RESUMO
Jellyfish exhibit innovative swimming patterns that contribute to exploring the origins of animal locomotion. However, the genetic and cellular basis of these patterns remains unclear. Herein, we generated chromosome-level genome assemblies of two jellyfish species, Turritopsis rubra and Aurelia coerulea, which exhibit straight and free-swimming patterns, respectively. We observe positive selection of numerous genes involved in statolith formation, hair cell ciliogenesis, ciliary motility, and motor neuron function. The lineage-specific absence of otolith morphogenesis- and ciliary movement-related genes in T. rubra may be associated with homeostatic structural statocyst loss and straight swimming pattern. Notably, single-cell transcriptomic analyses covering key developmental stages reveal the enrichment of diapause-related genes in the cyst during reverse development, suggesting that the sustained diapause state favours the development of new polyps under favourable conditions. This study highlights the complex relationship between genetics, locomotion patterns and survival strategies in jellyfish, thereby providing valuable insights into the evolutionary lineages of movement and adaptation in the animal kingdom.
Assuntos
Cifozoários , Análise de Célula Única , Natação , Animais , Cifozoários/genética , Cifozoários/fisiologia , Diapausa/genética , Genômica/métodos , Genoma/genética , Transcriptoma , Perfilação da Expressão GênicaRESUMO
mTORC1 is aberrantly activated in renal cell carcinoma (RCC) and is targeted by rapalogs. As for other targeted therapies, rapalogs clinical utility is limited by the development of resistance. Resistance often results from target mutation, but mTOR mutations are rarely found in RCC. As in humans, prolonged rapalog treatment of RCC tumorgrafts (TGs) led to resistance. Unexpectedly, explants from resistant tumors became sensitive both in culture and in subsequent transplants in mice. Notably, resistance developed despite persistent mTORC1 inhibition in tumor cells. In contrast, mTORC1 became reactivated in the tumor microenvironment (TME). To test the role of the TME, we engineered immunocompromised recipient mice with a resistance mTOR mutation (S2035T). Interestingly, TGs became resistant to rapalogs in mTORS2035T mice. Resistance occurred despite mTORC1 inhibition in tumor cells and could be induced by coculturing tumor cells with mutant fibroblasts. Thus, enforced mTORC1 activation in the TME is sufficient to confer resistance to rapalogs. These studies highlight the importance of mTORC1 inhibition in nontumor cells for rapalog antitumor activity and provide an explanation for the lack of mTOR resistance mutations in RCC patients.
Assuntos
Carcinoma de Células Renais , Resistencia a Medicamentos Antineoplásicos , Neoplasias Renais , Alvo Mecanístico do Complexo 1 de Rapamicina , Serina-Treonina Quinases TOR , Animais , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Camundongos , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Sirolimo/farmacologia , Mutação , Inibidores de MTOR/farmacologia , Inibidores de MTOR/uso terapêuticoRESUMO
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
Assuntos
Técnicas Biossensoriais , Proteínas Proto-Oncogênicas c-akt , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas Biossensoriais/métodosRESUMO
PURPOSE: HIF2α is a key driver of kidney cancer. Using a belzutifan analogue (PT2399), we previously showed in tumorgrafts (TG) that â¼50% of clear cell renal cell carcinomas (ccRCC) are HIF2α dependent. However, prolonged treatment induced resistance mutations, which we also identified in humans. Here, we evaluated a tumor-directed, systemically delivered, siRNA drug (siHIF2) active against wild-type and resistant-mutant HIF2α. EXPERIMENTAL DESIGN: Using our credentialed TG platform, we performed pharmacokinetic and pharmacodynamic analyses evaluating uptake, HIF2α silencing, target gene inactivation, and antitumor activity. Orthogonal RNA-sequencing studies of siHIF2 and PT2399 were pursued to define the HIF2 transcriptome. Analyses were extended to a TG line generated from a study biopsy of a siHIF2 phase I clinical trial (NCT04169711) participant and the corresponding patient, an extensively pretreated individual with rapidly progressive ccRCC and paraneoplastic polycythemia likely evidencing a HIF2 dependency. RESULTS: siHIF2 was taken up by ccRCC