RESUMEN
Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.
Asunto(s)
Carcinoma de Células Pequeñas/genética , Neoplasias Pulmonares/genética , Proteínas Oncogénicas/genética , Factores de Empalme Serina-Arginina/genética , Adulto , Anciano , Variaciones en el Número de Copia de ADN , Daño del ADN , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
OBJECTIVE: To assess the pharmacodynamic effects of sifalimumab, an investigational anti-IFN-α monoclonal antibody, in the blood and muscle of adult dermatomyositis and polymyositis patients by measuring neutralisation of a type I IFN gene signature (IFNGS) following drug exposure. METHODS: A phase 1b randomised, double-blinded, placebo controlled, dose-escalation, multicentre clinical trial was conducted to evaluate sifalimumab in dermatomyositis or polymyositis patients. Blood and muscle biopsies were procured before and after sifalimumab administration. Selected proteins were measured in patient serum with a multiplex assay, in the muscle using immunohistochemistry, and transcripts were profiled with microarray and quantitative reverse transcriptase PCR assays. A 13-gene IFNGS was used to measure the pharmacological effect of sifalimumab. RESULTS: The IFNGS was suppressed by a median of 53-66% across three time points (days 28, 56 and 98) in blood (p=0.019) and 47% at day 98 in muscle specimens post-sifalimumab administration. Both IFN-inducible transcripts and proteins were prevalently suppressed following sifalimumab administration. Patients with 15% or greater improvement from baseline manual muscle testing scores showed greater neutralisation of the IFNGS than patients with less than 15% improvement in both blood and muscle. Pathway/functional analysis of transcripts suppressed by sifalimumab showed that leucocyte infiltration, antigen presentation and immunoglobulin categories were most suppressed by sifalimumab and highly correlated with IFNGS neutralisation in muscle. CONCLUSIONS: Sifalimumab suppressed the IFNGS in blood and muscle tissue in myositis patients, consistent with this molecule's mechanism of action with a positive correlative trend between target neutralisation and clinical improvement. These observations will require confirmation in a larger trial powered to evaluate efficacy.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Dermatomiositis/tratamiento farmacológico , Dermatomiositis/inmunología , Inmunosupresores/administración & dosificación , Polimiositis/tratamiento farmacológico , Polimiositis/inmunología , Adulto , Anciano , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Método Doble Ciego , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Humanos , Inmunosupresores/efectos adversos , Interferón Tipo I/sangre , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón-alfa/sangre , Interferón-alfa/genética , Interferón-alfa/inmunología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Masculino , Persona de Mediana Edad , Músculo Esquelético/inmunología , Placebos , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: MicroRNAs (miRNAs) function to fine-tune the control of immune cell signaling. It is well established that there are abnormalities in the interleukin-2 (IL-2)-related signaling pathways in systemic lupus erythematosus (SLE). The miR-31 microRNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-31 in IL-2 defects in lupus T cells. METHODS: Expression levels of miR-31 were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-31. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-31 targets and effects. RESULTS: The expression of miR-31 was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-31 in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-31 reduced IL-2 production. RhoA expression was directly repressed by miR-31 in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-31 in lupus T cells. Manipulation of miR-31 expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels. CONCLUSION: MicroRNA-31 is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-31 and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.
