RESUMEN
Sepsis is a prevalent health issue that can lead to central nervous system (CNS) inflammation with long-term behavioral and cognitive alterations. Using unbiased proteomic profiling of over 100 different cytokines, we found that Lipocalin-2 (LCN2) was the most substantially elevated protein in the CNS after peripheral administration of lipopolysaccharide (LPS). To determine whether the high level of LCN2 in the CNS is protective or deleterious, we challenged Lcn2-/- mice with peripheral LPS and determined effects on behavior and neuroinflammation. At a time corresponding to peak LCN2 induction in wild-type (WT) mice injected with LPS, Lcn2-/- mice challenged with LPS had exacerbated levels of pro-inflammatory cytokines and exhibited significantly worsened behavioral phenotypes. To determine the extent of global inflammatory changes dependent upon LCN2, we performed an RNAseq transcriptomic analysis. Compared with WT mice injected with LPS, Lcn2-/- mice injected with LPS had unique transcriptional profiles and significantly elevated levels of multiple pro-inflammatory molecules. Several LCN2-dependent pathways were revealed with this analysis including, cytokine and chemokine signaling, nucleotide-binding oligomerization domain-like receptor signaling and Janus kinase-signal transducer and activator of transcription signaling. These findings demonstrate that LCN2 serves as a potent protective factor in the CNS in response to systemic inflammation and may be a potential candidate for limiting sepsis-related CNS sequelae.
Asunto(s)
Lipocalina 2/fisiología , Animales , Encéfalo , Sistema Nervioso Central , Citocinas , Femenino , Inflamación/metabolismo , Lipocalina 2/genética , Lipocalina 2/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteómica , Sepsis/metabolismo , Sepsis/prevención & control , Transducción de SeñalAsunto(s)
Linfoma Anaplásico de Células Grandes/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular , Femenino , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Tumor-specific mutations can result in immunogenic neoantigens, both of which have been correlated with responsiveness to immune checkpoint inhibitors in highly mutagenic cancers. However, early results of single-agent checkpoint inhibitors in multiple myeloma (MM) have been underwhelming. Therefore, we sought to understand the relationship between mutation and neoantigen landscape of MM patients and responsiveness to therapies. Somatic mutation burden, neoantigen load, and response to therapy were determined using interim data from the MMRF CoMMpass study (NCT01454297) on 664 MM patients. In this population, the mean somatic and missense mutation loads were 405.84(s=608.55) and 63.90(s=95.88) mutations per patient, respectively. There was a positive linear relationship between mutation and neoantigen burdens (R2=0.862). The average predicted neoantigen load was 23.52(s=52.14) neoantigens with an average of 9.40(s=26.97) expressed neoantigens. Survival analysis revealed significantly shorter progression-free survival (PFS) in patients with greater than average somatic missense mutation load (N=163, 0.493 vs 0.726 2-year PFS, P=0.0023) and predicted expressed neoantigen load (N=214, 0.555 vs 0.729 2-year PFS, P=0.0028). This pattern is maintained when stratified by disease stage and cytogenetic abnormalities. Therefore, high mutation and neoantigen load are clinically relevant risk factors that negatively impact survival of MM patients under current standards of care.
