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Idiopathic pulmonary fibrosis (IPF) is an incurable interstitial lung disease characterized by fibrosis. Two FDA-approved drugs, pirfenidone and nintedanib, only modestly prolong survival. In this study, we asked whether levels of select circulating biomarkers in patients with IPF demonstrated changes in response to treatment over time and whether treatment with pirfenidone and nintedanib led to differential biomarker expression. Serial plasma samples from 48 patients with IPF on usual treatment and six healthy volunteers were analyzed to identify differentially expressed blood protein. Hypothesis-driven potential biomarker selection was based on recent literature, internal preclinical data, and the PROLIFIC Consortium (Schafer P. 6th Annual IPF Summit. Boston, MA, 2022) proposed biomarkers of pulmonary fibrosis. We compared our findings to public databases to provide insights into relevant signaling pathways in IPF. Of the 26 proteins measured, we found that 11 (SP-D, TIMP1, MMP7, CYFRA21-1, YKL40, CA125, sICAM, IP-10, MDC, CXCL13) were significantly elevated in patients with IPF compared with healthy volunteers but their levels did not significantly change over time. In the IPF samples, seven proteins were elevated in the treatment group compared with the no-treatment group. However, protein profiles were not distinguishable between patients on pirfenidone versus nintedanib. We demonstrated that most proteins differentially detected in our samples were predicted to be secreted from the lung epithelial or interstitial compartments. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. Understanding the contributions of the systemic response in IPF may be important as new therapeutics are developed.NEW & NOTEWORTHY In this study, we confirmed protein expression differences in only a subset of predicted biomarkers from IPF and control subjects. Most differentially expressed proteins were predicted to be secreted from lung cells. However, a significant minority of the proteins are not known to be transcriptionally expressed by lung cells, suggesting an ongoing systemic response. The contributions of the systemic response in IPF may be important as new therapeutics are developed.
Assuntos
Antígenos de Neoplasias , Fibrose Pulmonar Idiopática , Queratina-19 , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Fibrose , BiomarcadoresRESUMO
Immune checkpoint blockade (ICB) has led to durable clinical responses in multiple cancer types. However, biomarkers that identify which patients are most likely to respond to ICB are not well defined. Many putative biomarkers developed from a small number of samples often fail to maintain their predictive status in larger validation cohorts. We show across multiple human malignancies and syngeneic murine tumor models that neither pretreatment T cell receptor (TCR) clonality nor changes in clonality after ICB correlate with response. Dissection of tumor infiltrating lymphocytes pre- and post-ICB by paired single-cell RNA sequencing and single-cell TCR sequencing reveals conserved and distinct transcriptomic features in expanded TCR clonotypes between anti-PD1 responder and nonresponder murine tumor models. Overall, our results indicate a productive anti-tumor response is agnostic of TCR clonal expansion. Further, we used single-cell transcriptomics to develop a CD8+ T cell specific gene signature for a productive anti-tumor response and show the response signature to be associated with overall survival (OS) on nivolumab monotherapy in CheckMate-067, a phase 3 clinical trial in metastatic melanoma. These results highlight the value of leveraging single-cell assays to dissect heterogeneous tumor and immune subsets and define cell-type specific transcriptomic biomarkers of ICB response.
