RESUMO
RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.
Assuntos
Actinas/metabolismo , Linfócitos B/imunologia , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Adolescente , Inibidores da Angiogênese/farmacologia , Linfócitos B/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Criança , Citotoxicidade Imunológica/genética , Análise Mutacional de DNA , Dineínas/metabolismo , Feminino , Células HEK293 , Humanos , Switching de Imunoglobulina/genética , Síndromes de Imunodeficiência/tratamento farmacológico , Células Jurkat , Células Matadoras Naturais/efeitos dos fármacos , Lenalidomida , Masculino , Mutação/genética , Linhagem , RNA Interferente Pequeno/genética , Linfócitos T/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologiaRESUMO
Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.
Assuntos
Pontos de Checagem do Ciclo Celular , Citotoxicidade Imunológica , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Animais , Humanos , Sinapses Imunológicas/imunologiaRESUMO
Recent discoveries have led to the testing of novel targeted therapies for the treatment of acute myeloid leukemia (AML). To better inform the results of clinical trials, there is a need to identify and systematically assess biomarkers of response and pharmacodynamic markers of successful target engagement. Spleen tyrosine kinase (SYK) is a candidate therapeutic target in AML. Small-molecule inhibitors of SYK induce AML differentiation and impair leukemia progression in preclinical studies. However, tools to predict response to SYK inhibition and to routinely evaluate SYK activation in primary patient samples have been lacking. In this study we quantified phosphorylated SYK (P-SYK) in AML cell lines and establish that increasing levels of baseline P-SYK are correlated with an increasing sensitivity to small-molecule inhibitors targeting SYK. In addition, we found that pharmacological inhibition of SYK activity extinguishes P-SYK expression as detected by an immunohistochemical (IHC) test. Quantitative analysis of P-SYK expression by the IHC test in a series of 70 primary bone marrow biopsy specimens revealed a spectrum of P-SYK expression across AML cases and that high P-SYK expression is associated with unfavourable outcome independent of age, cytogenetics, and white blood cell count. This study thus establishes P-SYK as a critical biomarker in AML that identifies tumors sensitive to SYK inhibition, identifies an at-risk patient population, and allows for the monitoring of target inhibition during treatment.
Assuntos
Biomarcadores Tumorais/metabolismo , Medula Óssea/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/enzimologia , Proteínas Tirosina Quinases/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Exame de Medula Óssea , Relação Dose-Resposta a Droga , Ativação Enzimática , Células HL-60 , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Fosforilação , Valor Preditivo dos Testes , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Fatores de Risco , Transdução de Sinais , Quinase Syk , Células U937 , Regulação para CimaRESUMO
PURPOSE: B-cell receptor (BCR)-mediated signaling is important in the pathogenesis of a subset of diffuse large B-cell lymphomas (DLBCL) and the BCR-associated kinases SYK and BTK have recently emerged as potential therapeutic targets. We sought to identify a signature of activated BCR signaling in DLBCL to aid the identification of tumors that may be most likely to respond to BCR-pathway inhibition. EXPERIMENTAL DESIGN: We applied quantitative immunofluorescence (qIF) using antibodies to phosphorylated forms of proximal BCR signaling kinases LYN, SYK, and BTK and antibody to BCR-associated transcription factor FOXO1 on BCR-cross-linked formalin-fixed paraffin-embedded (FFPE) DLBCL cell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n = 154). RESULTS: A robust signature of active BCR signaling was identified and validated in BCR-cross-linked DLBCL cell lines and in 71/154 (46%) of the primary DLBCL patient specimens. Further analysis of the primary biopsy samples revealed increased nuclear exclusion of FOXO1 among DLBCL with qIF evidence of active BCR signaling compared with those without (P = 0.004). Nuclear exclusion of FOXO1 was also detected in a subset of DLBCL without evidence of proximal BCR signaling suggesting that alternative mechanisms for PI3K/AKT activation may mediate FOXO1 subcellular localization in these cases. CONCLUSION: This study establishes the feasibility of detecting BCR activation in primary FFPE biopsy specimens of DLBCL. It lays a foundation for future dissection of signal transduction networks in DLBCL and provides a potential platform for evaluating individual tumors in patients receiving novel therapies targeting the BCR pathway.
