Interleucina-1alfa/sangre , Receptores de Lipopolisacáridos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/trasplante , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/trasplante , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal , Factores de Tiempo , Células U937RESUMEN
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which accumulate in cancer, infection and chronic inflammation. These cells suppress T-cell function and the immune response. Angiotensin-converting enzyme (ACE) is a peptidase that is now known to regulate aspects of myelopoiesis. Here, we show that ACE expression correlates with myeloid maturation in vitro. Forced ACE overexpression in monocytic cells reduces the generation of MDSCs. In vivo, mice with a genetic change resulting in myeloid cell ACE overexpression have reduced numbers of blood and splenic MDSCs in a tumor model and in a model of chronic inflammation induced by complete Freund's adjuvant. In contrast, ACE-null mice produce large numbers of MDSCs during chronic inflammation. Macrophages from mice with myeloid ACE overexpressing are more pro-inflammatory and have more tumor-killing activity than cells from wild-type mice. Thus, manipulating myeloid ACE activity can interfere with MDSC development and the maturation of myeloid cells.
Asunto(s)
Células Progenitoras Mieloides/fisiología , Mielopoyesis , Peptidil-Dipeptidasa A/metabolismo , Animales , Melanoma Experimental/enzimología , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Progenitoras Mieloides/enzimología , FenotipoRESUMEN
Increased ßcatenin activity correlates with leukemia stem cell expansion and disease progression in chronic myeloid leukemia (CML). We found previously that expression of the CML-related Bcr-abl oncoprotein in myeloid progenitor cells increases expression of Fas-associated phosphatase 1 (Fap1). This resulted in Fap1-dependent resistance to Fas-induced apoptosis in these cells. Fap1 also interacts with the adenomatous polyposis coli (Apc) protein, but the functional significance of this interaction is unknown. Apc participates in a complex that includes glycogen synthase kinase ß (Gsk3ß) and ßcatenin. Assembly of this complex results in phosphorylation of ßcatenin by Gsk3ß, which facilitates ßcatenin ubiquitination and degradation by the proteasome. In this study, we found increased association of Fap1 with the Apc complex in Bcr-abl(+) myeloid progenitor cells. We also found Fap1-dependent inactivation of Gsk3ß and consequent stabilization of ßcatenin in these cells. Consistent with this, Bcr-abl(+) cells exhibited a Fap1-dependent increase in ßcatenin activity. Our studies identified Fap1-dependent Gsk3ß inactivation as a molecular mechanism for increased ßcatenin activity in CML.
Asunto(s)
Proteínas de Fusión bcr-abl/biosíntesis , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/metabolismo , Células Madre Neoplásicas/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 13/metabolismo , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Proteínas de Fusión bcr-abl/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación Leucémica de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Células Madre Neoplásicas/patología , Fosforilación/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 13/genética , Células U937 , Ubiquitinación/genética , beta Catenina/genéticaRESUMEN
Neutrophils are critically involved in host defense and tissue damage. Intrinsic molecular mechanisms controlling neutrophil differentiation and activities are poorly defined. Herein we found that p53-induced phosphatase 1(Wip1) is preferentially expressed in neutrophils among immune cells. The Wip1 expression is gradually up-regulated during the differentiation of myeloid precursors into mature neutrophils. Wip1-deficient mice and chimera mice with Wip1(-/-) hematopoietic cells had an expanded pool of neutrophils with hypermature phenotypes in the periphery. The in vivo and in vitro studies showed that Wip1 deficiency mainly impaired the developing process of myeloid progenitors to neutrophils in an intrinsic manner. Mechanism studies showed that the enhanced development and maturation of neutrophils caused by Wip1 deficiency were mediated by p38 MAPK-STAT1 but not p53-dependent pathways. Thus, our findings identify a previously unrecognized p53-independent function of Wip1 as a cell type-specific negative regulator of neutrophil generation and homeostasis through limiting the p38 MAPK-STAT1 pathway.
Asunto(s)
Sistema de Señalización de MAP Quinasas/inmunología , Neutrófilos/citología , Neutrófilos/enzimología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/inmunología , Animales , Diferenciación Celular/inmunología , Femenino , Homeostasis/inmunología , Trastornos Leucocíticos/congénito , Trastornos Leucocíticos/genética , Trastornos Leucocíticos/inmunología , Leucopoyesis/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/inmunología , Neutrófilos/inmunología , Proteína Fosfatasa 2C , Estallido Respiratorio/inmunología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.
Asunto(s)
Hematopoyesis , Células Progenitoras Mieloides/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/fisiología , Antígenos CD34/metabolismo , Línea Celular , Proliferación Celular , Células Cultivadas , Sangre Fetal/citología , Proteína Adaptadora GRB2/metabolismo , Humanos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Análisis de la Célula IndividualRESUMEN
Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 µM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 µM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 µM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies.