Asunto(s)
Interleucina-2/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , MicroARNs/inmunología , Linfocitos T/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-2/deficiencia , Interleucina-2/inmunología , Células Jurkat , Lupus Eritematoso Sistémico/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/inmunología , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/inmunologíaRESUMEN
Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD19/inmunología , Leucemia de Células B/inmunología , Leucemia de Células B/terapia , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones SCID , Ingeniería de Proteínas/métodos , Receptores Fc/inmunología , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Type I interferons (IFNs) appear to play a central role in disease pathogenesis in systemic lupus erythematosus (SLE), making them potential therapeutic targets. METHODS: Safety profile, pharmacokinetics, immunogenicity, pharmacodynamics and clinical activity of sifalimumab, an anti-IFNα monoclonal antibody, were assessed in a phase I, multicentre, randomised, double-blind, dose-escalation study with an open-label extension in adults with moderately active SLE. SUBJECTS: received one intravenous dose of sifalimumab (n=33 blinded phase, 0.3, 1, 3, 10 or 30 mg/kg; n=17 open-label, 1, 3, 10 or 30 mg/kg) or placebo (n=17). Each phase lasted 84 days. RESULTS: Adverse events (AEs) were similar between groups; about 97% of AEs were grade 1 or 2. All grade 3 and 4 AEs and all serious AEs (2 placebo, 1 sifalimumab) were deemed unrelated to the study drug. No increase in viral infections or reactivation was observed. Sifalimumab caused dose-dependent inhibition of type I IFN-induced mRNAs (type I IFN signature) in whole blood and corresponding changes in related proteins in affected skin. Exploratory analyses showed consistent trends toward improvement in disease activity in sifalimumab-treated versus placebo-treated subjects. A lower proportion of sifalimumab-treated subjects required new or increased immunosuppressive treatments (12% vs 41%; p=0.03) and had fewer Systemic Lupus Erythematosus Disease Activity Index flares (3% vs 29%; p=0.014). CONCLUSIONS: Sifalimumab had a safety profile that supports further clinical development. This trial demonstrated that overexpression of type I IFN signature in SLE is at least partly driven by IFNα, and exploratory analyses suggest that IFNα inhibition may be associated with clinical benefit in SLE. Trial registration number NCT00299819.
Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Inmunosupresores/efectos adversos , Interferón-alfa/antagonistas & inhibidores , Lupus Eritematoso Sistémico/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/sangre , Inmunosupresores/uso terapéutico , Inyecciones Intravenosas , Interferón Tipo I/biosíntesis , Interferón Tipo I/genética , Interferón-alfa/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues. METHODS: Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements. RESULTS: A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc. CONCLUSIONS: The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy.
Asunto(s)
Interferón Tipo I/biosíntesis , Enfermedades Reumáticas/inmunología , Adulto , Artritis Reumatoide/inmunología , Biomarcadores/sangre , Proteínas Sanguíneas/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Interferón Tipo I/genética , Interferón-alfa/antagonistas & inhibidores , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , Miositis/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Esclerodermia Sistémica/inmunología , Índice de Severidad de la Enfermedad , Transducción de Señal/inmunologíaRESUMEN
OBJECTIVE: We investigated interferon-stimulated gene 15 (ISG15), a poorly understood ubiquitin-like modifier, and its enzymatic pathway in dermatomyositis (DM), an autoimmune disease primarily involving muscle and skin. METHODS: We generated microarray data measuring transcript abundance for approximately 18,000 genes in each of 113 human muscle biopsy specimens, and studied biopsy specimens and cultured skeletal muscle using immunohistochemistry, immunoblotting proteomics, real-time quantitative polymerase chain reaction, and laser-capture microdissection. RESULTS: Transcripts encoding ISG15-conjugation pathway proteins were markedly upregulated in DM with perifascicular atrophy (DM-PFA) muscle (ISG15 339-fold, HERC5 62-fold, and USP18 68-fold) compared with 99 non-DM samples. Combined analysis with publicly available microarray datasets showed that >50-fold ISG15 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM patients compared with 199 muscle samples from other muscle diseases. Free ISG15 and ISG15-conjugated proteins were only found on immunoblots from DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and ISG15 protein and conjugates. Laser-capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers. INTERPRETATION: A large-scale microarray study of muscle samples demonstrated that among a diverse group of muscle diseases DM was uniquely associated with upregulation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produced a molecular picture highly similar to that seen in human DM muscle. Perifascicular atrophic myofibers in DM were deficient in a number of skeletal muscle proteins including titin.