Assuntos
Linfoma Difuso de Grandes Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Adulto , Tirosina Quinase da Agamaglobulinemia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinase Syk , Análise Serial de Tecidos , Quinases da Família src/metabolismoRESUMO
PURPOSE: Programmed cell death ligand 1 (PD-L1) is a molecule expressed on antigen-presenting cells that engages the PD-1 receptor on T cells and inhibits T-cell receptor signaling. The PD-1 axis can be exploited by tumor cells to dampen host antitumor immune responses and foster tumor cell survival. PD-1 blockade has shown promise in multiple malignancies but should be directed toward patients in whom it will be most effective. In recent studies, we found that the chromosome 9p24.1 amplification increased the gene dosage of PD-L1 and its induction by JAK2 in a subset of patients with classical Hodgkin lymphoma (cHL). However, cHLs with normal 9p24.1 copy numbers also expressed detectable PD-L1, prompting analyses of additional PD-L1 regulatory mechanisms. EXPERIMENTAL DESIGN: Herein, we utilized immunohistochemical, genomic, and functional analyses to define alternative mechanisms of PD-L1 activation in cHL and additional EBV(+) lymphoproliferative disorders. RESULTS: We identified an AP-1-responsive enhancer in the PD-L1 gene. In cHL Reed-Sternberg cells, which exhibit constitutive AP-1 activation, the PD-L1 enhancer binds AP-1 components and increases PD-L1 promoter activity. In addition, we defined Epstein-Barr virus (EBV) infection as an alternative mechanism for PD-L1 induction in cHLs with diploid 9p24.1. PD-L1 was also expressed by EBV-transformed lymphoblastoid cell lines as a result of latent membrane protein 1-mediated, JAK/STAT-dependent promoter and AP-1-associated enhancer activity. In addition, more than 70% of EBV(+) posttransplant lymphoproliferative disorders expressed detectable PD-L1. CONCLUSIONS: AP-1 signaling and EBV infection represent alternative mechanisms of PD-L1 induction and extend the spectrum of tumors in which to consider PD-1 blockade.
Assuntos
Antígeno B7-H1/metabolismo , Infecções por Vírus Epstein-Barr/complicações , Doença de Hodgkin/genética , Transtornos Linfoproliferativos/genética , Fator de Transcrição AP-1/genética , Linhagem Celular Tumoral , Transformação Celular Viral/genética , Cromossomos Humanos Par 9 , Elementos Facilitadores Genéticos , Doença de Hodgkin/imunologia , Doença de Hodgkin/virologia , Humanos , Terapia de Imunossupressão , Proteínas da Matriz Viral/farmacologiaRESUMO
Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. A small subset of DLBCLs has translocations involving the MYC locus and an additional group has a molecular signature resembling Burkitt lymphoma (mBL). Presently, identification of such cases by morphology is unreliable and relies on cytogenetic or complex molecular methods such as gene transcriptional profiling. Herein, we describe an immunohistochemical (IHC) method for identifying DLBCLs with increased MYC protein expression. We tested 77 cases of DLBCL and identified 15 cases with high MYC protein expression (nuclear staining in >50% of tumor cells). All MYC translocation positive cases had increased MYC protein expression by this IHC assay. In addition, gene set enrichment analysis (GSEA) of the DLBCL transcriptional profiles revealed that tumors with increased MYC protein expression (regardless of underlying MYC translocation status) had coordinate upregulation of MYC target genes, providing molecular confirmation of the IHC results. We then generated a molecular classifier derived from the MYC IHC results in our cases and employed it to successfully classify mBLs from two previously reported independent case series, providing additional confirmation that the MYC IHC results identify clinically important subsets of DLBCLs. Lastly, we found that DLBCLs with high MYC protein expression had inferior overall survival when treated with R-CHOP. In conclusion, the IHC method described herein can be used to readily identify the biologically and clinically distinct cases of MYC-driven DLBCL, which represent a clinically significant subset of DLBCL cases due to their inferior overall survival.
Assuntos
Imuno-Histoquímica/métodos , Linfoma Difuso de Grandes Células B/diagnóstico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/mortalidade , Prognóstico , Translocação GenéticaRESUMO
PURPOSE: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. EXPERIMENTAL DESIGN: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. RESULTS: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. CONCLUSIONS: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.