RESUMEN
The chromatin-associated AAA+ ATPases Tip48 and Tip49 are the core components of various complexes implicated in diverse nuclear events such as DNA repair and gene regulation. Although they are frequently overexpressed in many human cancers, their functional significance remains unclear. Here, we show that loss of Tip49 triggered p53-dependent apoptosis and inhibited leukemia development in vivo. To examine the impact of chemical inhibition of this complex on leukemia, we have developed the novel compound DS-4950, which interferes with the ATPase activity of the Tip48/49. Administration of DS-4950 was well-tolerated in healthy mice, and the drug effectively reduced tumor burden and improved survival. We also provide evidence that the dependency on Tip48/49 is widely conserved in non-hematologic malignancies with wild type p53. These results demonstrated that the Tip48/49 ATPases are functionally necessary and therapeutically targetable for the treatment of human cancers.
Asunto(s)
ADN Helicasas , Leucemia Mieloide Aguda , Humanos , Ratones , Animales , ADN Helicasas/genética , Proteínas Portadoras/genética , Proteína p53 Supresora de Tumor/genética , Adenosina Trifosfatasas/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMEN
Disease-risk stratification and development of intensified chemotherapy protocols have substantially improved the outcome of acute lymphoblastic leukemia (ALL). However, outcomes of relapsed or refractory cases remain poor. Previous studies have discussed the oncogenic role of enhancer of zeste homolog 1 and 2 (EZH1/2), and the efficacy of dual inhibition of EZH1/2 as a treatment for hematological malignancy. Here, we investigated whether an EZH1/2 dual inhibitor, DS-3201 (valemetostat), has antitumor effects on B cell ALL (B-ALL). DS-3201 inhibited growth of B-ALL cell lines more significantly and strongly than the EZH2-specific inhibitor EPZ-6438, and induced cell cycle arrest and apoptosis in vitro. RNA-seq analysis to determine the effect of DS-3201 on cell cycle arrest-related genes expressed by B-ALL cell lines showed that DS-3201 upregulated CDKN1C and TP53INP1. CRIPSR/Cas9 knockout confirmed that CDKN1C and TP53INP1 are direct targets of EZH1/2 and are responsible for the antitumor effects of DS-3201 against B-ALL. Furthermore, a patient-derived xenograft (PDX) mouse model showed that DS-3201 inhibited the growth of B-ALL harboring MLL-AF4 significantly. Thus, DS-3201 provides another option for treatment of B-ALL.
Asunto(s)
Linfoma de Burkitt , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Ratones , Animales , Complejo Represivo Polycomb 2 , Regulación hacia Arriba , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacología , Puntos de Control del Ciclo Celular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Portadoras/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismoRESUMEN
Monocytic leukemia zinc finger protein (MOZ, MYST3, or KAT6A) is a MYST-type acetyltransferase involved in chromosomal translocation in acute myelogenous leukemia (AML) and myelodysplastic syndrome. MOZ is established as essential for hematopoiesis; however, the role of MOZ in AML has not been addressed. We propose that MOZ is critical for AML development induced by MLL-AF9, MLL-AF10, or MOZ-TIF2 fusions. Moz-deficient hematopoietic stem/progenitor cells (HSPCs) transduced with an MLL-AF10 fusion gene neither formed colonies in methylcellulose nor induced AML in mice. Moz-deficient HSPCs bearing MLL-AF9 also generated significantly reduced colony and cell numbers. Moz-deficient HSPCs expressing MOZ-TIF2 could form colonies in vitro but could not induce AML in mice. By contrast, Moz was dispensable for colony formation by HOXA9-transduced cells and AML development caused by HOXA9 and MEIS1, suggesting a specific requirement for MOZ in AML induced by MOZ/MLL fusions. Expression of the Hoxa9 and Meis1 genes was decreased in Moz-deficient MLL fusion-expressing cells, while expression of Meis1, but not Hoxa9, was reduced in Moz-deficient MOZ-TIF2 AML cells. AML development induced by MOZ-TIF2 was rescued by introducing Meis1 into Moz-deficient cells carrying MOZ-TIF2. Meis1 deletion impaired MOZ-TIF2-mediated AML development. Active histone modifications were also severely reduced at the Meis1 locus in Moz-deficient MOZ-TIF2 and MLL-AF9 AML cells. These results suggest that endogenous MOZ is critical for MOZ/MLL fusion-induced AML development and maintains active chromatin signatures at target gene loci.