Asunto(s)
Antígenos de Neoplasias/genética , Mieloma Múltiple/genética , Mieloma Múltiple/mortalidad , Mutación Missense , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapia , Tasa de SupervivenciaRESUMEN
Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9-3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTKC481S and CXCR4WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTKC481S and CXCR4WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/farmacología , Mutación Missense , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores CXCR4 , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Macroglobulinemia de Waldenström , Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Piperidinas , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Regulación hacia Arriba/genética , Macroglobulinemia de Waldenström/tratamiento farmacológico , Macroglobulinemia de Waldenström/genética , Macroglobulinemia de Waldenström/metabolismo , Macroglobulinemia de Waldenström/patologíaRESUMEN
Lack of remission or early relapse remains a major clinical issue in diffuse large B-cell lymphoma (DLBCL), with 30% of patients failing standard of care. Although clinical factors and molecular signatures can partially predict DLBCL outcome, additional information is needed to identify high-risk patients, particularly biologic factors that might ultimately be amenable to intervention. Using whole-exome sequencing data from 51 newly diagnosed and immunochemotherapy-treated DLBCL patients, we evaluated the association of somatic genomic alterations with patient outcome, defined as failure to achieve event-free survival at 24 months after diagnosis (EFS24). We identified 16 genes with mutations, 374 with copy number gains and 151 with copy number losses that were associated with failure to achieve EFS24 (P<0.05). Except for FOXO1 and CIITA, known driver mutations did not correlate with EFS24. Gene losses were localized to 6q21-6q24.2, and gains to 3q13.12-3q29, 11q23.1-11q23.3 and 19q13.12-19q13.43. Globally, the number of gains was highly associated with poor outcome (P=7.4 × 10(-12)) and when combined with FOXO1 mutations identified 77% of cases that failed to achieve EFS24. One gene (SLC22A16) at 6q21, a doxorubicin transporter, was lost in 54% of EFS24 failures and our findings suggest it functions as a doxorubicin transporter in DLBCL cells.
Asunto(s)
Exoma/genética , Linfoma de Células B Grandes Difuso/genética , Proteínas de Transporte de Catión Orgánico/genética , Anciano , Anciano de 80 o más Años , Transporte Biológico , Terapia Combinada , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Doxorrubicina/metabolismo , Femenino , Estudios de Asociación Genética , Genoma Humano , Humanos , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/mortalidad , Linfoma de Células B Grandes Difuso/terapia , Masculino , Persona de Mediana Edad , Eliminación de Secuencia , Resultado del TratamientoRESUMEN
OBJECTIVES: To identify venous thromboembolism (VTE) disease-susceptibility genes. PATIENTS AND METHODS: We performed in silico genome wide association scan (GWAS) analyses using genotype data imputed to approximately 2.5 million single-nucleotide polymorphisms (SNPs) from adults with objectively-diagnosed VTE (n=1503), and controls frequency matched on age and gender (n=1459; discovery population). Single-nucleotide polymorphisms exceeding genome-wide significance were replicated in a separate population (VTE cases, n=1407; controls, n=1418). Genes associated with VTE were re-sequenced. RESULTS: Seven SNPs exceeded genome-wide significance (P<5×10(-8)): four on chromosome 1q24.2 (F5 rs6025 [factor V Leiden], BLZF1 rs7538157, NME7 rs16861990 and SLC19A2 rs2038024) and three on chromosome 9q34.2 (ABO rs2519093 [ABO intron 1], rs495828, rs8176719 [ABO blood type O allele]). The replication study confirmed a significant association of F5, NME7 and ABO with VTE. However, F5 was the main signal on 1q24.2 as only ABO SNPs remained significantly associated with VTE after adjusting for F5 rs6025. This 1q24.2 region was shown to be inherited as a haplotype block. ABO re-sequencing identified 15 novel single nucleotide variations (SNV) in ABO intron 6 and the ABO 3' UTR that were strongly associated with VTE (P<10(-4)) and belonged to three distinct linkage disequilibrium (LD) blocks; none were in LD with ABO rs8176719 or rs2519093. Our sample size provided 80% power to detect odds ratios (ORs)=2.0 and 1.51 for minor allele frequencies=0.05 and 0.5, respectively (α=1×10(-8); 1% VTE prevalence). CONCLUSIONS: Apart from F5 rs6025, ABO rs8176719, rs2519093 and F2 rs1799963, additional common and high VTE-risk SNPs among whites are unlikely.