Asunto(s)
Citocinas/metabolismo , Dermatomiositis/metabolismo , Músculo Esquelético/metabolismo , Ubiquitinas/metabolismo , Células Cultivadas , Conectina , Citocinas/genética , Bases de Datos Genéticas , Dermatomiositis/diagnóstico , Dermatomiositis/genética , Humanos , Immunoblotting , Inmunohistoquímica , Interferón Tipo I/metabolismo , Rayos Láser , Microdisección/métodos , Proteínas Musculares/deficiencia , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/deficiencia , Proteómica/métodos , Sensibilidad y Especificidad , Ubiquitinas/genética , Regulación hacia ArribaRESUMEN
To determine the potential consequences of plasmacytoid dendritic cell (pDC) accumulation in tissue sites observed in several autoimmune diseases, we measured type 1 interferon production from circulating human pDCs as a function of pDC concentration. The effects of interferon-alpha and blockade of the type 1 interferon receptor (IFNAR) on human pDC type 1 interferon and interferon-inducible transcription and protein production were measured. Human pDCs became far more efficient producers of interferon-alpha at concentrations beyond those normally present in blood, through an IFNAR-dependent mechanism. Extracellular interferon-alpha increased pDC production of type 1 interferons. The accumulation of pDCs in diseased tissue sites allows marked non-linear amplification of type 1 interferon production locally. The role of the IFNAR-dependent mechanism of interferon production by human pDCs is greater than previously suggested. IFNAR blockade has potential for diminishing type 1 interferon production by all human cells.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/metabolismo , Interferón Tipo I/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/inmunología , Proteínas Portadoras/genética , Recuento de Células , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Endopeptidasas/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Interferón Tipo I/genética , Interferón alfa-2 , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Interferón beta/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Cinética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Proteínas de Resistencia a Mixovirus , Oligodesoxirribonucleótidos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Isoformas de Proteínas/genética , Proteínas/genética , Proteínas de Unión al ARN , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/inmunología , Proteínas Recombinantes , Receptor Toll-Like 9/agonistas , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Ubiquitina Tiolesterasa , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.
Asunto(s)
Antígenos CD19/inmunología , Linfocitos B/fisiología , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD19/genética , Proliferación Celular/efectos de los fármacos , Fucosa/química , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Ingeniería de Proteínas , RituximabRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder characterized by the surface expression of CD20, CD5 antigens, as well as the receptor CD40. Activation of CD40 by its ligand (CD40L) induces proliferation and rescues the cells from spontaneous and chemotherapy-induced apoptosis. CD40 activation also induces secretion of cytokines, such as IL-6, IL-10, TNF-alpha, IL-8, and GM-CSF, which are involved in tumor cell survival, migration, and interaction with cells in the tumor microenvironment. Here we demonstrate that in primary B-CLL tumor cells, the novel antagonist anti-CD40 monoclonal antibody, HCD122, inhibits CD40L-induced activation of signaling pathways, proliferation and survival, and secretion of cytokines. Furthermore, HCD122 is also a potent mediator of antibody-dependent cellular cytotoxicity (ADCC), lysing B-CLL cells more efficiently than rituximab in vitro, despite a significantly higher number of cell surface CD20 binding sites compared with CD40. Unlike rituximab, however, HCD122 (formerly CHIR-12.12) does not internalize upon binding to the cells. Our data suggest that HCD122 may inhibit B-CLL growth by blocking CD40 signaling and by ADCC-mediated cell lysis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Antígenos CD40/inmunología , Leucemia de Células B/inmunología , Adulto , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Sitios de Unión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Rituximab , Transducción de Señal/efectos de los fármacosRESUMEN
Myositis muscle contains antigen-matured B-cells and plasma cells. Myositis muscle biopsy specimens were examined for nodular collections of T-cells, B-cells, myeloid dendritic cells, plasma cells, and follicular dendritic cells. Immunoglobulin and B-cell-activating factor (BAFF) transcripts were quantitated. Laser-capture microdissection was used to isolate single plasma cells, and their immunoglobulin transcripts were sequenced. Dense inflammatory infiltrates contained histological elements of ectopic lymphoid tissue but not B-cell follicles. Immunoglobulin transcript sequence analysis demonstrated spatially distributed, clonally related B-cells and plasma cells, suggesting local maturation of B-cells into plasma cells in myositis muscle. Regions of dense cellular infiltrates in myositis muscle are sometimes areas of B-cell maturation into antibody-producing plasma cells. An atypical lymphoid histology, lacking concentrated collections of germinal-center-like B-cell follicles, is capable of antigen-stimulated clonal maturation of antibody-producing plasma cells.