Asunto(s)
Leucemia Mieloide Aguda , Animales , Cromatina , Hematopoyesis , Histona Acetiltransferasas/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Metilcelulosa , RatonesRESUMEN
Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR-S1 (a close analog of the clinical-stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model, OR-S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2-specific inhibitor GSK126, OR-S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR-S1-sensitive or insensitive MCL cell lines and showed that OR-S1 treatment modulated B-cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib-resistant MCL via CDKN1C-mediated cell cycle arrest.
Asunto(s)
Adenina/análogos & derivados , Antineoplásicos/farmacología , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Linfoma de Células del Manto/tratamiento farmacológico , Piperidinas/farmacología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Adenina/farmacología , Adenina/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Ratones , Piperidinas/uso terapéutico , Sindecano-1/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of α-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood-brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Glioblastoma/tratamiento farmacológico , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isoxazoles/farmacología , Administración Oral , Animales , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Glioblastoma/enzimología , Glioblastoma/patología , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Isoxazoles/química , Isoxazoles/farmacocinética , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chondrosarcoma is the second most common malignant bone tumor. It is characterized by low vascularity and an abundant extracellular matrix, which confer these tumors resistance to chemotherapy and radiotherapy. There are currently no effective treatment options for relapsed or dedifferentiated chondrosarcoma, and new targeted therapies need to be identified. Isocitrate dehydrogenase (IDH) mutations, which are detected in ~50% of chondrosarcoma patients, contribute to malignant transformation by catalyzing the production of 2-hydroxyglutarate (2-HG), a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Mutant IDH inhibitors are therefore potential novel anticancer drugs in IDH mutant tumors. Here, we examined the efficacy of the inhibition of mutant IDH1 as an antitumor approach in chondrosarcoma cells in vitro and in vivo, and investigated the association between the IDH mutation and chondrosarcoma cells. DS-1001b, a novel, orally bioavailable, selective mutant IDH1 inhibitor, impaired the proliferation of chondrosarcoma cells with IDH1 mutations in vitro and in vivo, and decreased 2-HG levels. RNA-seq analysis showed that inhibition of mutant IDH1 promoted chondrocyte differentiation in the conventional chondrosarcoma L835 cell line and caused cell cycle arrest in the dedifferentiated JJ012 cell line. Mutant IDH1-mediated modulation of SOX9 and CDKN1C expression regulated chondrosarcoma tumor progression, and DS-1001b upregulated the expression of these genes via a common mechanism involving the demethylation of H3K9me3. DS-1001b treatment reversed the epigenetic changes caused by aberrant histone modifications. The present data strongly suggest that inhibition of mutant IDH1 is a promising therapeutic approach in chondrosarcoma, particularly for the treatment of relapsed or dedifferentiated chondrosarcoma.
Asunto(s)
Neoplasias Óseas/patología , Condrosarcoma/patología , Inhibidores Enzimáticos/farmacología , Código de Histonas , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Mutación , Neoplasias Óseas/metabolismo , Puntos de Control del Ciclo Celular , Diferenciación Celular , Proliferación Celular , Condrosarcoma/metabolismo , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug-resistant myeloma stem cells. Side population (SP) cells show cancer stem cell-like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non-SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR-S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient-derived xenograft model. Moreover, long-term continuous dosing of OR-S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR-S1, leading to significant advances in the treatment of MM.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mieloma Múltiple/prevención & control , Células Madre Neoplásicas/efectos de los fármacos , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Vía de Señalización Wnt/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Células de Población Lateral/efectos de los fármacos , Células de Población Lateral/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Eradication of chemotherapy-resistant leukemia stem cells is expected to improve treatment outcomes in patients with acute myelogenous leukemia (AML). In a mouse model of AML expressing the MOZ-TIF2 fusion, we found that Ring1A and Ring1B, components of Polycomb repressive complex 1, play crucial roles in maintaining AML stem cells. Deletion of Ring1A and Ring1B (Ring1A/B) from MOZ-TIF2 AML cells diminished self-renewal capacity and induced the expression of numerous genes, including Glis2 Overexpression of Glis2 caused MOZ-TIF2 AML cells to differentiate into mature cells, whereas Glis2 knockdown in Ring1A/B-deficient MOZ-TIF2 cells inhibited differentiation. Thus, Ring1A/B regulate and maintain AML stem cells in part by repressing Glis2 expression, which promotes their differentiation. These findings provide new insights into the mechanism of AML stem cell homeostasis and reveal novel targets for cancer stem cell therapy.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Histona Acetiltransferasas/biosíntesis , Factores de Transcripción de Tipo Kruppel/biosíntesis , Leucemia Mieloide Aguda/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Coactivador 2 del Receptor Nuclear/biosíntesis , Proteínas de Fusión Oncogénica/biosíntesis , Complejo Represivo Polycomb 1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Histona Acetiltransferasas/genética , Factores de Transcripción de Tipo Kruppel/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Coactivador 2 del Receptor Nuclear/genética , Proteínas de Fusión Oncogénica/genética , Complejo Represivo Polycomb 1/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Animales , Hipoxia de la Célula , Regulación Leucémica de la Expresión Génica , Humanos , Isocitrato Deshidrogenasa/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ratones Endogámicos C57BL , Mutación Missense , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , Nucleofosmina , Regulación hacia ArribaRESUMEN
Acute myeloid leukemia is a clonal malignant disorder derived from a small number of leukemic stem cells (LSCs). Rearrangements of the mixed lineage leukemia (MLL) gene are found in acute myeloid leukemia associated with poor prognosis. The upregulation of Hox genes is critical for LSC induction and maintenance, but is unlikely to support malignancy and the high LSC frequency observed in MLL leukemias. The present study shows that MLL fusion proteins interact with the transcription factor PU.1 to activate the transcription of CSF-1R, which is critical for LSC activity. Acute myeloid leukemia is cured by either deletion of PU.1 or ablation of cells expressing CSF-1R. Kinase inhibitors specific for CSF-1R prolong survival time. These findings indicate that PU.1-mediated upregulation of CSF-1R is a critical effector of MLL leukemogenesis.
Asunto(s)
Carcinogénesis/genética , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Proto-Oncogénicas/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Transactivadores/genética , Animales , Regulación Leucémica de la Expresión Génica , Genes Homeobox , Leucemia Mieloide Aguda/mortalidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre Neoplásicas , Compuestos de Fenilurea/farmacología , Pronóstico , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transducción de Señal , Tacrolimus/análogos & derivados , Tacrolimus/farmacología , Tiazoles/farmacología , Transcripción Genética , Activación Transcripcional , Regulación hacia ArribaRESUMEN
The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.
Asunto(s)
Eritropoyesis , Histona Acetiltransferasas/fisiología , Hígado/embriología , Procesamiento Proteico-Postraduccional , Transactivadores/fisiología , Acetilación , Anemia/embriología , Anemia/genética , Animales , Proteínas Portadoras/fisiología , Daño del ADN , Replicación del ADN , Muerte Fetal/sangre , Muerte Fetal/etiología , Muerte Fetal/genética , Factor de Transcripción GATA1/metabolismo , Genes Letales , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Células K562 , Hígado/fisiología , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos , Neoplasias/genética , Neoplasias/metabolismo , Mapeo de Interacción de Proteínas , ARN Interferente Pequeño/farmacología , Transactivadores/deficiencia , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44+CD24- population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10(4)sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.
Asunto(s)
Antígenos CD/metabolismo , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Doxorrubicina/farmacología , Células Madre Neoplásicas/patología , Esferoides Celulares/patología , Antígeno AC133 , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Biomarcadores de Tumor/metabolismo , Antígeno CD24/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Perros , Femenino , Genes MDR , Glicoproteínas/metabolismo , Receptores de Hialuranos/metabolismo , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/trasplante , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores Notch/metabolismo , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/trasplante , Regulación hacia ArribaRESUMEN
Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony-stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1R(high) cells), but not those expressing low amounts of CSF1R (CSF1R(low) cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the CSF1R promoter, we cured AML by ablation of CSF1R(high) cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2-induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1R(high) cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach.
