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INTRODUCTION: Respiratory syncytial virus (RSV) leads to significant morbidity in newborn infants in the United Kingdom (UK). Nirsevimab, a long-acting monoclonal antibody, received approval from the European Medicines Agency and has been licensed by the Medicines and Healthcare products Regulatory Agency for preventing RSV lower respiratory tract disease (LRTD) in neonates and infants during their first RSV season. The objective of this study was to assess the potential impact of nirsevimab on RSV-associated LRTDs, related costs, and loss of quality-adjusted life years (QALYs) in infants experiencing their first RSV season. METHODS: The impact of administering nirsevimab across all infant populations compared to palivizumab in the high-risk palivizumab-eligible population was assessed via a static decision-analytic model specified for a UK birth cohort experiencing their first RSV season. The RSV-related health events of interest included primary care (PC), accident and emergency (A&E) visits, hospitalizations [including hospitalizations alone and those resulting in intensive care unit (ICU) admissions], recurrent wheezing in infants who were previously hospitalized, and all-cause LRTD hospitalizations. RESULTS: Under the current standard of practice (SoP), RSV was estimated to result in 329,425 RSV LRTDs annually, including 24,381 hospitalizations and ICU admissions, representing £117.8 million (2024 GBP) in costs. Comparatively, universal immunization of all infants with nirsevimab could avoid 198,886 RSV LRTDs, including 16,657 hospitalizations and ICU admissions, resulting in savings of £77.2 million in RSV treatment costs. Considering the impact on all-cause LRTD of a universal immunization strategy, nirsevimab could be valued between £243 and £274, assuming willingness-to-pay (WTP) thresholds of £20,000 and £30,000 per QALY saved, respectively. CONCLUSIONS: This analysis demonstrated that the health and economic burden of RSV would be substantially reduced in all infants experiencing their first RSV season in the UK (including term, preterm, and palivizumab-eligible infants) as a result of a universal immunization strategy with nirsevimab.
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Background: Glioblastoma (GBM) is one of the most aggressive human brain tumors, with a median survival of 15-18 months. There is a desperate need to find novel therapeutic targets. Various receptor protein kinases have been identified as potential targets; however, response rates in clinical studies have been somewhat disappointing. Targeting the spleen tyrosine kinase (SYK), which acts downstream of a range of oncogenic receptors, may therefore show more promising results. Methods: Kinase expression of brain tumor samples including GBM and low-grade tumors were compared with normal brain and normal human astrocytes by microarray analysis. Furthermore, SYK, LYN, SLP76, and PLCG2 protein expressions were analyzed by immunohistochemistry, western blot, and immunofluorescence of additional GBM patient samples, murine glioma samples, and cell lines. SYK was then blocked chemically and genetically in vitro and in vivo in 2 different mouse models. Multiphoton intravital imaging and multicolor flow cytometry were performed in a syngeneic immunocompetent C57BL/6J mouse GL261 glioma model to study the effect of these inhibitors on the tumor microenvironment. Results: SYK, LYN, SLP76, and PLCG2 were found expressed in human and murine glioma samples and cell lines. SYK inhibition blocked proliferation, migration, and colony formation. Flow cytometric and multiphoton imaging imply that targeting SYK in vivo attenuated GBM tumor growth and invasiveness and reduced B and CD11b+ cell mobility and infiltration. Conclusions: Our data suggest that gliomas express a SYK signaling network important in glioma progression, inhibition of which results in reduced invasion with slower tumor progression.
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Biomarcadores de Tumor/metabolismo , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Glioblastoma/patología , Quinasa Syk/metabolismo , Microambiente Tumoral , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Pronóstico , Quinasa Syk/genética , Células Tumorales CultivadasRESUMEN
Current standard-of-care treatment for malignant cancers includes radiotherapy and adjuvant chemotherapy. Here, we report increased MAP kinase-interacting kinase (MNK)-regulated phosphorylation of translation initiation factor 4E (eIF4E) in glioma cells upon temozolomide (TMZ) treatment and in medullary thyroid carcinoma (MTC) cells in response to targeted radionuclide therapy. Depletion of MNK activity by using two MNK inhibitors, CGP57380 or cercosporamide, as well as by MNK1-specific knockdown sensitized glioblastoma (GBM) cells and GBM-derived spheres to TMZ. Furthermore, CGP57380 treatment enhanced response of MTC cells to (177)Lu-labeled gastrin analogue. In order to understand how MNK signaling pathways support glioma survival we analyzed putative MNK substrates by quantitative phosphoproteomics in normal condition and in the presence of TMZ. We identified MNK inhibitor-sensitive phosphorylation sites on eIF4G1, mutations of which either influenced eIF4E phosphorylation or glioma cell response to TMZ, pointing to altered regulation of translation initiation as a resistance mechanism. Pharmacological inhibition of overexpressed MNK1 by CGP57380 reduced eIF4E phosphorylation and induced association of inactive MNK1 with eIF4G1. Taken together, our data show an activation of MNK-mediated survival mechanisms in response to either glioma chemotherapy or MTC targeted radiation and suggest that inhibition of MNK activity represents an attractive sensitizing strategy for cancer treatments.
