RESUMEN
PURPOSE: Tumors activate protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK, also called EIF2AK3) in response to hypoxia and nutrient deprivation as a stress-mitigation strategy. Here, we tested the hypothesis that inhibiting PERK with HC-5404 enhances the antitumor efficacy of standard-of-care VEGF receptor tyrosine kinase inhibitors (VEGFR-TKI). EXPERIMENTAL DESIGN: HC-5404 was characterized as a potent and selective PERK inhibitor, with favorable in vivo properties. Multiple renal cell carcinoma (RCC) tumor models were then cotreated with both HC-5404 and VEGFR-TKI in vivo, measuring tumor volume across time and evaluating tumor response by protein analysis and IHC. RESULTS: VEGFR-TKI including axitinib, cabozantinib, lenvatinib, and sunitinib induce PERK activation in 786-O RCC xenografts. Cotreatment with HC-5404 inhibited PERK in tumors and significantly increased antitumor effects of VEGFR-TKI across multiple RCC models, resulting in tumor stasis or regression. Analysis of tumor sections revealed that HC-5404 enhanced the antiangiogenic effects of axitinib and lenvatinib by inhibiting both new vasculature and mature tumor blood vessels. Xenografts that progress on axitinib monotherapy remain sensitive to the combination treatment, resulting in â¼20% tumor regression in the combination group. When tested across a panel of 18 RCC patient-derived xenograft (PDX) models, the combination induced greater antitumor effects relative to monotherapies. In this single animal study, nine out of 18 models responded with ≥50% tumor regression from baseline in the combination group. CONCLUSIONS: By disrupting an adaptive stress response evoked by VEGFR-TKI, HC-5404 presents a clinical opportunity to improve the antitumor effects of well-established standard-of-care therapies in RCC.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Animales , Humanos , Carcinoma de Células Renales/patología , Axitinib/farmacología , Axitinib/uso terapéutico , Neoplasias Renales/patología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Grape (Vitis vinifera L.) in production is frequently exposed to inadequate light, which significantly affects its agronomic traits via inhibiting their physiological, metabolic and developmental processes. To explore the mechanism how the grape plants respond to the weak light stress, we used 'Yinhong' grape and examined their physiology-biochemistry characteristics and transcriptional profile under different levels of weak light stress. The results showed that grape seedlings upon low intensity shading treatments were not significantly affected. As the shading stress intensity was strengthened, the epidermis cells, palisade tissue, and spongy tissue in the leaves were thinner, the intercellular space between the palisade tissue and spongy tissue was larger compared with that of the control, and the activities of superoxide dismutase, catalase and peroxidase were decreased gradually. Additionally, the soluble protein content increased and the free proline content decreased gradually. Compared with the control, significant changes in plant photosynthetic characteristics and physiology-biochemistry characteristics were observed under high intensity of shading (80%). RNA-seq data showed that the differentially expressed genes between CK and T2, CK and T4, T2 and T4 were 13 913, 13 293 and 14 943, respectively. Most of the enrichment pathways were closely related with the plant's response to stress. Several signaling pathways in response to stress-resistance, e.g. JA/MYC2 pathway and MAPK signal pathway, were activated under weak light stress. The expression level of a variety of genes related to antioxidation (such as polyphenol oxidase and thioredoxin), photosynthesis (such as phytochrome) was altered under weak light stress, indicating that 'Yinhong' grape may activate the antioxidation related pathways to cope with reactive oxygen species (ROS). In addition, it may activate the expression of photosynthetic pigment and light reaction structural protein to maintain the photosynthesis activity. This research may help better understand the relevant physiological response mechanism and facilitate cultivation of grape seedlings under weak light.
Asunto(s)
Vitis , Vitis/genética , Vitis/metabolismo , Regulación de la Expresión Génica de las Plantas , Fotosíntesis/genética , Hojas de la Planta , Luz , Plantones/metabolismoRESUMEN
The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Activación de Linfocitos/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Apoptosis , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. RESULTS: Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. CONCLUSIONS: LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.
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Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Inmunoglobulina G/inmunología , Neoplasias/inmunología , Animales , Línea Celular , Cricetulus , Femenino , Humanos , Macaca fascicularis , Ratones , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Unfortunately, after publication of this article [1], it was noticed that corrections to the legends of Figs. 1 and 2 were not correctly incorporated. The correct legends can be seen below.