Asunto(s)
Linfocitos B , Senescencia Celular , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miositis/fisiopatología , Presentación de Antígeno , Factor Activador de Células B/genética , Antígeno de Maduración de Linfocitos B/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Senescencia Celular/efectos de los fármacos , Coristoma/patología , Células Dendríticas/inmunología , Células Dendríticas/patología , Centro Germinal , Humanos , Inmunoglobulinas/genética , Técnicas In Vitro , Tejido Linfoide , Microdisección/métodos , Enfermedades Musculares/patología , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , ARN Mensajero/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
HGK (hepatocyte progenitor kinase-like/germinal center kinase-like kinase) is a member of the human STE20/mitogen-activated protein kinase kinase kinase kinase family of serine/threonine kinases and is the ortholog of mouse NIK (Nck-interacting kinase). We have cloned a novel splice variant of HGK from a human tumor line and have further identified a complex family of HGK splice variants. We showed HGK to be highly expressed in most tumor cell lines relative to normal tissue. An active role for this kinase in transformation was suggested by an inhibition of H-Ras(V12)-induced focus formation by expression of inactive, dominant-negative mutants of HGK in both fibroblast and epithelial cell lines. Expression of an inactive mutant of HGK also inhibited the anchorage-independent growth of cells yet had no effect on proliferation in monolayer culture. Expression of HGK mutants modulated integrin receptor expression and had a striking effect on hepatocyte growth factor-stimulated epithelial cell invasion. Together, these results suggest an important role for HGK in cell transformation and invasiveness.
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Glioblastoma/enzimología , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Células 3T3 , Empalme Alternativo , Animales , Secuencia de Bases , Adhesión Celular/fisiología , Transformación Celular Neoplásica/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Clonación Molecular , Inducción Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Fibroblastos/enzimología , Regulación Neoplásica de la Expresión Génica , Biblioteca de Genes , Genes Dominantes , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Integrinas/biosíntesis , Integrinas/genética , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/fisiología , Sistema de Señalización de MAP Quinasas , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas/enzimologíaRESUMEN
PURPOSE: The ectopically expressed and deregulated fibroblast growth factor receptor 3 (FGFR3) results from a t(4;14) chromosomal translocation that occurs in approximately 15% of multiple myeloma (MM) patients and confers a particularly poor prognosis. This study assesses the antimyeloma activity of CHIR-258, a small-molecule inhibitor of multiple receptor tyrosine kinases that is currently in phase I trials, in a newly developed FGFR3-driven preclinical MM animal model. EXPERIMENTAL DESIGN: We developed an orthotopic MM model in mice using a luciferase-expressing human KMS-11-luc line that expresses mutant FGFR3 (Y373C). The antimyeloma activity of CHIR-258 was evaluated at doses that inhibited FGFR3 signaling in vivo in this FGFR3-driven animal model. RESULTS: Noninvasive bioluminescence imaging detected MM lesions in nearly all mice injected with KMS-11-luc cells, which were mainly localized in the spine, skull, and pelvis, resulting in frequent development of paralysis. Daily oral administration of CHIR-258 at doses that inhibited FGFR3 signaling in KMS-11-luc tumors in vivo resulted in a significant inhibition of KMS-11-luc tumor growth, which translated into a significant improvement in animal survival. CONCLUSIONS: Our data provide a relevant preclinical basis for clinical trials of CHIR-258 in FGFR3-positive MM patients.