Asunto(s)
Leucemia Mieloide Aguda/genética , Coactivador 2 del Receptor Nuclear/metabolismo , Proteínas Proto-Oncogénicas/genética , Receptores del Factor Estimulante de Colonias/genética , Transactivadores/genética , Regulación hacia Arriba , Animales , Diferenciación Celular/genética , Factores Estimulantes de Colonias/genética , Genes , Humanos , Leucemia/genética , Factor Estimulante de Colonias de Macrófagos/genética , Ratones , Ratones Transgénicos , Coactivador 2 del Receptor Nuclear/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Proteínas Recombinantes de Fusión/metabolismo , Dedos de Zinc/genéticaRESUMEN
The epigenetic mechanism involving chromatin modification plays a critical role in the maintenance of the expression of Hox genes. Here, we characterize a mutant of the medaka fish, named biaxial symmetries (bis), in which brpf1, a subunit of the MOZ histone acetyl transferase (HAT) complex, is mutated. The bis mutant displayed patterning defects both in the anterior-posterior axis of the craniofacial skeleton and the dorsal-ventral axis of the caudal one. In the anterior region, the bis mutant exhibited craniofacial cartilage homeosis. The expression of Hox genes was decreased in the pharyngeal arches, suggesting that the pharyngeal segmental identities were altered in the bis mutant. In the posterior region, the bis mutant exhibited abnormal patterning of the caudal skeleton, which ectopically formed at the dorsal side of the caudal fin. The expression of Zic genes was decreased at the posterior region, suggesting that the dorsal-ventral axis formation of the posterior trunk was disrupted in the bis mutant. We also found that the MOZ-deficient mice exhibited an abnormal patterning of their craniofacial and cervical skeletons and a decrease of Hox transcripts. We propose a common role of the MOZ HAT complex in vertebrates, a complex which is required for the proper patterning for skeletal development.
Asunto(s)
Tipificación del Cuerpo , Cara/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Homeobox , Histona Acetiltransferasas/fisiología , Cráneo/embriología , Cola (estructura animal)/embriología , Animales , Secuencia de Bases , Cartilla de ADN , Histona Acetiltransferasas/genética , Ratones , Mutación , OryziasRESUMEN
Histone-modified enzymes are involved in various cell functions, including proliferation, differentiation, cell death and carcinogenesis. The protein MOZ (monocytic leukemia zinc finger protein) is a Myst (MOZ, Ybf2 (Sas3), Sas2, Tip60)-type histone acetyltranseferase (HAT) that generates fusion genes, such as MOZ-TIF2, MOZ-CBP and MOZ-p300, in acute myeloid leukemia (AML) by chromosomal translocation. MOZ associates with AML1 (RUNX1), PU.1, and p53, and cooperatively activates target gene transcription. Gene targeting in mice has revealed that MOZ is essential for the generation and maintenance of hematopoietic stem cells (HSC) and for the appropriate development of myeloid, erythroid and B-lineage cell progenitors. In AML, MOZ fusion genes lead to repressed differentiation, hyper-proliferation, and self-renewal of myeloid progenitors through deregulation of MOZ-regulated target gene expression. It is therefore necessary to analyze the roles of MOZ and MOZ fusion genes in normal and malignant hematopoiesis to elucidate the mechanisms underlying and develop therapies for MOZ-related AML.
Asunto(s)
Fusión Génica , Hematopoyesis/genética , Histona Acetiltransferasas/metabolismo , Leucemia Mieloide/metabolismo , Animales , Muerte Celular/genética , Diferenciación Celular/genética , Proliferación Celular , Expresión Génica , Humanos , Leucemia Mieloide/genética , RatonesRESUMEN
Runx1, one of three mammalian runt-domain transcription factor family proteins, is essential for definitive hematopoiesis. Based on transfection assays, phosphorylation of Runx1 at the three serine residues, Ser249, Ser266, and Ser276, was thought to be important for trans-activation activity of Runx1. By using "knock-in" gene targeting, we generated mouse strains expressing mutant Runx1 protein that harbored a combined serine-to-alanine substitution at either of two residues, Ser249/Ser266 or Ser249/Ser276. Either mutation resulted in a lack of major phosphorylated form of Runx1. However, while loss of definitive hematopoiesis and impaired thymocyte differentiation was observed following the loss of Runx1, these phenotypes were rescued in those mice lacking the major phosphorylated form of Runx1. These results not only challenge the predicted regulation of Runx1 activity by phosphorylation at these serine residues, but also reaffirm the effectiveness of "knock-in" mutagenesis as a powerful tool for addressing the physiological relevance of post-translation modifications.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Hematopoyesis/fisiología , Linfocitos/citología , Linfocitos/metabolismo , Serina/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Ratones , Ratones Noqueados , Fosforilación , Serina/química , Timo/citología , Timo/metabolismoRESUMEN
