RESUMEN
The Myb transcription factor is involved in the proliferation of hematopoietic cells, and deregulation of its expression can lead to cancers such as leukemia. Myb interacts with various proteins, including the histone acetyltransferases p300 and CBP. Myb binds to a small domain of p300, the KIX domain (p300KIX), and inhibiting this interaction is a potential new drug discovery strategy in oncology. The available structures show that Myb binds to a very shallow pocket of the KIX domain, indicating that it might be challenging to identify inhibitors of this interaction. Here, we report the design of Myb-derived peptides which interact with p300KIX. We show that by mutating only two Myb residues that bind in or near a hotspot at the surface of p300KIX, it is possible to obtain single-digit nanomolar peptidic inhibitors of the Myb/p300KIX interaction that bind 400-fold tighter to p300KIX than wildtype Myb. These findings suggest that it might also be possible to design potent low molecular-weight compounds to disrupt the Myb/p300KIX interaction.
Asunto(s)
Proteína p300 Asociada a E1A , Péptidos , Proteínas Proto-Oncogénicas c-myb , Péptidos/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/química , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Proteína p300 Asociada a E1A/químicaRESUMEN
BACKGROUND: Tubulin is the biochemical target for several clinically used anticancer drugs as it helps in the formation of mitotic spindle during mitosis stage of cell division. Many of the anti-cancer drugs are known to interact with tubulin and microtubules including some plant alkaloids, such as paclitaxel, etoposide and topotecan. In silico drug design of these molecules were performed prior to testing these drugs in vitro. In silico drug design of these anti-cancer drugs becomes a challenge due to the complex structure of target protein. This challenge was overcome by predicting the structure of the target protein (tubulin) by homology modeling. METHODS: In this study, computer aided drug designing approach was applied to predict the suitable docking site in target protein and the interaction of tubulin protein with paclitaxel, etoposide and topotecan was explored by molecular docking using Schrödinger software. Docking score and glide energy were determined with ligands to validate their anticancer properties. RESULTS: The results indicate that etoposide is the best drug for tubulin with a docking score of - 4.916 and glide energy of -46.470 kcal/mol compared to paclitaxel and topotecan. CONCLUSION: The testing of these drugs in silico provides an alternate to in vitro testing of these molecules on cancer cell lines which is a time and cost intensive process. The in silico study of parameters, such as docking score and glide energy, will help pharmacists in developing new molecules as targets for cancers in a time and cost-effective manner.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Diseño de Fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/uso terapéutico , Etopósido/química , Etopósido/farmacología , Etopósido/uso terapéutico , Humanos , Lactonas/química , Lactonas/farmacología , Lactonas/uso terapéutico , Ligandos , Simulación del Acoplamiento Molecular , Neoplasias/genética , Paclitaxel/química , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas c-myb/ultraestructura , Sesquiterpenos/química , Sesquiterpenos/farmacología , Sesquiterpenos/uso terapéutico , Relación Estructura-Actividad , Topotecan/química , Topotecan/farmacología , Topotecan/uso terapéuticoRESUMEN
Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target MYB-driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of c-myb expression. Retinoic acid treatment also decreased c-myb gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor MYB are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient-derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.
