RESUMEN
Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC-Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Megacariocitos/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Megacariocitos/patología , Ratones , Ratones Noqueados , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.
Asunto(s)
Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Animales , Linfocitos B/citología , Línea Celular , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Fosforilación , Células Precursoras de Linfocitos B/citología , Transducción de Señal , Activación TranscripcionalRESUMEN
A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.
Asunto(s)
Apoptosis/efectos de los fármacos , Daño del ADN , Doxorrubicina/farmacología , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Hidroxiurea/farmacología , Proteínas WT1/metabolismo , Animales , Apoptosis/genética , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Replicación del ADN/efectos de los fármacos , Trompas Uterinas/efectos de los fármacos , Femenino , Humanos , Células K562 , Ratones Noqueados , Cultivo Primario de Células , Proteínas WT1/genéticaRESUMEN
Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P = .05), improved PFS (HR, .393; P = .01), and OS (HR, .448; P = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [P = .008], and HR, 5.451 [P = .002], respectively), whereas no impact was observed for "triple negative" patients. Molecular genetics, especially CALR, IDH2, and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Biología Molecular/métodos , Mielofibrosis Primaria/genética , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo/métodos , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The activity of antagonizing transcription factors represents a mechanistic paradigm of bidirectional lineage-fate control during hematopoiesis. At the megakaryocytic/erythroid bifurcation, the cross-antagonism of krueppel-like factor 1 (KLF1) and friend leukemia integration 1 (FLI1) has such a decisive role. However, how this antagonism is resolved during lineage specification is poorly understood. We found that runt-related transcription factor 1 (RUNX1) inhibits erythroid differentiation of murine megakaryocytic/erythroid progenitors and primary human CD34(+) progenitor cells. We show that RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation by epigenetic repression of the erythroid master regulator KLF1. RUNX1 binding to the KLF1 locus is increased during megakaryocytic differentiation and counterbalances the activating role of T-cell acute lymphocytic leukemia 1 (TAL1). We found that corepressor recruitment by RUNX1 contributes to a block of the KLF1-dependent erythroid gene expression program. Our data indicate that the repressive function of RUNX1 influences the balance between erythroid and megakaryocytic differentiation by shifting the balance between KLF1 and FLI1 in the direction of FLI1. Taken together, we show that RUNX1 is a key player within a network of transcription factors that represses the erythroid gene expression program.
Asunto(s)
Diferenciación Celular/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica/fisiología , Megacariocitos/metabolismo , Trombopoyesis/fisiología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Eritropoyesis/fisiología , Humanos , Células K562 , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Progenitoras de Megacariocitos/citología , Células Progenitoras de Megacariocitos/metabolismo , Megacariocitos/citología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T AgudaRESUMEN
The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdc(scid) and Il2rg(null) alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation.
Asunto(s)
Retrovirus Endógenos/genética , Leucemia Experimental/genética , Leucemia Mieloide Aguda/genética , Mielofibrosis Primaria/genética , Anciano , Animales , Southern Blotting , Femenino , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Virus de la Leucemia Murina/genética , Leucemia Experimental/patología , Leucemia Experimental/virología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Persona de Mediana Edad , Datos de Secuencia Molecular , Mielofibrosis Primaria/patología , Mielofibrosis Primaria/virología , Provirus/genética , Trasplante Heterólogo , Integración Viral/genética , Replicación Viral/genética , Adulto JovenRESUMEN
The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small-animal models that mimick human disease. Here we show that mice with transplanted human hematopoetic stem cells reproduce features typical of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small-animal model with human components for the development of EVD therapies.
Asunto(s)
Modelos Animales de Enfermedad , Ebolavirus/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/transmisión , Xenoinjertos/inmunología , Ratones Endogámicos NOD , Animales , Hígado Graso/patología , Hemorragia/patología , Fiebre Hemorrágica Ebola/patología , Humanos , Estimación de Kaplan-Meier , Ratones , Microscopía Fluorescente , Viremia/patologíaRESUMEN
Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)-induced proliferation with diminished myeloid differentiation of hematopoietic CD34(+) cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.