TGs, effectively depleted HIF2α, deactivated orthogonally defined effector pathways (including Myc and novel E2F pathways), downregulated cell cycle genes, and inhibited tumor growth. Effects on the study subject TG mimicked those in the patient, where HIF2α was silenced in tumor biopsies, circulating erythropoietin was downregulated, polycythemia was suppressed, and a partial response was induced. CONCLUSIONS: To our knowledge, this is the first example of functional inactivation of an oncoprotein and tumor suppression with a systemic, tumor-directed, RNA-silencing drug. These studies provide a proof-of-principle of HIF2α inhibition by RNA-targeting drugs in ccRCC and establish a paradigm for tumor-directed RNA-based therapeutics in cancer.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Policitemia , Animais , Humanos , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/patologia , RNA Interferente Pequeno/genética , Ensaios Clínicos Fase I como AssuntoRESUMO
Metastasis is the principal cause of cancer related deaths. Tumor invasion is essential for metastatic spread. However, determinants of invasion are poorly understood. We addressed this knowledge gap by leveraging a unique attribute of kidney cancer. Renal tumors invade into large vessels forming tumor thrombi (TT) that migrate extending sometimes into the heart. Over a decade, we prospectively enrolled 83 ethnically-diverse patients undergoing surgical resection for grossly invasive tumors at UT Southwestern Kidney Cancer Program. In this study, we perform comprehensive histological analyses, integrate multi-region genomic studies, generate in vivo models, and execute functional studies to define tumor invasion and metastatic competence. We find that invasion is not always associated with the most aggressive clone. Driven by immediate early genes, invasion appears to be an opportunistic trait attained by subclones with diverse oncogenomic status in geospatial proximity to vasculature. We show that not all invasive tumors metastasize and identify determinants of metastatic competency. TT associated with metastases are characterized by higher grade, mTOR activation and a particular immune contexture. Moreover, TT grade is a better predictor of metastasis than overall tumor grade, which may have implications for clinical practice.
Assuntos
Carcinoma de Células Renais/secundário , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Trombose/genética , Idoso , Animais , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Feminino , Humanos , Rim/irrigação sanguínea , Rim/patologia , Neoplasias Renais/complicações , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Estudos Prospectivos , RNA-Seq , Fatores de Risco , Trombose/patologia , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.
Assuntos
Nanopartículas , Difusão , Portadores de Fármacos , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Dióxido de SilícioRESUMO
T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Células COS , Cálcio/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Análise por Conglomerados , Citosol/metabolismo , Difusão , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Luz , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Optogenética , Fosforilação , Receptores de Antígenos de Linfócitos T/química , Espectrometria de FluorescênciaRESUMO
Renal cell carcinoma (RCC) is characterized by a particularly broad metastatic swath, and, enigmatically, when the pancreas is a destination, the disease is associated with improved survival. Intrigued by this observation, we sought to characterize the clinical behavior, therapeutic implications, and underlying biology. While pancreatic metastases (PM) are infrequent, we identified 31 patients across 2 institutional cohorts and show that improved survival is independent of established prognostic variables, that these tumors are exquisitely sensitive to antiangiogenic agents and resistant to immune checkpoint inhibitors (ICIs), and that they are characterized by a distinctive biology. Primary tumors of patients with PM exhibited frequent PBRM1 mutations, 3p loss, and 5q amplification, along with a lower frequency of aggressive features such as BAP1 mutations and loss of 9p, 14q, and 4q. Gene expression analyses revealed constrained evolution with remarkable uniformity, reduced effector T cell gene signatures, and increased angiogenesis. Similar findings were observed histopathologically. Thus, RCC metastatic to the pancreas is characterized by indolent biology, heightened angiogenesis, and an uninflamed stroma, likely underlying its good prognosis, sensitivity to antiangiogenic therapies, and refractoriness to ICI. These data suggest that metastatic organotropism may be an indicator of a particular biology with prognostic and treatment implications for patients.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/secundário , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Receptores de Antígenos de Linfócitos T , Humanos , Células Jurkat , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