Asunto(s)
Blastómeros/inmunología , Carcinoma Adenoide Quístico/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Tretinoina/farmacología , Proteínas de Pez Cebra/antagonistas & inhibidores , Pez Cebra/inmunología , Animales , Blastómeros/patología , Carcinoma Adenoide Quístico/genética , Carcinoma Adenoide Quístico/inmunología , Carcinoma Adenoide Quístico/patología , Humanos , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/inmunología , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/inmunología , Neoplasias de las Glándulas Salivales/patología , Células U937 , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/inmunologíaRESUMEN
The transcription factor MYB plays key roles in hematopoietic cells and has been implicated the development of leukemia. MYB has therefore emerged as an attractive target for drug development. Recent work has suggested that targeting MYB by small-molecule inhibitors is feasible and that inhibition of MYB has potential as a therapeutic approach against acute myeloid leukemia. To facilitate the identification of small-molecule MYB inhibitors we have re-designed and improved a previously established cell-based screening assay and have employed it to screen a natural product library for potential inhibitors. Our work shows that teniposide and etoposide, chemotherapeutic agents causing DNA-damage by inhibiting topoisomerase II, potently inhibit MYB activity and induce degradation of MYB in AML cell lines. MYB inhibition is suppressed by caffeine, suggesting that MYB is inhibited indirectly via DNA-damage signalling. Importantly, ectopic expression of an activated version of MYB in pro-myelocytic NB4 cells diminished the anti-proliferative effects of teniposide, suggesting that podophyllotoxins disrupt the proliferation of leukemia cells not simply by inducing general DNA-damage but that their anti-proliferative effects are boosted by inhibition of MYB. Teniposide and etoposide therefore act like double-edged swords that might be particularly effective to inhibit tumor cells with deregulated MYB.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Etopósido/farmacología , Regulación Leucémica de la Expresión Génica , Podofilotoxina/farmacología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Tenipósido/farmacología , Línea Celular Tumoral , Daño del ADN , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/patología , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/farmacologíaAsunto(s)
MicroARNs/genética , Neovascularización Patológica/genética , Proteínas Proto-Oncogénicas c-myb/genética , Neoplasias Gástricas/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Células Endoteliales/patología , Exosomas/genética , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Neovascularización Patológica/patología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Transducción de Señal/genética , Neoplasias Gástricas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The regulation of natural killer (NK) cell activity is an important research goal for the development of immunotherapies. In this study, we identified transcription factors affecting NK cell activity. In particular, we screened transcription factors affected by interleukin-2 (IL-2) and transforming growth factor-beta (TGF-ß) by protein/DNA arrays using primary NK cells. We found that celastrol, a c-Myb inhibitor, inhibited NK-92â¯cells more strongly than any other inhibitors of transcription factor candidates. In addition, c-Myb and c-Myb-related signaling molecules, e.g., Nemo-like kinase (NLK) and c-Myc, were regulated by the activation status of NK cells, suggesting that c-Myb is a key regulator of NK cell activity. We also found that celastrol inhibits NK-92-cell-mediated cytotoxicity via the downregulation of NKG2D and granzyme B. Knockdown studies also showed that c-Myb is important for NK cell activation. In particular, the knockdown of c-Myb did not significantly affect NK cell proliferation and survival but decreased the secretion of IFN-γ and the cytotoxicity of NK cells. Our data demonstrate that c-Myb plays a critical role in the activation of NK cells and therefore is a therapeutic target for cancer and viral diseases.
Asunto(s)
Citotoxicidad Inmunológica/efectos de los fármacos , Factores Inmunológicos/farmacología , Células Asesinas Naturales/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myb/genética , Triterpenos/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica , Granzimas/genética , Granzimas/inmunología , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-2/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Triterpenos Pentacíclicos , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/inmunología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacologíaAsunto(s)
Antiinflamatorios no Esteroideos/farmacología , Diseño Asistido por Computadora , Descubrimiento de Drogas , Lactonas/farmacología , Vacunas/metabolismo , Algoritmos , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Química Farmacéutica , Humanos , Lactonas/síntesis química , Lactonas/química , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Relación Estructura-Actividad Cuantitativa , Vacunas/síntesis química , Vacunas/químicaRESUMEN