Asunto(s)
Transformación Celular Neoplásica/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide/genética , Mutación , Neutropenia/congénito , Receptores del Factor Estimulante de Colonias/genética , Adolescente , Adulto , Niño , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Análisis Citogenético , Femenino , Humanos , Masculino , Neutropenia/genética , Neutropenia/patología , Transducción de Señal/genética , Adulto JovenRESUMEN
The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. This study revealed 2 critical functions of Runx1: (1) to promote survival and development of progenitors specified to the B-cell lineage, a function that can be substituted by ectopic Bcl2 expression, and (2) to enable the developmental transition through the pre-B stage triggered by the pre-B-cell antigen receptor (pre-BCR). Gene expression analysis and genomewide Runx1 occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network governing early B-cell survival and development and specifically regulates genes encoding members of the Lyn kinase subfamily (key integrators of interleukin-7 and pre-BCR signaling) and the stage-specific transcription factors SpiB and Aiolos (critical downstream effectors of pre-BCR signaling). Interrogation of expression databases of 257 ALL samples demonstrated the specific down-regulation of the SPIB and IKZF3 genes (the latter encoding AIOLOS) in t(12;21) ALL, providing novel insight into the mechanism by which the translocation blocks B-cell development and promotes leukemia.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Animales , Apoptosis/genética , Sitios de Unión , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Proliferación Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 21/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Marcación de Gen , Genoma/genética , Humanos , Factor de Transcripción Ikaros , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Translocación GenéticaRESUMEN
Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133(+) stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133(+) but not CD34(+)CD133(-) cells, with a variable burden for JAK2 (V617F) mutation, had multipotent cloning capacity in vitro. CD133(+) cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F(+) myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F(+) megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133(+) cells harboring EZH2 (D265H) but lacking a secondary JAK2 (V617F) mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms.
Asunto(s)
Antígenos CD/sangre , Biomarcadores de Tumor/sangre , Glicoproteínas/sangre , Células Madre Hematopoyéticas/metabolismo , Péptidos/sangre , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/diagnóstico , Antígeno AC133 , Adulto , Anciano , Animales , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Persona de Mediana EdadRESUMEN
Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in approximately one-third of AML patients, mostly by internal tandem duplications (ITDs). Adaptor protein Lnk is a negative regulator of hematopoietic cytokine signaling. In the present study, we show that Lnk interacts physically with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through the SH2 domains. We have identified the tyrosine residues 572, 591, and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Lnk itself was tyrosine phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Both shRNA-mediated depletion and forced overexpression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Moreover, Lnk inhibited 32D cell proliferation driven by different FLT3 variants. Analysis of primary BM cells from Lnk-knockout mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors. The results of the present study show that through direct binding to FLT3, Lnk suppresses FLT3-WT/ITD-dependent signaling pathways involved in the proliferation of hematopoietic cells. Therefore, modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic disease.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Células Madre Hematopoyéticas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales , Animales , Proliferación Celular , Transformación Celular Neoplásica/patología , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Leucemia Mieloide Aguda/patología , Proteínas de la Membrana , Ratones , Ratones Noqueados , Mutación/genética , Fosforilación , ARN Interferente Pequeño/genética , Transducción de Señal , Secuencias Repetidas en Tándem/genética , Tirosina/metabolismo , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Dominios Homologos srcRESUMEN
The transcription factor runt-related transcription factor 1 (Runx1) is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cells. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. In the present study, we describe a genome-scale RNA interference screen to detect genes that cooperate with Runx1 in regulating hematopoietic stem and progenitor cells. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation and show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of lineage marker-negative cells in vitro. We found an up-regulation of HoxA cluster genes on Pcgf1 knock-down that possibly accounts for the increase in self-renewal. Moreover, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1, together with the epigenetic repressor Pcgf1, is necessary for terminal differentiation. The results of the present study uncover a link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Proteínas de Unión al ADN/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Animales , Trasplante de Médula Ósea , División Celular , Células Cultivadas/citología , Inmunoprecipitación de Cromatina , Ensayo de Unidades Formadoras de Colonias , Subunidad alfa 2 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Epigénesis Genética , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Complejo Represivo Polycomb 1 , ARN Interferente Pequeño/farmacología , Quimera por Radiación , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/fisiología , Organismos Libres de Patógenos Específicos , Transducción GenéticaRESUMEN
Brain metastasis leads to increased mortality and is a major site of relapse for several cancers, yet the molecular mechanisms of brain metastasis are not well understood. In this study, we established and characterized a new leukemic cell line, FIA10, that metastasizes into the central nervous system (CNS) following injection into the tail vein of syngeneic mice. Mice injected with FIA10 cells developed neurological symptoms such as loss of balance, tremor, ataxic gait and seizures, leading to death within 3 months. Histopathology coupled with PCR analysis clearly showed infiltration of leukemic FIA10 cells into the brain parenchyma of diseased mice, with little involvement of bone marrow, peripheral blood and other organs. To define pathways that contribute to CNS metastasis, global transcriptome and proteome analysis was performed on FIA10 cells and compared with that of the parental stem cell line FDCP-Mix and the related FIA18 cells, which give rise to myeloid leukemia without CNS involvement. 188 expressed genes (RNA level) and 189 proteins were upregulated (log2 ratio FIA10/FIA18 ≥ 1) and 120 mRNAs and 177 proteins were downregulated (log2 ratio FIA10/FIA18 ≤ 1) in FIA10 cells compared with FIA18 cells. Major upregulated pathways in FIA10 cells revealed by biofunctional analyses involved immune response components, adhesion molecules and enzymes implicated in extracellular matrix remodeling, opening and crossing the blood-brain barrier (BBB), molecules supporting migration within the brain parenchyma, alterations in metabolism necessary for growth within the brain microenvironment, and regulators for these functions. Downregulated RNA and protein included several tumor suppressors and DNA repair enzymes. In line with the function of FIA10 cells to specifically infiltrate the brain, FIA10 cells have acquired a phenotype that permits crossing the BBB and adapting to the brain microenvironment thereby escaping immune surveillance. These data and our model system FIA10 will be valuable resources to study the occurrence of brain metastases and may help in the development of potential therapies against brain invasion.