Asunto(s)
Células Progenitoras Mieloides/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/toxicidad , Ticlopidina/análogos & derivados , Ticlopidina/toxicidad , Supervivencia Celular/efectos de los fármacos , Clopidogrel , Ensayo de Unidades Formadoras de Colonias , Citocromo P-450 CYP3A/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/metabolismo , Peroxidasa/metabolismoRESUMEN
During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B-lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B-cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)-α in developing B cells transformed B-lineage cells. Here, we demonstrate that PKC-α modulation in committed CD19(+) B lymphocytes also promoted lineage conversion toward myeloid, NK-, and T-cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T-cell lineage commitment was indicated by the expression of T-cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage-converted T cells carried Igh gene rearrangements reminiscent of their B-cell origin. Our findings suggest that modulation of PKC-α induces hematopoietic-lineage plasticity in committed B-lineage cells by perturbing expression of critical B-lineage transcription factors, and deregulation of PKC-α activity/expression represents a potential mechanism for lineage trans-differentiation during malignancies.
Asunto(s)
Linfocitos B/inmunología , Desdiferenciación Celular/inmunología , Células Progenitoras Linfoides/inmunología , Células Progenitoras Mieloides/inmunología , Factor de Transcripción PAX5/inmunología , Proteína Quinasa C-alfa/inmunología , Animales , Linfocitos B/enzimología , Desdiferenciación Celular/genética , Línea Celular , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Femenino , Reordenamiento Génico de Linfocito B/genética , Reordenamiento Génico de Linfocito B/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Progenitoras Linfoides/enzimología , Linfoma de Células B/enzimología , Linfoma de Células B/inmunología , Masculino , Ratones , Ratones Endogámicos ICR , Células Progenitoras Mieloides/enzimología , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Receptores Notch/genética , Receptores Notch/inmunología , Receptores Notch/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
We previously showed that the TLR7/8 agonist, R-848, activated HIV from cells of myeloid-monocytic origin. In this work, we show that this effect was solely due to triggering TLR8 and that NF-κB was involved in the TLR8-mediated activation of HIV from latently infected cells of myeloid-monocytic origin. Inhibition of Erk1/2 or p38α resulted in attenuation of TLR8-mediated activation of NF-κB. Western blots confirmed that TLR8 triggering activated Erk1/2 and p38α but, surprisingly, not JNK. Although the Erk1/2 inhibitors resulted in a less attenuated TLR8-mediated NF-κB response than did p38α inhibitors, they had a more pronounced effect on blocking TLR8-mediated HIV replication, indicating that other transcription factors controlled by Erk1/2 are involved in TLR8-mediated HIV activation from latently infected cells. TNF-α, which was secreted subsequent to TLR8 triggering, contributed to the activation of HIV from the latently infected cells in an autocrine manner, revealing a bimodal mechanism by which the effect of TLR8 triggering can be sustained. We also found that TNF-α secreted by myeloid dendritic cells acted in a paracrine manner in the activation of HIV from neighboring latently infected CD4(+) T cells, which do not express TLR8. Notably, monocytes from highly active antiretroviral therapy-treated HIV(+) patients with suppressed HIV RNA showed a robust TNF-α secretion in response to TLR8 agonists, pointing to a functional TLR8 signaling axis in HIV infection. Thus, triggering TLR8 represents a very promising strategy for attacking the silent HIV from its reservoir in HIV(+) patients treated successfully with highly active antiretroviral therapy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , VIH-1/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Monocitos/inmunología , Monocitos/virología , Receptor Toll-Like 8/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Activación Viral/inmunología , Linfocitos T CD4-Positivos/enzimología , Línea Celular , Células Cultivadas , Células Clonales , Células HEK293 , Humanos , Monocitos/enzimología , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/virología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/agonistasRESUMEN
IFNγ exhibits potent antitumor effects and plays important roles in the innate immunity against cancer. However, the mechanisms accounting for the antiproliferative effects of IFNγ still remain to be elucidated. We examined the role of Mnk1 (MAPK-interacting protein kinase 1) in IFNγ signaling. Our data demonstrate that IFNγ treatment of sensitive cells results in engagement of Mnk1, activation of its kinase domain, and downstream phosphorylation of the cap-binding protein eIF4E on Ser-209. Such engagement of Mnk1 plays an important role in IFNγ-induced IRF-1 (IFN regulatory factor 1) gene mRNA translation/protein expression and is essential for generation of antiproliferative responses. In studies aimed to determine the role of Mnk1 in the induction of the suppressive effects of IFNs on primitive hematopoietic progenitors, we found that siRNA-mediated Mnk1/2 knockdown results in partial reversal of the suppressive effects of IFNγ on human CD34+-derived myeloid (CFU-GM) and erythroid (BFU-E) progenitors. These findings establish a key role for the Mnk/eIF4E pathway in the regulatory effects of IFNγ on normal hematopoiesis and identify Mnk kinases as important elements in the control of IFNγ-inducible ISG mRNA translation.