Myb is a key regulator of hematopoietic progenitor cell proliferation and differentiation and has emerged as a potential target for the treatment of acute leukemia. Using a myeloid cell line with a stably integrated Myb-inducible reporter gene as a screening tool we have previously identified Celastrol, a natural compound with anti-tumor activity, as a potent Myb inhibitor that disrupts the interaction of Myb with the co-activator p300. We showed that Celastrol inhibits the proliferation of acute myeloid leukemia (AML) cells and prolongs the survival of mice in an in vivo model of AML, demonstrating that targeting Myb with a small-molecule inhibitor is feasible and might have potential as a therapeutic approach against AML. Recently we became aware that the reporter system used for Myb inhibitor screening also responds to inhibition of C/EBPß, a transcription factor known to cooperate with Myb in myeloid cells. By re-investigating the inhibitory potential of Celastrol we have found that Celastrol also strongly inhibits the activity of C/EBPß by disrupting its interaction with the Taz2 domain of p300. Together with previous studies our work reveals that Celastrol independently targets Myb and C/EBPß by disrupting the interaction of both transcription factors with p300. Myb, C/EBPß and p300 cooperate in myeloid-specific gene expression and, as shown recently, are associated with so-called super-enhancers in AML cells that have been implicated in the maintenance of the leukemia. We hypothesize that the ability of Celastrol to disrupt the activity of a transcriptional Myb-C/EBPß-p300 module might explain its promising anti-leukemic activity.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Triterpenos/farmacología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Células 3T3-L1 , Secuencia de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Pollos , Cisteína/química , Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Triterpenos Pentacíclicos , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myb/química , Codorniz , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/genéticaRESUMEN
BACKGROUND: Protein c-Myb is a therapeutic target. Some sesquiterpene lactones suppress Myb-dependent gene expression, which results in their potential anti-cancer activity. MATERIAL & METHODS: Database ChEMBL is a representative of lactones for physicochemical and physiochemical properties. Data presented for 31 natural lactones are discussed in terms of quantitative structureactivity relationships with the objective to predict inhibitors of Myb-induced gene expression. Several constitutional descriptors are related to structure-activity. α-Methylene-γ-lactone groups enhance while OH functions worsen potency. The latter feature is in agreement with the fact that the more lipophilic the lactone, the greater the cytotoxicity because of the ability to cross lipoidal biomembranes. In general, numbers of π-systems and atoms, and polarizability enhance activity. Linear and nonlinear structure-activity models are developed, between lactones of a great structural diversity, to predict inhibitors of Myb-induced gene expression. Four variables (ML, UNC, TCO+OCOR, UNC+UNA) related to ATOM show a positive correlation because of the partial anionic and H-acceptor characters of O-atom. In most, CO group is conjugated. RESULT AND CONCLUSION: Term OH shows negative coefficients because of the partial cationic quality of H-atom and because OH forms H-bonds with CO, causing them to be less H-acceptor. s-trans-s-trans-Germacranolide structure is the most active. Coefficients standard errors result acceptable in almost all equations. After cross-validation, linear equations for lactones, pseudoguaianolides and germacranolides are the most predictive. Most descriptors are constitutional variables.
Asunto(s)
Productos Biológicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Lactonas/farmacología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Relación Estructura-Actividad Cuantitativa , Sesquiterpenos/farmacología , Productos Biológicos/química , Relación Dosis-Respuesta a Droga , Humanos , Lactonas/química , Modelos Moleculares , Estructura Molecular , Proteínas Proto-Oncogénicas c-myb/genética , Sesquiterpenos/químicaRESUMEN
Background: Adenoid cystic carcinoma (ACC) is an aggressive cancer with no curative treatment for patients with recurrent/metastatic disease. The MYB-NFIB gene fusion is the main genomic hallmark and a potential therapeutic target. Methods: Oncogenic signaling pathways were studied in cultured cells and/or tumors from 15 ACC patients. Phospho-receptor tyrosine kinase (RTK) arrays were used to study the activity of RTKs. Effects of RTK inhibition on cell proliferation were analyzed with AlamarBlue, sphere assays, and two ACC xenograft models (n = 4-9 mice per group). The molecular effects of MYB-NFIB knockdown and IGF1R inhibition were studied with quantitative polymerase chain reaction, immunoblot, and gene expression microarrays. All statistical tests were two-sided. Results: The MYB-NFIB fusion drives proliferation of ACC cells and is crucial for spherogenesis. Intriguingly, the fusion is regulated through AKT-dependent signaling induced by IGF1R overexpression and is downregulated upon IGF1R-inhibition (% expression of control ± SD = 27.2 ± 1.3, P < .001). MYB-NFIB regulates genes involved in cell cycle control, DNA replication/repair, and RNA processing. The transcriptional program induced by MYB-NFIB affects critical oncogenic mediators normally controlled by MYC and is reversed by pharmacological inhibition of IGF1R. Co-activation of epidermal growth factor receptor (EGFR) and MET promoted proliferation of ACC cells, and combined targeting of IGFR1/EGFR/MET induced differentiation and synergistically inhibited the growth of patient-derived xenografted ACCs (ACCX5M1, % growth of control ± SD = 34.9 ± 20.3, P = .006; ACCX6, % growth of control ± SD = 24.1 ± 17.5, P = .04). Conclusions: MYB-NFIB is an oncogenic driver and a key therapeutic target in ACC that is regulated by AKT-dependent IGF1R signaling. Our studies uncover a new strategy to target an oncogenic transcriptional master regulator and provide new important insights into the biology and treatment of ACC.