PURPOSE: The heterodimeric transcription factor HIF-2 is arguably the most important driver of clear cell renal cell carcinoma (ccRCC). Although considered undruggable, structural analyses at the University of Texas Southwestern Medical Center (UTSW, Dallas, TX) identified a vulnerability in the α subunit, which heterodimerizes with HIF1ß, ultimately leading to the development of PT2385, a first-in-class inhibitor. PT2385 was safe and active in a first-in-human phase I clinical trial of patients with extensively pretreated ccRCC at UTSW and elsewhere. There were no dose-limiting toxicities, and disease control ≥4 months was achieved in 42% of patients. PATIENTS AND METHODS: We conducted a prospective companion substudy involving a subset of patients enrolled in the phase I clinical trial at UTSW (n = 10), who were treated at the phase II dose or above, involving multiparametric MRI, blood draws, and serial biopsies for biochemical, whole exome, and RNA-sequencing studies. RESULTS: PT2385 inhibited HIF-2 in nontumor tissues, as determined by a reduction in erythropoietin levels (a pharmacodynamic marker), in all but one patient, who had the lowest drug concentrations. PT2385 dissociated HIF-2 complexes in ccRCC metastases, and inhibited HIF-2 target gene expression. In contrast, HIF-1 complexes were unaffected. Prolonged PT2385 treatment resulted in the acquisition of resistance, and we identified a gatekeeper mutation (G323E) in HIF2α, which interferes with drug binding and precluded HIF-2 complex dissociation. In addition, we identified an acquired TP53 mutation elsewhere, suggesting a possible alternate mechanism of resistance. CONCLUSIONS: These findings demonstrate a core dependency on HIF-2 in metastatic ccRCC and establish PT2385 as a highly specific HIF-2 inhibitor in humans. New approaches will be required to target mutant HIF-2 beyond PT2385 or the closely related PT2977 (MK-6482).
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Indanos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Sulfonas/uso terapêutico , Idoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Ensaios Clínicos Fase I como Assunto , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Imageamento por Ressonância Magnética Multiparamétrica/métodos , Estudos ProspectivosRESUMO
Advances in single-cell RNA sequencing (scRNA-Seq) have allowed for comprehensive analyses of single cell data. However, current analyses of scRNA-Seq data usually start from unsupervised clustering or visualization. These methods ignore prior knowledge of transcriptomes and the probable structures of the data. Moreover, cell identification heavily relies on subjective and possibly inaccurate human inspection afterwards. To address these analytical challenges, we developed SCINA (Semi-supervised Category Identification and Assignment), a semi-supervised model that exploits previously established gene signatures using an expectation-maximization (EM) algorithm. SCINA is applicable to scRNA-Seq and flow cytometry/CyTOF data, as well as other data of similar format. We applied SCINA to a wide range of datasets, and showed its accuracy, stability and efficiency, which exceeded most popular unsupervised approaches. SCINA discovered an intermediate stage of oligodendrocytes from mouse brain scRNA-Seq data. SCINA also detected immune cell population changes in cytometry data in a genetically-engineered mouse model. Furthermore, SCINA performed well with bulk gene expression data. Specifically, we identified a new kidney tumor clade with similarity to FH-deficient tumors (FHD), which we refer to as FHD-like tumors (FHDL). Overall, SCINA provides both methodological advances and biological insights from perspectives different from traditional analytical methods.