Asunto(s)
Neoplasias Encefálicas , Neoplasias del Sistema Nervioso Central , Ratones , Animales , Transcriptoma , Proteómica , Encéfalo/metabolismo , Barrera Hematoencefálica/metabolismo , Neoplasias del Sistema Nervioso Central/patología , Neoplasias Encefálicas/patología , Perfilación de la Expresión Génica , ARN/metabolismo , Línea Celular , Microambiente TumoralRESUMEN
Prion diseases are fatal neurodegenerative disorders. An important step in disease pathophysiology is the conversion of cellular prion protein (PrP(C)) to disease-associated misfolded conformers (PrP(Sc)). These misfolded PrP variants are a common component of prion infectivity and are detectable in diseased brain and lymphoreticular organs such as spleen. In the latter, PrP(Sc) is thought to replicate mainly in follicular dendritic cells within spleen follicles. Although the presence of PrP(Sc) is a hallmark for prion disease and serves as a main diagnostic criterion, in certain instances the amount of PrP(Sc) does not correlate well with neurotoxicity or prion infectivity. Therefore, it has been proposed that prions might be a mixture of different conformers and aggregates with differing properties. This study investigated the impact of disruption of spleen architecture by neoplasia on the abundance of different PrP species in spleens of prion-infected mice. Although follicular integrity was completely disturbed, titres of prion infectivity in neoplastic spleens were not significantly altered, yet no protease-resistant PrP(Sc) was detectable. Instead, unique protease-sensitive prion species could be detected in neoplastic spleens. These results indicate the dissociation of PrP(Sc) and prion infectivity and showed the presence of non-PrP(Sc) PrP species in spleen with divergent biochemical properties that become apparent after tissue architecture disruption.
Asunto(s)
Péptido Hidrolasas/metabolismo , Enfermedades por Prión/complicaciones , Priones/aislamiento & purificación , Priones/metabolismo , Bazo/patología , Neoplasias del Bazo/complicaciones , Animales , Modelos Animales de Enfermedad , Ratones , Enfermedades por Prión/patología , Proteínas Priónicas , Priones/clasificación , Neoplasias del Bazo/patologíaRESUMEN
The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein, which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis, we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells, as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients, even in the absence of t(8;21). On a functional level, knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly, self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies, serial replating capacity of primary cells, and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Proteínas Co-Represoras , Células HeLa , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteína 1 Compañera de Translocación de RUNX1 , Proteínas Represoras/metabolismoRESUMEN
BACKGROUND: Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. METHODS: We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. RESULTS: We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. CONCLUSIONS: The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.
RESUMEN
Although the potential role of Pim2 as a cooperative oncogene has been well described in lymphoma, its role in leukemia has remained largely unexplored. Here we show that high expression of Pim2 is observed in patients with acute promyelocytic leukemia (APL). To further characterize the cooperative role of Pim2 with promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha), we used a well-established PML-RARalpha (PRalpha) mouse model. Pim2 coexpression in PRalpha-positive hematopoietic progenitor cells (HPCs) induces leukemia in recipient mice after a short latency. Pim2-PRalpha cells were able to repopulate mice in serial transplantations and to induce disease in all recipients. Neither Pim2 nor PRalpha alone was sufficient to induce leukemia upon transplantation in this model. The disease induced by Pim2 overexpression in PRalpha cells contained a slightly higher fraction of immature myeloid cells, compared with the previously described APL disease induced by PRalpha. However, it also clearly resembled an APL-like phenotype and showed signs of differentiation upon all-trans retinoic acid (ATRA) treatment in vitro. These results support the hypothesis that Pim2, which is also a known target of Flt3-ITD (another gene that cooperates with PML-RARalpha), cooperates with PRalpha to induce APL-like disease.