Asunto(s)
Carcinoma Adenoide Quístico/genética , Carcinoma Adenoide Quístico/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores de Somatomedina/metabolismo , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Biomarcadores de Tumor , Carcinoma Adenoide Quístico/tratamiento farmacológico , Carcinoma Adenoide Quístico/patología , Ciclo Celular , Proliferación Celular/genética , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Receptor IGF Tipo 1 , Transducción de Señal/efectos de los fármacos , Transcripción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic driver mutations are those that provide a proliferative or survival advantage to neoplastic cells, resulting in clonal selection. Although most cancer-causing mutations have been detected in the protein-coding regions of the cancer genome; driver mutations have recently also been discovered within noncoding genomic sequences. Thus, a current challenge is to gain precise understanding of how these unique genomic elements function in cancer pathogenesis, while clarifying mechanisms of gene regulation and identifying new targets for therapeutic intervention. Here we report a C-to-T single nucleotide transition that occurs as a somatic mutation in noncoding sequences 4 kb upstream of the transcriptional start site of the LMO1 oncogene in primary samples from patients with T-cell acute lymphoblastic leukaemia. This single nucleotide alteration conforms to an APOBEC-like cytidine deaminase mutational signature, and generates a new binding site for the MYB transcription factor, leading to the formation of an aberrant transcriptional enhancer complex that drives high levels of expression of the LMO1 oncogene. Since APOBEC-signature mutations are common in a broad spectrum of human cancers, we suggest that noncoding nucleotide transitions such as the one described here may activate potent oncogenic enhancers not only in T-lymphoid cells but in other cell lineages as well.
Asunto(s)
Desaminasas APOBEC/metabolismo , Proteínas de Unión al ADN/biosíntesis , Elementos de Facilitación Genéticos/genética , Regulación Leucémica de la Expresión Génica/genética , Proteínas con Dominio LIM/biosíntesis , Proteínas de Neoplasias/biosíntesis , Mutación Puntual , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Factores de Transcripción/biosíntesis , Transcriptoma , Regiones no Traducidas 5'/genética , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Niño , Inmunoprecipitación de Cromatina , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Genes myb , Humanos , Células Jurkat , Proteínas con Dominio LIM/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
c-Myb is a transcription factor that plays a key role in cell proliferation, differentiation, and apoptosis. It has been reported that c-Myb is expressed within the chicken otic placode, but whether c-Myb exists in the mammalian cochlea, and how it exerts its effects, has not been explored yet. Here, we investigated the expression of c-Myb in the postnatal mouse cochlea and HEI-OC1 cells and found that c-Myb was expressed in the hair cells (HCs) of mouse cochlea as well as in cultured HEI-OC1 cells. Next, we demonstrated that c-Myb expression was decreased in response to neomycin treatment in both cochlear HCs and HEI-OC1 cells, suggesting an otoprotective role for c-Myb. We then knocked down c-Myb expression with shRNA transfection in HEI-OC1 cells and found that c-Myb knockdown decreased cell viability, increased expression of pro-apoptotic factors, and enhanced cell apoptosis after neomycin insult. Mechanistic studies revealed that c-Myb knockdown increased cellular levels of reactive oxygen species and decreased Bcl-2 expression, both of which are likely to be responsible for the increased sensitivity of c-Myb knockdown cells to neomycin. This study provides evidence that c-Myb might serve as a new target for the prevention of aminoglycoside-induced HC loss.
Asunto(s)
Células Ciliadas Auditivas/patología , Mitocondrias/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-myb/genética , Aminoglicósidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Ciliadas Auditivas/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Neomicina/farmacología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The transcription factor Myb is a key regulator of hematopoietic cell proliferation, differentiation, and survival and has been implicated in the development of leukemia and several other human cancers. Pharmacological inhibition of Myb is therefore emerging as a potential therapeutic strategy. Recently, the first low-molecular-weight compounds that show Myb inhibitory activity have been identified. Characterization of these compounds suggests disruption of the protein-protein-interaction of Myb and the coactivator p300 as a suitable strategy to inhibit Myb.
Asunto(s)
Antineoplásicos/farmacología , Terapia Molecular Dirigida , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia/tratamiento farmacológico , Leucemia/etiología , Leucemia/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myb/química , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
SETBP1 missense mutations have been frequently identified in multiple myeloid neoplasms; however, their oncogenic potential remains unclear. Here we show that expression of Setbp1 mutants carrying two such mutations in mouse bone marrow progenitors efficiently induced development of acute myeloid leukemias (AMLs) in irradiated recipient mice with significantly shorter latencies and greater penetrance than expression of wild-type Setbp1, suggesting that these mutations are highly oncogenic. The increased oncogenicity of Setbp1 missense mutants could be due in part to their capability to drive significantly higher target gene transcription. We further identify Myb as a critical mediator of Setbp1-induced self-renewal as its knockdown caused efficient differentiation of myeloid progenitors immortalized by wild-type Setbp1 and Setbp1 missense mutants. Interestingly, Myb is also a direct transcriptional target of Setbp1 and Setbp1 missense mutants as they directly bind to the Myb locus in immortalized cells and dramatically activate a critical enhancer/promoter region of Myb in luciferase reporter assays. Furthermore, Myb knockdown in Setbp1 and Setbp1 missense mutations-induced AML cells also efficiently induced their differentiation in culture and significantly prolonged the survival of their secondary recipient mice, suggesting that targeting MYB pathway could be a promising strategy for treating human myeloid neoplasms with SETBP1 activation.
Asunto(s)
Proteínas Portadoras/fisiología , Leucemia Mieloide Aguda/etiología , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas c-myb/fisiología , Animales , Proteínas Portadoras/genética , Femenino , Proteínas Homeobox A10 , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidoresRESUMEN
The transcription factor c-Myb is essential for the proliferation of hematopoietic cells and has been implicated in the development of leukemia and other human cancers. Pharmacologic inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. By using a Myb reporter cell line, we have identified plumbagin and several naphthoquinones as potent low-molecular weight Myb inhibitors. We demonstrate that these compounds inhibit c-Myb by binding to the c-Myb transactivation domain and disrupting the cooperation of c-Myb with the coactivator p300, a major driver of Myb activity. Naphthoquinone-induced inhibition of c-Myb suppresses Myb target gene expression and induces the differentiation of the myeloid leukemia cell line HL60. We demonstrate that murine and human primary acute myeloid leukemia cells are more sensitive to naphthoquinone-induced inhibition of clonogenic proliferation than normal hematopoietic progenitor cells. Overall, our work demonstrates for the first time the potential of naphthoquinones as small-molecule Myb inhibitors that may have therapeutic potential for the treatment of leukemia and other tumors driven by deregulated Myb. Mol Cancer Ther; 15(12); 2905-15. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Proteína p300 Asociada a E1A/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Antineoplásicos/química , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Naftoquinonas/química , Naftoquinonas/farmacología , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We have recently demonstrated that the transcription factor MYB can modulate several cancer-associated phenotypes in pancreatic cancer. In order to understand the molecular basis of these MYB-associated changes, we conducted deep-sequencing of transcriptome of MYB-overexpressing and -silenced pancreatic cancer cells, followed by in silico pathway analysis. We identified significant modulation of 774 genes upon MYB-silencing (p < 0.05) that were assigned to 25 gene networks by in silico analysis. Further analyses placed genes in our RNA sequencing-generated dataset to several canonical signalling pathways, such as cell-cycle control, DNA-damage and -repair responses, p53 and HIF1α. Importantly, we observed downregulation of the pancreatic adenocarcinoma signaling pathway in MYB-silenced pancreatic cancer cells exhibiting suppression of EGFR and NF-κB. Decreased expression of EGFR and RELA was validated by both qPCR and immunoblotting and they were both shown to be under direct transcriptional control of MYB. These observations were further confirmed in a converse approach wherein MYB was overexpressed ectopically in a MYB-null pancreatic cancer cell line. Our findings thus suggest that MYB potentially regulates growth and genomic stability of pancreatic cancer cells via targeting complex gene networks and signaling pathways. Further in-depth functional studies are warranted to fully understand MYB signaling in pancreatic cancer.