Assuntos
Algoritmos , Carcinoma de Células Renais/genética , Técnicas Citológicas , Neoplasias Renais/genética , RNA Neoplásico , Análise de Sequência de RNA/métodos , Animais , Carcinoma de Células Renais/patologia , Simulação por Computador , Humanos , Neoplasias Renais/patologia , Camundongos , Camundongos KnockoutRESUMO
The essential function of the T cell receptor (TCR) is to translate the engagement of peptides on the major histocompatibility complex (pMHC) into appropriate intracellular signals through the associated cluster of differentiation 3 (CD3) complex. The spatial organization of the TCR-CD3 complex in the membrane is thought to be a key regulatory element of signal transduction, raising the question of how receptor clustering impacts on TCR triggering. How signal transduction at the TCR-CD3 complex encodes the quality and quantity of pMHC molecules is not fully understood. This question can be approached by reconstituting T cell signaling in model and cell membranes and addressed by single-molecule imaging of endogenous proteins in T cells. We highlight such methods and further discuss how TCR clustering could affect pMHC rebinding rates, the local balance between kinase and phosphatase activity and/or the lipid environment to regulate the signal efficiency of the TCR-CD3 complex. We also examine whether clustering could affect the conformation of cytoplasmic CD3 tails through a biophysical mechanism. Taken together, we highlight how the spatial organization of the TCR-CD3 complex - addressed by reconstitution approaches - has emerged as a key regulatory element in signal transduction of this archetypal immune receptor.
Assuntos
Complexo CD3/imunologia , Complexo Principal de Histocompatibilidade , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/química , Complexo CD3/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Modelos Biológicos , Ligação Proteica , Transporte Proteico , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem Individual de Molécula/métodos , Linfócitos T/metabolismo , Linfócitos T/ultraestruturaRESUMO
The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (FeII-IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an FeII-nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M-1 s-1 (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M-1 s-1; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1's nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1's nitrite reductase activity may have important implications for the enzyme's biological actions when expressed within hypoxic tissues.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Nitrito Redutases/metabolismo , Anaerobiose , Espectroscopia de Ressonância de Spin Eletrônica , Heme/química , Heme/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Nitrito Redutases/química , Nitritos/química , Nitritos/metabolismo , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1ß) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1ß, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Indanos/farmacologia , Indanos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Sulfonas/farmacologia , Sulfonas/uso terapêutico , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Eritropoetina/antagonistas & inibidores , Eritropoetina/sangue , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Indanos/administração & dosagem , Indóis/farmacologia , Indóis/uso terapêutico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Terapia de Alvo Molecular , Mutação , Pirróis/farmacologia , Pirróis/uso terapêutico , Reprodutibilidade dos Testes , Sulfonas/administração & dosagem , Sunitinibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.
Assuntos
Antígenos/imunologia , Complexo CD3/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Imunidade Adaptativa/genética , Animais , Antígenos/genética , Humanos , Camundongos , Peptídeos/imunologia , Fosforilação/imunologia , Transdução de Sinais , Imagem Individual de MoléculaRESUMO
During vertebrate embryogenesis, the neuroectoderm is induced from dorsal ectoderm and then partitioned into anterior and posterior neuroectodermal domains by posteriorizing signals, such as Wnt and fibroblast growth factor. However, little is known about epigenetic regulation of posteriorizing gene expression. Here, we report a requirement of the chromatin remodeling protein Bptf for neuroectodermal posteriorization in zebrafish embryos. Knockdown of bptf leads to an expansion of the anterior neuroectoderm at the expense of the posterior ectoderm. Bptf functionally and physically interacts with p-Smad2, which is activated by non-Nodal TGF-ß signaling, to promote the expression of wnt8a, a critical gene for neural posteriorization. Bptf and Smad2 directly bind to and activate the wnt8a promoter through recruiting NURF remodeling complex. When bptf function or TGF-ß signal transduction is inhibited, the nucleosome density on the wnt8a promoter is increased. We propose that Bptf and TGF-ß/Smad2 mediate nucleosome remodeling to regulate wnt8a expression and hence neural posteriorization.