Asunto(s)
Células Precursoras Eritroides/enzimología , Hematopoyesis/efectos de los fármacos , Interferón gamma/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Progenitoras Mieloides/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular Transformada , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Células Precursoras Eritroides/citología , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Hematopoyesis/fisiología , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Células Progenitoras Mieloides/citología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiologíaRESUMEN
Cancer vaccines aim to induce antitumor CTL responses, which require cross-presentation of tumor Ag to CTLs by dendritic cells (DCs). Adjuvants that facilitate cross-presentation of vaccine Ag are therefore key for inducing antitumor immunity. We previously reported that human DCs could not efficiently cross-present the full-length cancer/testis Ag NY-ESO-1 to CTL unless formulated as either an immune complex (NY-ESO-1/IC) or with ISCOMATRIX adjuvant. We now demonstrate that NY-ESO-1/ICs induce cross-presentation of HLA-A2- and HLA-Cw3-restricted epitopes via a proteasome-dependent pathway. In contrast, cross-presentation of NY-ESO-1/ISCOMATRIX vaccine was proteasome independent and required the cytosolic protease tripeptidyl peptidase II. Trafficking studies revealed that uptake of ICs and ISCOMATRIX vaccine by DCs occurred via endocytosis with delivery to lysosomes. Interestingly, ICs were retained in lysosomes, whereas ISCOMATRIX adjuvant induced rapid Ag translocation into the cytosol. Ag translocation was dependent on endosomal acidification and IL-4-driven differentiation of monocytes into DCs. This study demonstrates that Ag formulation determines Ag processing and supports a role for tripeptidyl peptidase II in cross-presentation of CTL epitopes restricted to diverse HLA alleles.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Colesterol/administración & dosificación , Reactividad Cruzada/inmunología , Citosol/inmunología , Células Dendríticas/inmunología , Proteínas de la Membrana/inmunología , Fosfolípidos/administración & dosificación , Saponinas/administración & dosificación , Serina Endopeptidasas/metabolismo , Aminopeptidasas , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/metabolismo , Diferenciación Celular/inmunología , Citosol/enzimología , Citosol/metabolismo , Células Dendríticas/enzimología , Células Dendríticas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Combinación de Medicamentos , Endocitosis/inmunología , Endosomas/enzimología , Endosomas/inmunología , Endosomas/metabolismo , Humanos , Hidrólisis , Lisosomas/enzimología , Lisosomas/inmunología , Lisosomas/metabolismo , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/metabolismo , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Transporte de Proteínas/inmunología , Quillaja/inmunología , Transducción de Señal/inmunologíaRESUMEN
Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.
Asunto(s)
Transformación Celular Neoplásica , Glucógeno Sintasa Quinasa 3/metabolismo , Leucemia Linfoide/tratamiento farmacológico , Leucemia Linfoide/patología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , División Celular , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Modelos Animales de Enfermedad , Fase G1 , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3/genética , N-Metiltransferasa de Histona-Lisina , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/metabolismo , Leucemia Linfoide/enzimología , Leucemia Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Células Progenitoras Mieloides/enzimología , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , Células Precursoras de Linfocitos B/enzimología , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Proto-Oncogenes MasRESUMEN
Menin displays the unique ability to either promote oncogenic function in the hematopoietic lineage or suppress tumorigenesis in the endocrine lineage; however, its molecular mechanism of action has not been defined. We demonstrate here that these discordant functions are unified by menin's ability to serve as a molecular adaptor that physically links the MLL (mixed-lineage leukemia) histone methyltransferase with LEDGF (lens epithelium-derived growth factor), a chromatin-associated protein previously implicated in leukemia, autoimmunity, and HIV-1 pathogenesis. LEDGF is required for both MLL-dependent transcription and leukemic transformation. Conversely, a subset of menin mutations in multiple endocrine neoplasia type 1 patients abrogate interaction with LEDGF while preserving MLL interaction but nevertheless compromise MLL/menin-dependent functions. Thus, LEDGF critically associates with MLL and menin at the nexus of transcriptional pathways that are recurrently targeted in diverse diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transformación Celular Neoplásica/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Células HeLa , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia/enzimología , Leucemia/genética , Leucemia/patología , Ratones , Ratones Endogámicos C57BL , Neoplasia Endocrina Múltiple Tipo 1/genética , Neoplasia Endocrina Múltiple Tipo 1/metabolismo , Mutación , Células Progenitoras Mieloides/enzimología , Proteína de la Leucemia Mieloide-Linfoide/genética , Unión Proteica , Proteína Metiltransferasas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Factores de Tiempo , Factores de Transcripción/genética , Transcripción Genética , Transducción Genética , Proteínas Supresoras de Tumor/genética , Células U937RESUMEN
Menin binds to the N terminus of the chromatin-remodeling histone methyltransferase MLL and is essential for the transforming activity of at least several oncogenic MLL fusion proteins. In this issue, Yokoyama and Cleary (2008) show that menin's essential, and perhaps only, contribution to leukemia is to tether a third protein, LEDGF--a chromatin-associated protein implicated in leukemia and several other disease states--to MLL. Thus, this study identifies a new, critical player in leukemias caused by MLL fusion proteins and defines the biochemical function of menin in the MLL complex.