RESUMEN
Upon viral infection, NF-kappaB translocates to the nucleus and activates the IFN-beta gene by binding to the PRDII element. Strikingly, NF-kappaB loses its ability to activate the IFN-beta gene when the PRDII element is substituted by closely related sites. We report here the crystal structure of NF-kappaB p50/p65 heterodimer bound to the PRDII element from the IFN-beta promoter. The structure reveals an unexpected alteration in configuration, in which the p50 specificity domain moves by as much as approximately 9 A when compared to NF-kappaB heterodimer bound to the immunoglobulin kappaB site (Ig-kappaB) while maintaining the same base-specific contacts with the DNA. Taken together, the structure offers new insights into the allosteric effects of closely related DNA sites on the configuration of NF-kappaB and its transcriptional selectivity.
Asunto(s)
ADN/química , Interferón beta/genética , FN-kappa B/química , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Estructura Cuaternaria de Proteína , Cristalización , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Interferón beta/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Subunidades de ProteínaRESUMEN
The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.
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Inhibidores de la Angiogénesis/química , Anticuerpos Monoclonales/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales Humanizados , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , RamucirumabRESUMEN
Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the assembly of the mammalian interferon-beta (IFN-beta) enhanceosome. We present here the structure of IRF-3 DNA binding domain in complex with the complete PRDIII-I regulatory element of the human IFN-beta enhancer. We show that four IRF-3 molecules bind in tandem to, variably spaced, consensus and nonconsensus IRF sites on the composite element. The ability of IRF-3 to bind these variable sites derives in part from two nonconserved arginines (Arg78 and Arg86) that partake in alternate protein-DNA contacts. We also show that the protein-DNA contacts are highly overlapped and that all four IRF sites are required for gene activation in vivo. In addition, we show that changing the nonconsensus IRF sites to consensus sites creates a more efficient enhancer in vivo. Together, the structure and accompanying biological data provide insights into the assembly of the IFN-beta enhanceosome in mammals.
Asunto(s)
Elementos de Facilitación Genéticos , Factor 3 Regulador del Interferón/química , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina/química , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Secuencia de Consenso , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Factor 3 Regulador del Interferón/genética , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The Ets and IRF transcription factor families contain structurally divergent members, PU.1, Spi-B and IRF-4 (Pip), IRF-8 (ICSBP), respectively, which have evolved to cooperatively assemble on composite DNA elements and regulate gene expression in the immune system. Whereas PU.1 recruits IRF-4 or IRF-8 to DNA, it exhibits an anticooperative interaction with IRF-1 and IRF-2. We report here the structure of the ternary complex formed with the DNA binding domains of PU.1 and IRF-4 on a composite DNA element. The DNA in the complex contorts into an unusual S shape that juxtaposes PU.1 and IRF-4 for selective electrostatic and hydrophobic interactions across the central minor groove. Together, the protein-protein and protein-DNA interactions provide insights into the stereochemical basis of cooperativity and anti-cooperativity between Ets and IRF factors.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Proteínas Proto-Oncogénicas/química , Transactivadores/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores Reguladores del Interferón , Ratones , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
PU.1 and IRF-4, members of the Ets and interferon regulatory families of transcription factors, respectively, form a cooperative ternary complex that is implicated in the regulation of B-cell-specific gene expression. A portion of the cooperativity involves interaction between the PU.1 and IRF-4 DNA-binding domains. We report here the crystallization and preliminary characterization of PU.1 and IRF-4 DNA-binding domains bound to a 21-mer DNA site from the lambdaB element of immunoglobulin light chain lambda(2-4) enhancer. The crystals belong to space group P2(1) with unit cell dimensions of a=40.7A, b=62.3A, c=67.9A, beta=95.6 degrees with one complex molecule per asymmetric unit. Crystals diffract to a resolution of 3A using X-rays from a rotating anode generator but improve to 2.3A with synchrotron radiation. The crystals are highly mosaic, but the mosaicity can be improved following a series of steps involving the addition of additives, use of peritone oil as a cryoprotectant, slow flash-cooling in the cryostream, and several cycles of crystal annealing.