Asunto(s)
Leucemia Mieloide Aguda/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Animales , Secuencia de Bases , Trasplante de Médula Ósea , Cartilla de ADN/genética , Femenino , Expresión Génica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Leucemia Promielocítica Aguda/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Oncogenes , Tretinoina/farmacología , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
A fundamental step in pathophysiology of prion diseases is the conversion of the host encoded prion protein (PrP(C)) into a misfolded isoform (PrP(Sc)) that accumulates mainly in neuronal but also non-neuronal tissues. Prion diseases are transmissible within and between species. In a subset of prion diseases, peripheral prion uptake and subsequent transport to the central nervous system are key to disease initiation. The involvement of retroviruses in this process has been postulated based on the findings that retroviral infections enhance the spread of prion infectivity and PrP(Sc) from cell to cell in vitro. To study whether retroviral infection influences the phenotype of prion disease or the spread of prion infectivity and PrP(Sc) in vivo, we developed a murine model with persistent Moloney murine leukemia retrovirus (MoMuLV) infection with and without additional prion infection. We investigated the pathophysiology of prion disease in MoMuLV and prion-infected mice, monitoring temporal kinetics of PrP(Sc) spread and prion infectivity, as well as clinical presentation. Unexpectedly, infection of MoMuLV challenged mice with prions did not change incubation time to clinical prion disease. However, clinical presentation of prion disease was altered in mice infected with both pathogens. This was paralleled by remarkably enhanced astrogliosis and pathognomonic astrocyte morphology in the brain of these mice. Therefore, we conclude that persistent viral infection might act as a disease modifier in prion disease.
Asunto(s)
Sistema Nervioso Central/patología , Virus de la Leucemia Murina de Moloney/patogenicidad , Enfermedades por Prión/patología , Priones/metabolismo , Infecciones por Retroviridae/patología , Infecciones Tumorales por Virus/patología , Animales , Proteínas de Unión al Calcio , Línea Celular Transformada , Sistema Nervioso Central/virología , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/patología , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Ratones , Proteínas de Microfilamentos , Fenotipo , Enfermedades por Prión/complicaciones , Enfermedades por Prión/metabolismo , Infecciones por Retroviridae/complicaciones , Bazo/metabolismo , Bazo/patología , Factores de Tiempo , Infecciones Tumorales por Virus/complicacionesRESUMEN
OBJECTIVES: : The symptom experience of community-dwelling persons with dementia adopting an open-ended approach has not been well documented. We sought to identify the most bothersome symptoms experienced using self and caregiver report, and to evaluate whether these symptoms are captured by commonly used symptom-assessment measures including the Edmonton Symptom Assessment System (ESAS, standard in palliative care), Neuropsychiatric Inventory (NPI), and End-of-Life Dementia Scale-Symptom Management (ELDS-SM). DESIGN: : The authors use data from the Palliative Excellence in Alzheimer Care Efforts (PEACE) study to characterize the symptom experience. SETTING: : PEACE included outpatient primary care geriatric patients in an urban setting affiliated with the University of Chicago. PARTICIPANTS: : Data were examined from the 150 patient-caregiver dyads. MEASUREMENTS: : The most bothersome symptoms of persons with dementia during the past week were reported separately by self and family caregiver. Symptoms were asked in an open-ended format and qualitative analysis using constant comparative technique was applied for each response. Reports were categorized and frequencies tabulated. RESULTS: : One hundred fifteen persons (35 could not respond to the interview questions) with dementia reported 135 symptoms (median 1, range: 0-3). The most frequently reported symptoms were pain (N = 48; 42%), depression (N = 13; 11%), cognitive deficit (N = 12; 10%), anxiety (N = 7; 6%), and ophthalmologic complaint (N = 5; 4%). One hundred fifty caregivers reported 259 symptoms (median 2, range: 0-5) with cognitive deficit (N = 71; 47%), pain (N = 46; 30%), depression (N = 26; 17%), activity disturbance (N = 23; 15%), and thought and perceptual disturbances (N = 12; 8%) being reported most frequently. The ESAS, NPI, and ELDS-SM missed two or more of the most commonly reported symptoms by dyads of persons with dementia and their family caregivers. CONCLUSION: : Symptoms were frequently reported by persons with dementia and their caregiver; however, commonly used symptom assessment measures overlooked important symptoms. All physicians should be vigilant about screening for both psychologic and physical symptoms in this population.
Asunto(s)
Cuidadores/psicología , Demencia/diagnóstico , Evaluación Geriátrica/métodos , Escalas de Valoración Psiquiátrica/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Femenino , Evaluación Geriátrica/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Características de la Residencia , AutoinformeRESUMEN
Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis, we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a myeloproliferation in vivo. Among the targeted genes, we identified Mef2c, encoding a MCM1-agamous-deficiens-serum response factor transcription factor, and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions, prompting an investigation of the causal interplay. Using a conditional mouse strain, we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however, its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to increased homing and motility. Thus, MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype.