Assuntos
Modelos Animais de Doenças , Síndrome de Down/complicações , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The transcriptional repressor BCL11A is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase Trib1 in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells in vitro and in vivo. Chromatin immunoprecipitation sequencing analysis showed that, upon DNA binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components HDAC and LSD1 reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor pracinostat and the LSD1 inhibitor GSK2879552 resulted in growth inhibition in vitro and in vivo. High BCL11A expression is associated with worse prognosis in humans with AML. Blocking of BCL11A expression upregulates the expression of PU.1 target genes and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.
Assuntos
Leucemia Mieloide Aguda , Adulto , DNA , Hemoglobina Fetal , Histona Desmetilases , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas Tipo C , Leucemia Mieloide Aguda/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Superfície Celular , Proteínas RepressorasRESUMO
BLNK (BASH/SLP-65) encodes an adaptor protein that plays an important role in B-cell receptor (BCR) signaling. Loss-of-function mutations in this gene are observed in human pre-B acute lymphoblastic leukemia (ALL), and a subset of Blnk knock-out (KO) mice develop pre-B-ALL. To understand the molecular mechanism of the Blnk mutation-associated pre-B-ALL development, retroviral tagging was applied to KO mice using the Moloney murine leukemia virus (MoMLV). The Blnk mutation that significantly accelerated the onset of MoMLV-induced leukemia and increased the incidence of pre-B-ALL Cebpb was identified as a frequent site of retroviral integration, suggesting that its upregulation cooperates with Blnk mutations. Transgenic expression of the liver-enriched activator protein (LAP) isoform of Cebpb reduced the number of mature B-lymphocytes in the bone marrow and inhibited differentiation at the pre-BI stage. Furthermore, LAP expression significantly accelerated leukemogenesis in Blnk KO mice and alone acted as a B-cell oncogene. Furthermore, an inverse relationship between BLNK and C/EBPß expression was also noted in human pre-B-ALL cases, and the high level of CEBPB expression was associated with short survival periods in patients with BLNK-downregulated pre-B-ALL. These results indicate the association between the C/EBPß transcriptional network and BCR signaling in pre-B-ALL development and leukemogenesis. This study gives insight into ALL progression and suggests that the BCR/C/EBPß pathway can be a therapeutic target.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Vírus da Leucemia Murina de Moloney/fisiologia , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/virologia , Regulação para Cima , Integração ViralRESUMO
The pseudokinase Trib1 functions as a myeloid oncogene that recruits the E3 ubiquitin ligase COP1 to C/EBPα and interacts with MEK1 to enhance extracellular signal-regulated kinase (ERK) phosphorylation. A close genetic effect of Trib1 on Hoxa9 has been observed in myeloid leukemogenesis, where Trib1 overexpression significantly accelerates Hoxa9-induced leukemia onset. However, the mechanism underlying how Trib1 functionally modulates Hoxa9 transcription activity is unclear. Herein, we provide evidence that Trib1 modulates Hoxa9-associated super-enhancers. Chromatin immunoprecipitation sequencing analysis identified increased histone H3K27Ac signals at super-enhancers of the Erg, Spns2, Rgl1, and Pik3cd loci, as well as increased messenger RNA expression of these genes. Modification of super-enhancer activity was mostly achieved via the degradation of C/EBPα p42 by Trib1, with a slight contribution from the MEK/ERK pathway. Silencing of Erg abrogated the growth advantage acquired by Trib1 overexpression, indicating that Erg is a critical downstream target of the Trib1/Hoxa9 axis. Moreover, treatment of acute myeloid leukemia (AML) cells with the BRD4 inhibitor JQ1 showed growth inhibition in a Trib1/Erg-dependent manner both in vitro and in vivo. Upregulation of ERG by TRIB1 was also observed in human AML cell lines, suggesting that Trib1 is a potential therapeutic target of Hoxa9-associated AML. Taken together, our study demonstrates a novel mechanism by which Trib1 modulates chromatin and Hoxa9-driven transcription in myeloid leukemogenesis.
Assuntos
Regulação Leucêmica da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Progressão da Doença , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição GênicaRESUMO
Trib1 has been identified as a myeloid oncogene in a murine leukemia model. Here we identified a TRIB1 somatic mutation in a human case of Down syndrome-related acute megakaryocytic leukemia. The mutation was observed at well-conserved arginine 107 residue in the pseudokinase domain. This R107L mutation remained in leukocytes of the remission stage in which GATA1 mutation disappeared, suggesting the TRIB1 mutation is an earlier genetic event in leukemogenesis. The bone marrow transfer experiment showed that acute myeloid leukemia development was accelerated by transducing murine bone marrow cells with the R107L mutant in which enhancement of ERK phosphorylation and C/EBPα degradation by Trib1 expression was even greater than in those expressing wild-type. These results suggest that TRIB1 may be a novel important oncogene for Down syndrome-related acute megakaryocytic leukemia.