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Bencimidazoles/farmacología , Mieloma Múltiple/tratamiento farmacológico , Quinolonas/farmacología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Ratones SCID , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mieloma Múltiple/enzimología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/sangre , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MST4, a member of the Sterile 20 serine/threonine kinase family, was found to be expressed in prostate carcinoma tumor samples and cell lines. In addition, expression levels appeared to correlate with tumorigenicity and androgen receptor status of the cells. Ectopic expression of wild-type and kinase-inactive MST4 was used to alter cellular MST4 activity levels in three widely studied human prostate tumor cell lines: LNCaP, DU 145, and PC-3. Overexpression of wild-type MST4 induced anchorage-independent growth of the LNCaP cell line, and increased both in vitro proliferation and in vivo tumorigenesis of the DU 145 cell line. On the other hand, expression of a kinase-inactive form reverted the anchorage-independent growth phenotype and highly tumorigenic behavior of the PC-3 cell line. MST4 kinase activity was stimulated significantly by epidermal growth factor receptor ligands, which are known to promote growth of prostate cancer cells. Together, our studies suggest a potential role for MST4 in the signal transduction pathways involved in prostate cancer progression.
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Adenocarcinoma/enzimología , Proteínas de Neoplasias/fisiología , Neoplasias de la Próstata/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Adenocarcinoma/patología , Adenocarcinoma/secundario , Adulto , Adhesión Celular , División Celular , Progresión de la Enfermedad , Inducción Enzimática , Factor de Crecimiento Epidérmico/farmacología , Matriz Extracelular/metabolismo , Humanos , Metástasis Linfática , Masculino , Proteínas de Neoplasias/genética , Especificidad de Órganos , Próstata/enzimología , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/fisiología , Transfección , Células Tumorales Cultivadas/enzimología , Células Tumorales Cultivadas/patologíaRESUMEN
OBJECTIVE: Diffuse alveolar hemorrhage (DAH) is a rare but life-threatening complication of systemic lupus erythematosus (SLE). Pristane-treated B6 mice develop severe DAH within 2 weeks of treatment. MicroRNA-155 (miR-155) is a pleiotropic microRNA that plays a crucial role in the regulation of immune responses. Recent studies have revealed a pathogenic role of miR-155 in various autoimmune disorders. The purpose of this study was to examine the role of miR-155 in the development of DAH in pristane-induced lupus using miR-155-knockout (miR-155(-/-)) mice and miR-155 antagomir to silence miR-155. METHODS: DAH was induced by an intraperitoneal injection of 0.5 ml of pristane. MicroRNA-155 antagomir was administered intravenously to silence miR-155 expression. Lung tissues were collected for RNA extraction and were embedded in paraffin for sectioning. Gene expression profiling data were analyzed using Ingenuity Pathway Analysis. Real-time quantitative polymerase chain reaction analysis was used for single-gene validation. Luciferase reporter assay and argonaute 2 immunoprecipitation were performed for target validation. RESULTS: MicroRNA-155 expression was significantly increased during the development of DAH. Disease progression was reduced in miR-155(-/-) mice as well as by in vivo silencing of miR-155 using a miR-155 antagomir. MicroRNA-155 silencing dampened pristane-induced ectopic activation of multiple inflammatory pathways and reduced the expression of proinflammatory cytokines. Several negative regulators of NF-κB signaling were inhibited by pristane and were reactivated in miR-155(-/-) mice. In particular, the antiinflammatory factor peroxisome proliferator-activated receptor α was identified as a direct target of miR-155. CONCLUSION: MicroRNA-155 promotes pristane-induced lung inflammation. It contributes to ectopic activation of NF-κB signaling pathways by targeting multiple negative regulators. MicroRNA-155 antagomir may be a promising therapeutic strategy for treating acute lung inflammation in lupus.