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Redes Reguladoras de Genes/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Transducción de Señal/genética , Línea Celular Tumoral , Regulación hacia Abajo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inestabilidad Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ADN , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Regulación hacia ArribaRESUMEN
Antisense oligonucleotides (ASOs) have a great therapeutic potential for the modulation of gene expression because of the high specificity. The major obstacles for clinical application are enzymatic degradation and low uptake into cells in vivo. In this study, we prepared the conjugate comprising Tat peptide and ASO with phosphorothioate linkages in a simple manner; azide alkyne Huisgen cycloaddition using a copper catalyst. The obtained conjugate showed a high stability in serum, compared with the conjugate with phosphodiester linkages. The conjugates with antisense for c-myb that is transcriptional factor concerning cell growth inhibited the cell proliferation in a dose dependent manner sequence-specifically. These findings suggest Tat-mediated ASOs delivery is useful for the treatment of various diseases.
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Productos del Gen tat/metabolismo , Oligonucleótidos Antisentido/química , Péptidos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Clic , Silenciador del Gen , Humanos , Fosfatos/química , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismoAsunto(s)
Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Imidazoles/farmacología , Indoles/farmacología , Mutación , Oximas/farmacología , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridonas/farmacología , Pirimidinonas/farmacología , Sulfonamidas/farmacología , Vemurafenib , beta Catenina/genéticaRESUMEN
The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3'UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.
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MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-maf/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Regiones no Traducidas 3' , Ancirinas/genética , Ancirinas/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Condrogénesis , Regulación hacia Abajo , Células Eritroides/citología , Células Eritroides/metabolismo , Genes Reporteros , Sitios Genéticos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Megacariocitos/citología , Megacariocitos/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Oligonucleótidos Antisentido/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-maf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-maf/genética , Proteínas Proto-Oncogénicas c-myb/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myb/genética , Interferencia de ARNRESUMEN
The transcription factor c-Myb is highly expressed in hematopoietic progenitor cells and controls the transcription of genes important for lineage determination, cell proliferation, and differentiation. Deregulation of c-Myb has been implicated in the development of leukemia and certain other types of human cancer. c-Myb activity is highly dependent on the interaction of the c-Myb with the KIX domain of the coactivator p300, making the disruption of this interaction a reasonable strategy for the development of Myb inhibitors. Here, we have used bacterial Autodisplay to develop an in vitro binding assay that mimics the interaction of Myb and the KIX domain of p300. We have used this binding assay to investigate the potential of Naphthol AS-E phosphate, a compound known to bind to the KIX domain, to disrupt the interaction between Myb and p300. Our data show that Naphthol AS-E phosphate interferes with the Myb-KIX interaction in vitro and inhibits Myb activity in vivo. By using several human leukemia cell lines, we demonstrate that Naphthol AS-E phosphate suppresses the expression of Myb target genes and induces myeloid differentiation and apoptosis. Our work identifies Naphthol AS-E phosphate as the first low molecular weight compound that inhibits Myb activity by disrupting its interaction with p300, and suggests that inhibition of the Myb-KIX interaction might be a useful strategy for the treatment of leukemia and other tumors caused by deregulated c-Myb.