Assuntos
Síndrome de Down/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Megacarioblástica Aguda/etiologia , Mutação/genética , Oncogenes/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Síndrome de Down/complicações , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Immunoblotting , Leucemia Megacarioblástica Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Trib1 is a myeloid oncogene that cooperates with Hoxa9 and Meis1. Although the MAPK pathway and C/EBP transcription factors are known to interact with Trib proteins, the mechanisms by which Trib1 contributes to myeloid leukemogenesis remains to be clarified. Here we report that interaction between Trib1 and MEK1 is required for Trib1-induced leukemogenesis. The C-terminal ILLHPWF motif that is well conserved among Trib family proteins is required for MEK1 binding, enhancement of ERK phosphorylation, enhanced self-renewal activity of bone marrow cells and leukemogenic activity by Trib1. The motif is also important for Trib1-induced C/EBPα degradation though interaction between Trib1 and C/EBPα is not necessary. Inhibition of ERK phosphorylation suppressed Trib1-induced C/EBPα degradation, indicating an important role for Trib1/MEK1 interaction. These results suggest that Trib1 may be a key mediator between the RTK-MAPK pathway and the C/EBP transcription factor in myeloid leukemogenesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide/etiologia , MAP Quinase Quinase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oncogenes , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Mutations of genes encoding alpha4, beta2, or alpha2 subunits (CHRNA4, CHRNB2, or CHRNA2, respectively) of nAChR [neuronal nicotinic ACh (acetylcholine) receptor] cause nocturnal frontal lobe epilepsy (NFLE) in human. NFLE-related seizures are seen exclusively during sleep and are characterized by three distinct seizure phenotypes: "paroxysmal arousals," "paroxysmal dystonia," and "episodic wandering." We generated transgenic rat strains that harbor a missense mutation S284L, which had been identified in CHRNA4 in NFLE. The transgenic rats were free of biological abnormalities, such as dysmorphology in the CNS, and behavioral abnormalities. The mRNA level of the transgene (mutant Chrna4) was similar to the wild type, and no distorted expression was detected in the brain. However, the transgenic rats showed epileptic seizure phenotypes during slow-wave sleep (SWS) similar to those in NFLE exhibiting three characteristic seizure phenotypes and thus fulfilled the diagnostic criteria of human NFLE. The therapeutic response of these rats to conventional antiepileptic drugs also resembled that of NFLE patients with the S284L mutation. The rats exhibited two major abnormalities in neurotransmission: (1) attenuation of synaptic and extrasynaptic GABAergic transmission and (2) abnormal glutamate release during SWS. The currently available genetically engineered animal models of epilepsy are limited to mice; thus, our transgenic rats offer another dimension to the epilepsy research field.
Assuntos
Epilepsia do Lobo Frontal/genética , Leucina/genética , Mutação/genética , Receptores Nicotínicos/genética , Serina/genética , Transmissão Sináptica/genética , Ácido gama-Aminobutírico/metabolismo , Fatores Etários , Análise de Variância , Animais , Comportamento Animal/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Relação Dose-Resposta a Droga , Eletroencefalografia , Epilepsia do Lobo Frontal/dietoterapia , Epilepsia do Lobo Frontal/fisiopatologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Ácido Glutâmico/metabolismo , Temperatura Alta/efeitos adversos , Técnicas In Vitro , Microscopia Imunoeletrônica , Atividade Motora/genética , Destreza Motora/fisiologia , Neurotransmissores/metabolismo , Nicotina/farmacologia , Medição da Dor/métodos , Pentilenotetrazol/farmacologia , Fenótipo , Ratos , Ratos Transgênicos , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/ultraestrutura , Transmissão Sináptica/efeitos dos fármacosRESUMO
The mutation of Apc is an important early genetic event in colon carcinogenesis. However, it remains to be clarified what kinds of cooperative genes are required for complete carcinogenesis. To identify cooperative genes for the Apc(Min) mutation the authors carried out retroviral insertional mutagenesis (RIM) using Min mouse-derived IMCE colon epithelial cells. Anchorage-independent transformed colonies were induced by retroviral infection only in IMCE cells, while no transformation was found in young adult mouse colon (YAMC) cells that are normal for Apc. One hundred and fifty-seven retroviral integration sites (RIS) were identified in 101 independent transformants, and four common integration sites (CIS), Dnah3, Ahnak, Stk17b and Rbm9, were observed. Upregulation of Dnah3 and Ahnak, and truncation of Dnah3 due to the viral integration, was revealed. In addition, Dnah3-overexpressing IMCE cells showed impairment of microtubule function. These data suggest the importance of cytoskeletal function in Apc-related tumor development and the usefulness of RIM in non-hematopoietic tissues, providing new insight into the early stage of colon carcinogenesis.
Assuntos
Transformação Celular Neoplásica/genética , Colo/patologia , Genes APC , Mutagênese Insercional , Mutação , Retroviridae/genética , Animais , Linhagem Celular , Colo/metabolismo , Dineínas/genética , Células Epiteliais/patologia , Proteínas de Membrana/genética , Camundongos , Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Regulação para CimaRESUMO
Cooperative activation of Meis1 and Hoxa9 perturbs myeloid differentiation and eventually leads myeloid progenitors to leukemia, yet it remains to be clarified what kinds of subsequent molecular processes are required for development of overt leukemia. To understand the molecular pathway in Hoxa9/Meis1-induced leukemogenesis, retroviral insertional mutagenesis was applied using retrovirus-mediated gene transfer. The mice that received Hoxa9/Meis1-transduced bone marrow cells developed acute myeloid leukemia (AML), and Trib1, Evi1, Ahi1, Raralpha, Pitpnb, and AK039950 were identified as candidate cooperative genes located near common retroviral integration sites. Trib1 and Evi1 were up-regulated due to retroviral insertions, and coexpression of these genes significantly accelerated the onset of Hoxa9/Meis1-induced AML, suggesting that Trib1 and Evi1 are the key collaborators. Furthermore, Trib1 by itself is a novel myeloid oncogene, enhancing phosphorylation of ERK, resulting in inhibition of apoptosis. These results demonstrate the importance of specific oncogene interaction in myeloid leukemogenesis.