RESUMEN
Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s, SMC3+/-, and MPLW515K, providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell-based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.
Asunto(s)
Síndrome de Down , Leucemia Megacarioblástica Aguda , Proteínas de Ciclo Celular/genética , Niño , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas Cromosómicas no Histona/genética , Síndrome de Down/genética , Factor de Transcripción GATA1/genética , Hematopoyesis , Humanos , Leucemia Megacarioblástica Aguda/complicaciones , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/metabolismo , Megacariocitos/metabolismo , Mutación , TrisomíaRESUMEN
EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss-of-function mutations have been found in myeloproliferative neoplasms, particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and short hairpin RNA induces megakaryocyte (MK) commitment by accelerating lineage marker acquisition without change in proliferation. Later in differentiation, EZH2 inhibition blocks proliferation and polyploidization and decreases proplatelet formation. EZH2 inhibitors similarly reduce MK polyploidization and proplatelet formation in vitro and platelet levels in vivo in a JAK2V617F background. In transcriptome profiling, the defect in proplatelet formation was associated with an aberrant actin cytoskeleton regulation pathway, whereas polyploidization was associated with an inhibition of expression of genes involved in DNA replication and repair and an upregulation of cyclin-dependent kinase inhibitors, particularly CDKN1A and CDKN2D. The knockdown of CDKN1A and to a lesser extent CDKN2D could partially rescue the percentage of polyploid MKs. Moreover, H3K27me3 and EZH2 chromatin immunoprecipitation assays revealed that CDKN1A is a direct EZH2 target and CDKN2D expression is not directly regulated by EZH2, suggesting that EZH2 controls MK polyploidization directly through CDKN1A and indirectly through CDKN2D.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Megacariocitos/citología , Trombopoyesis , Animales , Plaquetas/citología , Plaquetas/metabolismo , Células Cultivadas , Proteína Potenciadora del Homólogo Zeste 2/genética , Humanos , Megacariocitos/metabolismo , Ratones , Interferencia de ARN , TranscriptomaRESUMEN
Polymerization of the sickle hemoglobin (HbS) is a key determinant of sickle cell disease (SCD), an inherited blood disorder. Fetal hemoglobin (HbF) is a major modulator of the disease severity by both decreasing HbS intracellular concentration and inhibiting its polymerization. However, heterocellular distribution of HbF is common in SCD. For HbS polymerization inhibition, the hypothesis of an "HbF per red blood cell (HbF/RBC) threshold" requires accurate measurement of HbF in individual RBC. To date, HbF detection methods are limited to a qualitative measurement of RBC populations containing HbF - the F cells, which are variable. We developed an accurate method for HbF quantification in individual RBC. A linear association between mean HbF content and mean RBC fluorescence by flow cytometry, using an anti-Human-HbF antibody, was obtained from non-SCD subjects presenting homogeneous HbF distribution. This correlation was then used to measure HbF/RBC. Hydroxyurea (HU) improves SCD clinical manifestations, mainly through its ability to induce HbF synthesis. The HbF distribution was analyzed in 14 SCD patients before and during HU treatment. A significant decrease in RBC population containing less than 2 pg of HbF/RBC was observed. Therefore, we tested associations for %RBC above different HbF/RBC thresholds and showed a decrease in the pathognomonic vaso-occlusive crisis incidence from the threshold of 4 pg. This quantity was also correlated with the level of sickle RBC after in vitro deoxygenation. This new method allows the comparison of HbF/RBC distributions and could be a useful tool to characterize baseline patients HbF distribution and therapeutic response to HbF inducers.
Asunto(s)
Anemia de Células Falciformes , Eritrocitos Anormales/metabolismo , Hemoglobina Fetal/metabolismo , Citometría de Flujo , Hidroxiurea/administración & dosificación , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Femenino , Humanos , MasculinoRESUMEN
While enucleation is a critical step in the terminal differentiation of human red blood cells, the molecular mechanisms underlying this unique process remain unclear. To investigate erythroblast enucleation, we studied the erythroid differentiation of human embryonic stem cells (hESCs), which provide a unique model for deeper understanding of the development and differentiation of multiple cell types. First, using a two-step protocol, we demonstrated that terminal erythroid differentiation from hESCs is directly dependent on the age of the embryoid bodies. Second, by choosing hESCs in two extreme conditions of erythroid culture, we obtained an original differentiation model which allows one to study the mechanisms underlying the enucleation of erythroid cells by analyzing the gene and miRNA (miR) expression profiles of cells from these two culture conditions. Third, using an integrated analysis of mRNA and miR expression profiles, we identified five miRs potentially involved in erythroblast enucleation. Finally, by selective knockdown of these five miRs we found miR-30a to be a regulator of erythroblast enucleation in hESCs.