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Hemorragia/tratamiento farmacológico , Enfermedades Pulmonares/tratamiento farmacológico , Lupus Eritematoso Sistémico/tratamiento farmacológico , MicroARNs/antagonistas & inhibidores , Oligonucleótidos/uso terapéutico , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hemorragia/etiología , Hemorragia/genética , Inmunosupresores/toxicidad , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/genética , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Oligonucleótidos/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Terpenos/toxicidadRESUMEN
OBJECTIVE: Type I interferon (IFN) signaling is regarded as a central pathogenic pathway in systemic lupus erythematosus (SLE). Specific inhibition of this pathway is a core area for the development of new therapies for SLE. This study was undertaken to clarify the pathogenic mechanism involved and to identify new therapeutic targets, using a high-throughput screening platform to determine novel regulators that contribute to the overactivation of the type I IFN signaling pathway in SLE. METHODS: A high-throughput IFN-stimulated response element (ISRE)-luciferase assay was used to screen for candidate genes that regulate the IFN signaling pathway. Western blotting was used to confirm the regulatory function of CDK1. SYBR Green quantitative reverse transcriptase-polymerase chain reaction was used to detect the expression of individual IFN-stimulated genes (ISGs). The differential expression of CDK1 and ISGs in SLE patients and healthy controls was analyzed using RNA sequencing data and a microarray. RESULTS: The high-throughput ISRE-luciferase assay revealed that CDK1 enhanced type I IFN signaling. Consistent with this finding, CDK1 promoted the type I IFN-induced phosphorylation of STAT-1 and the up-regulated expression of ISGs. CDK1 expression was elevated in peripheral blood mononuclear cells (PBMCs) and kidney biopsy specimens from SLE patients and correlated positively with their IFN scores. A CDK1 inhibitor reduced the expression of ISGs in PBMCs from SLE patients and in renal cells from mice with lupus. CONCLUSION: Our findings indicate that CDK1 is a positive regulator of the IFN signaling pathway. The overexpression of CDK1 might contribute to the abnormally amplified type I IFN signaling in SLE, and the inhibition of CDK1 could be used to down-regulate type I IFN signaling in SLE.
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Quinasas Ciclina-Dependientes/metabolismo , Interferón Tipo I/inmunología , Riñón/metabolismo , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Nefritis Lúpica/metabolismo , Factor de Transcripción STAT1/metabolismo , Adulto , Animales , Western Blotting , Proteína Quinasa CDC2 , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/inmunología , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Interferón Tipo I/efectos de los fármacos , Riñón/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Masculino , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Ratones , Persona de Mediana Edad , Fosforilación , Quinolinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Tiazoles/farmacología , Regulación hacia ArribaRESUMEN
HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias/metabolismo , Receptor ErbB-3/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Ligandos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The receptor-like protein tyrosine phosphatase DEP1, also known as CD148, is expressed predominantly in epithelial cells, in a variety of tumor cell lines, and in lymphocytes. Expression of DEP1 is enhanced at high cell density, and this observation suggests that DEP1 may function in the regulation of cell adhesion and possibly contact inhibition of cell growth. In order to investigate the function of DEP1, substrate-trapping mutants of the phosphatase were used to identify potential substrates. GST-fusion proteins containing the DEP1 catalytic domain with a substrate-trapping D/A mutation were found to interact with p120(ctn), a component of adherens junctions. DEP1 also interacted with other members of the catenin gene family including beta-catenin and gamma-catenin. The interaction with p120(ctn) is likely to be direct, as the interaction occurs in K562 cells lacking functional adherens junctions and E-cadherin expression. Catalytic domains of the tyrosine phosphatases PTP-PEST, CD45, and PTPbeta did not interact with proteins of the catenin family to detectable levels, suggesting that the interaction of DEP1 with these proteins is specific. DEP1 expression was concentrated at sites of cell-cell contact in A549 cells. p120(ctn) was found to colocalize with these structures. Together these data suggest an important role for DEP-1 in the function of cell-cell contacts and adherens junctions.