Asunto(s)
Cromosomas Humanos Par 1 , Cromosomas Humanos Par 9 , Polimorfismo de Nucleótido Simple , Tromboembolia Venosa/genética , Sistema del Grupo Sanguíneo ABO/genética , Estudios de Casos y Controles , Simulación por Computador , Factor V/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Haplotipos , Humanos , Desequilibrio de Ligamiento , Modelos Logísticos , Minnesota/epidemiología , Modelos Genéticos , Oportunidad Relativa , Prevalencia , Protrombina/genética , Medición de Riesgo , Factores de Riesgo , Tromboembolia Venosa/etnología , Población Blanca/genéticaRESUMEN
MicroRNAs (miRNAs) are involved in the regulation of many cellular processes including hematopoiesis, with the aberrant expression of differentiation-stage specific miRNA associated with lymphomagenesis. miRNA profiling has been essential for understanding the underlying biology of many hematological malignancies; however the miRNA signature of the diverse tumor clone associated with Waldenstrom's macroglobulinemia (WM), consisting of B lymphocytes, plasmacytes and lymphoplasmacytic cells, has not been characterized. We have investigated the expression of over 13 000 known and candidate miRNAs in both CD19(+) and CD138(+) WM tumor cells, as well as in their malignant and non-malignant counterparts. Although neither CD19(+) nor CD138(+) WM cells were defined by a distinct miRNA profile, the combination of all WM cells revealed a unique miRNA transcriptome characterized by the dysregulation of many miRNAs previously identified as crucial for normal B-cell lineage differentiation. Specifically, miRNA-9(*)/152/182 were underexpressed in WM, whereas the expression of miRNA-21/125b/181a/193b/223/363 were notably increased (analysis of variance; P<0.0001). Future studies focusing on the effects of these dysregulated miRNAs will provide further insight into the mechanisms responsible for the pathogenesis of WM.
RESUMEN
The secretin receptor is prototypic of the class II family of G protein-coupled receptors, with a long extracellular amino-terminal domain containing six highly conserved Cys residues and one Cys residue (Cys(11)) that is present only in the most closely related family members. This domain is critical for function, with some component Cys residues believed to be involved in key disulfide bonds, although these have never been directly demonstrated. Here, we examine the functional importance of each of these residues and determine their involvement in disulfide bonds. Secretin binding was markedly diminished after treating cells with cell-impermeant reducing reagents, supporting the presence of important extracellular disulfide bonds. To determine whether the amino-terminal domain was covalently attached to the receptor body by disulfide linkage, a strategy was implemented that involved introduction of an acid-labile Asp-Pro sequence to enable specific cleavage at the boundary of these domains. Under nonreducing conditions, the amino terminus was released from the receptor body, supporting the absence of covalent association between these domains. Quantitative [(14)C]iodoacetamide incorporation into the isolated amino-terminal domain of the receptor in the absence and presence of chemical reduction established the ratio of free to total Cys residues as 1:7, consistent with three disulfide bonds. Mutagenesis of each of the amino-terminal Cys residues to Ala was tolerated only for Cys(11), suggesting that these bonds linked the conserved Cys residues. This was further supported by treatment of intact cells expressing wild-type or C11A mutant secretin receptor with a cell-impermeant sulfhydryl-reactive reagent. Thus, the functionally important amino terminus of the secretin receptor represents a structurally independent, highly folded, and disulfide-bonded domain, with a pattern that is likely critical and conserved throughout this receptor family.
Asunto(s)
Cisteína/química , Cistina/química , Disulfuros/metabolismo , Receptores de la Hormona Gastrointestinal/química , Animales , Células CHO , Cricetinae , Cisteína/genética , Cistina/genética , Disulfuros/química , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ratas , Receptores Acoplados a Proteínas G , Receptores de la Hormona Gastrointestinal/genéticaRESUMEN
The carboxyl-terminal domains of secretin family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within secretin and the prototypic secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant secretin receptors (V13M and V16M) and sequence analysis. We also developed a new probe incorporating a photolabile Bpa at position 26 of secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC(50) = 72 +/- 6 pm) and bound to the secretin receptor specifically and with high affinity (K(i) = 10.3 +/- 2.4 nm). It covalently labeled the secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36). These new insights, along with our recent report of contact between residue 6 within the amino-terminal half of secretin and this same amino-terminal region of this receptor (Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.