Asunto(s)
Diferenciación Celular , Eritroblastos/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , MicroARNs/biosíntesis , ARN Mensajero/biosíntesis , Células Cultivadas , Eritroblastos/citología , Células Madre Embrionarias Humanas/citología , HumanosRESUMEN
Lentiviral modification combined with ex vivo erythroid differentiation was used to stably inhibit RhAG expression, a critical component of the Rh(rhesus) membrane complex defective in the Rh(null) syndrome. The cultured red cells generated recapitulate the major alterations of native Rh(null) cells regarding antigen expression, membrane deformability, and gas transport function, providing the proof of principle for their use as model of Rh(null) syndrome and to investigate Rh complex biogenesis in human primary erythroid cells. Using this model, we were able to reveal for the first time that RhAG extinction alone is sufficient to explain ICAM-4 and CD47 loss observed on native Rh(null) RBCs. Together with the effects of RhAG forced expression in Rh(null) progenitors, this strongly strengthens the hypothesis that RhAG is critical to Rh complex formation. The strategy is also promising for diagnosis purpose in order to overcome the supply from rare blood donors and is applicable to other erythroid defects and rare phenotypes, providing models to dissect membrane biogenesis of multicomplex proteins in erythroid cells, with potential clinical applications in transfusion medicine.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Antígeno CD47/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Eritroides/metabolismo , Enfermedades Genéticas Congénitas/metabolismo , Glicoproteínas de Membrana/metabolismo , Sistema del Grupo Sanguíneo Rh-Hr/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Anemia Hemolítica Congénita/metabolismo , Anemia Hemolítica Congénita/patología , Anemia Hipoplástica Congénita/metabolismo , Anemia Hipoplástica Congénita/patología , Proteínas Sanguíneas/antagonistas & inhibidores , Proteínas Sanguíneas/genética , Diferenciación Celular , Línea Celular , Células Cultivadas , Células Eritroides/patología , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Femenino , Sangre Fetal , Células Madre Fetales/citología , Células Madre Fetales/metabolismo , Enfermedades Genéticas Congénitas/sangre , Enfermedades Genéticas Congénitas/patología , Humanos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/genética , Porfiria Eritropoyética/metabolismo , Porfiria Eritropoyética/patología , Embarazo , Interferencia de ARN , ARN Interferente Pequeño , Reticulocitos/metabolismo , Reticulocitos/patología , Sistema del Grupo Sanguíneo Rh-Hr/sangreRESUMEN
Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Eritrocitos/metabolismo , Janus Quinasa 2/metabolismo , Sistema del Grupo Sanguíneo Lutheran/metabolismo , Policitemia Vera/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Eritropoyetina/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Adhesión Celular/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Femenino , Humanos , Janus Quinasa 2/genética , Laminina/metabolismo , Sistema del Grupo Sanguíneo Lutheran/genética , Masculino , Ratones , Persona de Mediana Edad , Fosforilación , Policitemia Vera/genética , Transducción de SeñalRESUMEN
Although clinical benefits have been reported in several human hematopoietic gene therapy trials, a remaining important goal is the transition to nonmyeloablative pretransplantation conditioning to decrease toxicity. Previous attempts at reduced intensity conditioning in nonhuman primates have resulted in only temporary vector marking of autologous blood cells or their persistence at low levels, well below the thresholds for clinical efficacy. In addition, we reasoned that lentiviral vector particles displaying cytokines at their surface have the potential to preserve stem cell fitness better than current ex vivo transduction protocols, which involve exposure to cytokine overstimulation. Here we show that the classically nonmyeloablative agent fludarabine (30 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and the use of a stem cell factor-displaying simian immunodeficiency virus-based vector, resulted in sustained, single-copy vector marking of autologous blood cells in two macaques over 3 years posttransplantation at levels averaging 1% of all lineages. This percentage is within the range of anticipated efficacy levels for hemophilia and related diseases and forms a basis for further improvement.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Virus de la Inmunodeficiencia de los Simios/genética , Factor de Células Madre/genética , Acondicionamiento Pretrasplante , Animales , Antígenos CD34/metabolismo , Quimerismo , Terapia Genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Hemofilia A/terapia , Humanos , Recuento de Linfocitos , Macaca fascicularis , Masculino , Agonistas Mieloablativos/administración & dosificación , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción Genética , Trasplante Autólogo , Vidarabina/administración & dosificación , Vidarabina/análogos & derivados , Irradiación Corporal TotalRESUMEN
Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34(+) cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbalpha, GpIIb, or EF1alpha gene regulatory sequences. Both GpIbalpha and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbalpha promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbalpha gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Vectores Genéticos , Células Madre Hematopoyéticas/citología , Lentivirus , Megacariocitos/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Regiones Promotoras Genéticas/fisiología , Transducción Genética , Animales , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Megacariocitos/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Especificidad de Órganos , Inactivación de VirusRESUMEN
Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase alpha (ACCalpha), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACCalpha in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACCalpha or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)-resistant form of ACCalpha mRNA prevented the effect of ACCalpha-RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACCalpha and FAS siRNA-induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACCalpha or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy.
Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Neoplasias de la Mama/enzimología , Acetil-CoA Carboxilasa/biosíntesis , Acetil-CoA Carboxilasa/genética , Apoptosis/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Ácido Graso Sintasas/biosíntesis , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/biosíntesis , Silenciador del Gen , Humanos , Lipogénesis/fisiología , Ácido Palmítico/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
The mammalian gene (ACACA) encoding acetyl-CoA carboxylase-alpha, a key regulatory enzyme of fatty acid synthesis, is transcribed from multiple promoters. We have delineated the 5' boundary of ACACA in four species (human, mouse, rat, and ovine). The 5' end of ACACA is located within a 600- to 700-bp CpG island encompassing a bidirectional promoter shared with the divergently oriented TADA2L, which encodes a component of chromatin-modifying complexes. In mouse and rat, this promoter, now referred to as Acaca PI, is located 43 kb upstream of the previously known regulatory regions. The shared promoter coregulates transcripts for TADA2L and ACACA in an asymmetric fashion in human and mouse tissues. A higher concentration of RNA polymerase II (Pol II) within the intergenic region in brain compared to liver of mouse reflects the greater abundance of the two transcripts in brain. The concentration of Pol II tracking downstream, which is lower than at the promoter, is not significantly different in either gene in the two tissues and does not reflect the 10- and >200-fold greater abundance of Tada2l and Acaca PI transcripts, respectively, in brain. Thus, regulation of clearance of Pol II from the promoter and the rate of elongation may therefore be determinants of the asymmetric expression of these transcripts.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Acetil-CoA Carboxilasa/metabolismo , Animales , Secuencia de Bases , Islas de CpG/genética , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares , Especificidad de Órganos , ARN Polimerasa II/metabolismo , Ratas , Ovinos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
TAL-1 is a basic helix-loop-helix oncoprotein that is expressed in up to 30% of T-cell acute lymphoblastic leukemias but not in the T lineage. We have cloned a complementary DNA, called Human Immune Associated Nucleotide 1 (hIAN1), whose messenger RNA (mRNA) level expression is inversely correlated to the TAL-1 mRNA level in human leukemic T-cell lines. The hIAN1 encodes a 38-kd protein that belongs to a novel family of proteins conserved from plants to humans and characterized by motifs related to, but highly divergent from, the consensus motifs found in guanosine triphosphate (GTP)-binding proteins. Despite these divergent amino acids at positions involved in GTP/guanosine diphosphate (GDP) binding and guanosine triphosphatase (GTPase) activities, we found that hIAN1 specifically binds GDP (K(d) = 0.47 microM) and GTP (K(d) = 6 microM) and exhibits intrinsic GTPase activity. Among mature hematopoietic cells, hIAN1 is specifically expressed in resting T and B lymphocytes, and its expression level tremendously decreased at the protein but not the mRNA level during B- or T-lymphocyte activation, suggesting a specific role for this new type of GTPase during the immune response.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas Proto-Oncogénicas , Factores de Transcripción , Secuencia de Aminoácidos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cromosomas Humanos Par 7 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Células Jurkat/química , Activación de Linfocitos , Datos de Secuencia Molecular , Unión Proteica , ARN Mensajero/metabolismo , Alineación de Secuencia , Proteína 1 de la Leucemia Linfocítica T Aguda , Linfocitos T/inmunología , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
The initiation of a primary immune response requires contact between dendritic cells (DCs) and resting T cells. However, little is known about the proteins that mediate this initial contact. We show here that neuropilin-1, a receptor involved in axon guidance, was expressed by human DCs and resting T cells both in vitro and in vivo. The initial contact between DCs and resting T cells led to neuropilin-1 polarization on T cells. DCs and resting T cells specifically bound soluble neuropilin-1, and resting T cells formed clusters with neuropilin-1-transfected COS-7 cells in a neuropilin-1-dependent manner. Functionally, preincubation of DCs or resting T cells with blocking neuropilin-1 antibodies inhibited DC-induced proliferation of resting T cells. These data suggest that neuropilin-1 mediates interactions between DCs and T cells that are essential for initiation of the primary immune response and show parallels between the nervous and immune systems.