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Antineoplásicos/uso terapéutico , Dacarbazina/análogos & derivados , Glioma/tratamiento farmacológico , Glioma/radioterapia , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Radioisótopos/uso terapéutico , Transducción de Señal , Compuestos de Anilina , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Gastrinas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lutecio , Fosfoproteínas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica , Purinas , Transducción de Señal/efectos de los fármacos , TemozolomidaRESUMEN
MICA is a major ligand for the NKG2D immune receptor, which plays a key role in activating natural killer (NK) cells and cytotoxic T cells. We analyzed NKG2D ligand expression on a range of cell types and could demonstrate that MICA expression levels were closely linked to cellular growth mode. While the expression of other NKG2D ligands was largely independent of cell growth mode, MICA expression was mainly found on cells cultured as adherent cells. In addition, MICA surface expression was reduced through increase in cell-cell contact or loss of cell-matrix adherence. Furthermore, we found that the reduction in MICA expression was modulated by focal adhesion kinase (FAK)/Src signaling and associated with increased susceptibility to NK cell-mediated killing. While the mechanisms of tumor immune evasion are not fully understood, the reduction of MICA expression following loss of attachment poises a potential way by which metastasizing tumor cells avoid immune detection. The role of FAK/Src in this process indicates a potential therapeutic approach to modulate MICA expression and immune recognition of tumor cells during metastasis.
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High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.
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Neoplasias Encefálicas/tratamiento farmacológico , Factor 4E Eucariótico de Iniciación/metabolismo , Glioma/tratamiento farmacológico , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Compuestos de Anilina/química , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Humanos , Inmunohistoquímica , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Ratones Desnudos , Trasplante de Neoplasias , Fosforilación , Unión Proteica , Biosíntesis de Proteínas , Purinas/química , Transducción de Señal , Sirolimus/análogos & derivadosRESUMEN
Glioblastoma (GBM) is a highly malignant primary tumor of the central nervous system originating in glial cells. GBM results in more years of life lost than any other cancer type. Low levels of Notch receptor expression correlates with prolonged survival in various high grade gliomas independent of other markers. Different downstream pathways of Notch receptors have been identified. We tested if the Notch/Deltex pathway, which is distinct from the canonical, CSL-mediated pathway, has a role in GBM. We show that the alternative or non-canonical Notch pathway functioning through Deltex1 (DTX1) mediates key features of glioblastoma cell aggressiveness. For example, DTX1 activates the RTK/PI3K/PKB and the MAPK/ERK mitotic pathways and induces anti-apoptotic Mcl-1. The clonogenic and growth potential of established glioma cells correlated with DTX1 levels. Microarray gene expression analysis further identified a DTX1-specific, MAML1-independent transcriptional program - including microRNA-21- which is functionally linked to the changes in tumor cell aggressiveness. Over-expression of DTX1 increased cell migration and invasion correlating to ERK activation, miR-21 levels and endogenous Notch levels. In contrast to high and intermediate expressors, patients with low DTX1 levels have a more favorable prognosis. The alternative Notch pathway via DTX1 appears to be an oncogenic factor in glioblastoma and these findings offer new potential therapeutic targets.