PML and PU.1 play important roles in myeloid differentiation. PML-deficient mice have an impaired capacity for terminal maturation of their myeloid precursor cells. This finding has been explained, at least in part, by the lack of PML action to modulate retinoic acid-differentiating activities. In this study, we found that C/EBPepsilon expression is reduced in PML-deficient mice. We showed that PU.1 directly activates the transcription of the C/EBPepsilon gene that is essential for granulocytic differentiation. The type IV isoform of PML interacted with PU.1, promoted its association with p300, and then enhanced PU.1-induced transcription and granulocytic differentiation. In contrast to PML IV, the leukemia-associated PML-retinoic acid receptor alpha fusion protein dissociated the PU.1/PML IV/p300 complex and inhibited PU.1-induced transcription. These results suggest a novel pathogenic mechanism of the PML-retinoic acid receptor alpha fusion protein in acute promyelocytic leukemia.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Células Mieloides/citología , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Secuencia de Bases , Proteína p300 Asociada a E1A/metabolismo , Células Precursoras de Granulocitos/citología , Células Precursoras de Granulocitos/metabolismo , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Células Mieloides/metabolismo , Mielopoyesis , Células 3T3 NIH , Proteína de la Leucemia Promielocítica , Unión Proteica , Isoformas de Proteínas/metabolismo , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
Monocytic leukemia zinc-finger protein (MOZ), a MYST family histone acetyltransferase, is involved in the chromosome translocations associated with acute myeloid leukemia. MOZ acts as a transcriptional coactivator for AML1, which is essential for establishment of definitive hematopoiesis. To investigate the roles of MOZ in normal hematopoiesis, we generated MOZ-null mice. MOZ-/- mice died around embryonic day 15 (E15). In MOZ-/- E14.5 embryos, hematopoietic stem cells, lineage-committed progenitors, and B lineage cells were severely reduced. On the other hand, arrest of erythroid maturation and elevated myeloid lineage populations were observed. MOZ-deficient fetal liver cells could not reconstitute hematopoiesis of recipients after transplantation. Analysis using microarray and flow cytometry revealed that expression of thrombopoietin receptor (c-Mpl), HoxA9, and c-Kit was down-regulated. These results show that MOZ is required for maintenance of hematopoietic stem cells, and that it plays a role in differentiation of erythroid and myeloid cells. Some aspects of the MOZ-/- phenotype are similar to that observed in PU.1-deficient mice. MOZ was able to interact with PU.1 and activate PU.1-dependent transcription, thus suggesting a physical and functional link between PU.1 and MOZ.
Asunto(s)
Linaje de la Célula/genética , Eritropoyesis/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/fisiología , Histona Acetiltransferasas/fisiología , Mielopoyesis/genética , Animales , Linfocitos B/citología , Diferenciación Celular/genética , Regulación hacia Abajo , Desarrollo Embrionario/genética , Genes Letales , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/genética , Leucemia/genética , Hígado/citología , Hígado/crecimiento & desarrollo , Ratones , Ratones Mutantes , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Receptores de Citocinas/genética , Receptores de Trombopoyetina , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
NK cells differentiate into either NK1 or NK2 cells that produce IFN-gamma or IL-5 and IL-13, respectively. Little is known, however, about the molecular mechanisms that control NK1 and NK2 cell differentiation. To address these questions, we established an in vitro mouse NK1/NK2 cell differentiation culture system. For NK1/NK2 cell differentiation, initial stimulation with PMA and ionomycin was required. The in vitro differentiated NK2 cells produced IL-5 and IL-13, but the levels were 20 times lower than those of Th2 or T cytotoxic (Tc)2 cells. No detectable IL-4 was produced. Freshly prepared NK cells express IL-2Rbeta, IL-2RgammaC, and IL-4Ralpha. After stimulation with PMA and ionomycin, NK cells expressed IL-2Ralpha. NK1 cells displayed higher cytotoxic activity against Yac-1 target cells. The levels of GATA3 protein in developing NK2 cells were approximately one-sixth of those in Th2 cells. Both NK1 and NK2 cells expressed large amounts of repressor of GATA, the levels of which were equivalent to CD8 Tc1 and Tc2 cells and significantly higher than those in Th2 cells. The levels of histone hyperacetylation of the IL-4 and IL-13 gene loci in NK2 cells were very low and equivalent to those in naive CD4 T cells. The production of IL-5 and IL-13 in NK2 cells was found to be STAT6 dependent. Thus, similar to Th2 cells, NK2 cell development is dependent on STAT6, and the low level expression of GATA3 and the high level expression of repressor of GATA may influence the unique type 2 cytokine production profiles of NK2 cells.