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Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatasas/fisiología , Cateninas , Comunicación Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Humanos , Fosforilación , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Tirosina/metabolismo , alfa Catenina , Catenina deltaRESUMEN
OBJECTIVE: Following activation of T cells, phosphorylation of tyrosine residues occurs through a complex signaling process involving protein tyrosine kinases, phosphatases, and a variety of adapter molecules including Grb2. We have attempted to identify new signaling molecules that are important for the activation response. METHODS: Using a protein interaction screening protocol based on phage display, T-cell signaling components that associate with the adapter molecule, Grb2, the lymphoid-specific tyrosine phosphatase Lyp was identified. Using transcriptional reporter assays, the role of Lyp in T-cell activation was studied by overexpression of wild-type or catalytically inactive mutants of Lyp. RESULTS: A GST fusion containing the C-terminal SH3 domain of Grb2 bound to the nucleotide exchange factor Sos or Grb2-associated binder 2 (Gab2). In contrast, the N-terminal SH3-containing fusion bound to the protein tyrosine phosphatase Lyp. Grb2 was co-immunoprecipitated with Lyp in 293T cells overexpressing both proteins. Using Northern blot analysis, Lyp was found to be expressed predominantly in hematopoietic tissue, including spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver. Two human T-cell lines, Jurkat and HuT78, expressed both Lyp mRNA and protein. Overexpression of wild-type Lyp or a catalytically inactive, substrate-trapping mutant (D195A) in Jurkat cells inhibited transcriptional activity initiated by anti-CD3 and anti-CD28 antibodies. In contrast, two other catalytically inactive mutants (R233M or C227S) had no effect. CONCLUSION: These data demonstrate a novel interaction between the phosphatase Lyp and the adaptor Grb2 and are consistent with a negative regulatory role for Lyp in T-cell signaling.
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Proteínas Adaptadoras Transductoras de Señales , Homeostasis , Activación de Linfocitos , Proteínas Tirosina Fosfatasas/fisiología , Proteínas/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Northern Blotting , Línea Celular , Proteína Adaptadora GRB2 , Expresión Génica , Glutatión Transferasa/genética , Humanos , Técnicas de Inmunoadsorción , Células Jurkat , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Especificidad de Órganos , Fragmentos de Péptidos/genética , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa , Proteína Tirosina Fosfatasa no Receptora Tipo 22 , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/genética , Proteínas/genética , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , TransfecciónRESUMEN
OBJECTIVE: Systemic lupus erythematosus (SLE) is characterized by the uncontrolled production of inflammatory cytokines, among which type I interferon (IFN) is recognized as a crucial pathogenic factor. The expression of microRNA-146a (miR-146a) is reduced in the white blood cells of SLE patients and accounts for their overactivated inflammatory responses. However, the mechanism of the reduction of miR-146a is still not fully understood. This study was undertaken to test whether the key pathogenic cytokine, type I IFN, is responsible for the dysregulation of miR-146a in SLE. METHODS: Gene and protein expression was measured in all cells by reverse transcription-quantitative polymerase chain reaction, Northern blotting, or Western blotting. In THP-1 cells, expression of monocyte chemotactic protein-induced protein 1 (MCPIP-1) was knocked down with a lentivirus encoding a short hairpin RNA targeting MCPIP1. The cells were pretreated with type I IFN and assessed for gene expression levels of miR-146a. White blood cells from patients with SLE were analyzed for the expression of the IFN-inducible genes MCPIP1 and miR-146a, and the gene expression data were compared for correlation. RESULTS: Pretreatment of THP-1 cells with type I IFN attenuated the induction of miR-146a posttranscriptionally, by down-regulating the expression of pre-miR-146a but not pri-miR-146a or its original unspliced transcript. Expression of MCPIP-1, which was enhanced by type I IFN, was found to be responsible for the inhibition of miR-146a. In white blood cells from patients with SLE, MCPIP1 expression was elevated, and its expression correlated positively with the IFN score and negatively with the miR-146a transcript level. CONCLUSION: Type I IFN inhibits the maturation of miR-146a through the up-regulation of MCPIP-1, and thus contributes to the uncontrolled inflammation and excessive inflammatory gene expression in SLE.