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Proteínas de Unión al ADN/genética , Glioblastoma/genética , Glioblastoma/patología , Mitosis/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Expresión Génica , Glioblastoma/metabolismo , Humanos , MicroARNs/genética , Invasividad Neoplásica , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
Glioblastoma multiforme (GBM) is the most common aggressive brain cancer with a median survival of approximately 1 year. In a search for novel molecular targets that could be therapeutically developed, our kinome-focused microarray analysis identified the MAP (mitogen-activated protein) kinase-interacting kinase 1 (MNK1) as an attractive theranostic candidate. MNK1 overexpression was confirmed in both primary GBMs and glioma cell lines. Inhibition of MNK1 activity in GBM cells by the small molecule CGP57380 suppressed eIF4E phosphorylation, proliferation, and colony formation whereas concomitant treatment with CGP57380 and the mTOR inhibitor rapamycin accentuated growth inhibition and cell-cycle arrest. siRNA-mediated knockdown of MNK1 expression reduced proliferation of cells incubated with rapamycin. Conversely, overexpression of full-length MNK1 reduced rapamycin-induced growth inhibition. Analysis of polysomal profiles revealed inhibition of translation in CGP57380 and rapamycin-treated cells. Microarray analysis of total and polysomal RNA from MNK1-depleted GBM cells identified mRNAs involved in regulation of TGF-ß pathway. Translation of SMAD2 mRNA as well as TGF-ß-induced cell motility and vimentin expression was regulated by MNK1 signaling. Tissue microarray analysis revealed a positive correlation between the immunohistochemical staining of MNK1 and SMAD2. Taken together, our findings offer insights into how MNK1 pathways control translation of cancer-related mRNAs including SMAD2, a key component of the TGF-ß signaling pathway. Furthermore, they suggest MNK1-controlled translational pathways in targeted strategies to more effectively treat GBM.
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Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteína Smad2/genética , Factor de Crecimiento Transformador beta/metabolismo , Adolescente , Adulto , Anciano , Compuestos de Anilina/farmacología , Antibióticos Antineoplásicos/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Purinas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirolimus/farmacología , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The thymus serves as the primary site for the lifelong formation of new T lymphocytes; hence, it is essential for the maintenance of an effective immune system. Although thymocyte development has been widely studied, the mechanisms involved are incompletely defined. A comprehensive understanding of the molecular events that control regular thymocyte development will not only shed light on the physiological control of T cell differentiation but also probably provide insight into the pathophysiology of T cell immunodeficiencies, the molecular basis that underpins autoimmunity, and the mechanisms that instigate the formation of T cell lymphomas. Phosphatidylinositol 3-kinases (PI3Ks) play a critical role in thymocyte development, although not all of their downstream mediators have yet been identified. Here, we discuss experimental evidence that argues for a critical role of the PI3K-phosphoinositide-dependent protein kinase (PDK1)-protein kinase B (PKB) signaling pathway in the development of both normal and malignant thymocytes, and we highlight molecules that can potentially be targeted therapeutically.
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Células Madre Hematopoyéticas/fisiología , Modelos Inmunológicos , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Linfocitos T/fisiología , Timo/citología , Proliferación Celular , Células Madre Hematopoyéticas/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Linfocitos T/citologíaRESUMEN
The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.
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Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Multimerización de Proteína/fisiología , Línea Celular , Membrana Celular/metabolismo , Cromatografía en Gel , Cistina/análisis , Cistina/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoprecipitación , Células Jurkat , Lectinas Tipo C/genética , Luz , Espectrometría de Masas , Glicoproteínas de Membrana/genética , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/fisiología , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Dispersión de Radiación , Resonancia por Plasmón de Superficie , Transfección , Ultracentrifugación , Venenos de Víboras/químicaRESUMEN
The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.
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Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/análisis , Guanidina , Concentración de Iones de Hidrógeno , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Espectrometría de Fluorescencia/métodos , TermodinámicaRESUMEN
The chimaeric gene encoding a C-terminally truncated form of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 and the EGFP (enhanced green fluorescent protein) was ligated into plasmid pET28a and cloned and expressed in Escherichia coli. Just 1 h after induction of expression an intense EGFP fluorescence was detected in the cytoplasm of the host cells. Expression at 28 degrees C instead of 37 degrees C resulted in clearly increased fluorescence intensity, indicating that the folding process of the EGFP moiety was temperature sensitive. To maintain the EGFP fluorescence, isolation of the fusion protein from the host cells had to be performed in the presence of reducing agents. SDS/PAGE analysis, immunoblotting and N-terminal sequencing of the isolated and purified fusion protein confirmed the presence of both the S-layer protein and the EGFP moiety. The fusion protein had maintained the ability to self-assemble in suspension and to recrystallize on peptidoglycan-containing sacculi or on positively charged liposomes, as well as to fluoresce. Comparison of fluorescence excitation and emission spectra of recombinant EGFP and rSbpA(31-1068)/EGFP revealed identical maxima at 488 and 507 nm respectively. The uptake of liposomes coated with a fluorescent monomolecular protein lattice of rSbpA(31-1068)/EGFP into HeLa cells was studied by confocal laser-scanning microscopy. The major part of the liposomes was internalized within 2 h of incubation and entered the HeLa cells by endocytosis.