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Melanoma , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Humanos , Inibidores de Checkpoint Imunológico , Melanoma/tratamento farmacológico , Melanoma/genética , Camundongos , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
BACKGROUND: Single-cell (sc) sequencing performs unbiased profiling of individual cells and enables evaluation of less prevalent cellular populations, often missed using bulk sequencing. However, the scale and the complexity of the sc datasets poses a great challenge in its utility and this problem is further exacerbated when working with larger datasets typically generated by consortium efforts. As the scale of single cell datasets continues to increase exponentially, there is an unmet technological need to develop database platforms that can evaluate key biological hypotheses by querying extensive single-cell datasets. Large single-cell datasets like Human Cell Atlas and COVID-19 cell atlas (collection of annotated sc datasets from various human organs) are excellent resources for profiling target genes involved in human diseases and disorders ranging from oncology, auto-immunity, as well as infectious diseases like COVID-19 caused by SARS-CoV-2 virus. SARS-CoV-2 infections have led to a worldwide pandemic with massive loss of lives, infections exceeding 7 million cases. The virus uses ACE2 and TMPRSS2 as key viral entry associated proteins expressed in human cells for infections. Evaluating the expression profile of key genes in large single-cell datasets can facilitate testing for diagnostics, therapeutics, and vaccine targets, as the world struggles to cope with the on-going spread of COVID-19 infections. MAIN BODY: In this manuscript we describe REVEAL: SingleCell, which enables storage, retrieval, and rapid query of single-cell datasets inclusive of millions of cells. The array native database described here enables selecting and analyzing cells across multiple studies. Cells can be selected using individual metadata tags, more complex hierarchical ontology filtering, and gene expression threshold ranges, including co-expression of multiple genes. The tags on selected cells can be further evaluated for testing biological hypotheses. One such example includes identifying the most prevalent cell type annotation tag on returned cells. We used REVEAL: SingleCell to evaluate the expression of key SARS-CoV-2 entry associated genes, and queried the current database (2.2 Million cells, 32 projects) to obtain the results in < 60 s. We highlighted cells expressing COVID-19 associated genes are expressed on multiple tissue types, thus in part explains the multi-organ involvement in infected patients observed worldwide during the on-going COVID-19 pandemic. CONCLUSION: In this paper, we introduce the REVEAL: SingleCell database that addresses immediate needs for SARS-CoV-2 research and has the potential to be used more broadly for many precision medicine applications. We used the REVEAL: SingleCell database as a reference to ask questions relevant to drug development and precision medicine regarding cell type and co-expression for genes that encode proteins necessary for SARS-CoV-2 to enter and reproduce in cells.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/prevenção & controle , Receptores Virais/genética , SARS-CoV-2/genética , Serina Endopeptidases/genética , Análise de Célula Única/métodos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/epidemiologia , COVID-19/virologia , Células Cultivadas , Bases de Dados Genéticas , Epidemias , Perfilação da Expressão Gênica , Humanos , Receptores Virais/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Internalização do VírusRESUMO
The inflammatory CD40-CD40L pathway is implicated in various autoimmune diseases, but the activity status of this pathway in various stages of rheumatoid arthritis (RA) progression is unknown. In this study, we used gene signatures of CD40L stimulation derived from human immature dendritic cells and naive B cells to assess the expression of CD40-downstream genes in synovial tissues from anti-citrullinated protein Ab-positive arthralgia, undifferentiated arthritis (UA), early RA, and established RA cohorts in comparison with healthy donors. Interestingly, the expression of CD40LG and active full-length CD40 was increased in the disease tissues, whereas that of a dominant-negative CD40 isoform was decreased. Gene set variation analysis revealed that CD40L-responsive genes in immature dendritic cells and naive B cells were significantly enriched in synovial tissues from UA, early RA, and established RA patients. Additionally, CD40L-induced naive B cell genes were also significantly enriched in synovial tissues from arthralgia patients. In our efforts to characterize downstream mediators of CD40L signaling, we have identified GPR120 and KDM6B as novel components of the pathway. In conclusion, our data suggest that therapeutic CD40-CD40L blocking agents may prove efficacious not only in early and established RA, but also in inhibiting the progression of the disease from arthralgia or UA to RA.
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Artrite Reumatoide/imunologia , Artrite/imunologia , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Progressão da Doença , Transdução de Sinais , Adulto , Idoso , Artralgia/imunologia , Artralgia/fisiopatologia , Artrite Reumatoide/fisiopatologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Biópsia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/deficiência , Antígenos CD40/genética , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/farmacologia , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Feminino , Voluntários Saudáveis , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Líquido Sinovial/citologia , Líquido Sinovial/imunologia , TranscriptomaRESUMO
Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3' splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-of-function mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner.
Assuntos
Genes p53 , Metástase Neoplásica/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo , Animais , Antígeno CD24/metabolismo , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Transição Epitelial-Mesenquimal/genética , Humanos , Receptores de Hialuronatos/metabolismo , Íntrons , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Mitocôndrias/metabolismo , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNA , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The duration and specificity of epidermal growth factor receptor (EGFR) activation and signaling are determinants of cellular decision processes and are tightly regulated by receptor dephosphorylation, internalization and degradation. In addition, regulatory proteins that are upregulated or activated post-transcriptionally upon receptor activation may initiate feedback loops that play crucial roles in spatiotemporal regulation of signaling. We examined the roles of Sprouty2 (SPRY2) and mitogen-inducible gene 6 (MIG6), two feedback regulators of EGFR trafficking and signaling, in lung cancer cells with or without EGFR-activating mutations. These mutations are of interest because they confer unusual cellular sensitivity to EGFR inhibition through a mechanism involving an impairment of EGFR endocytosis. We found that the endocytosis of wild-type and mutant EGFR was promoted by SPRY2 knockdown and antagonized by MIG6 knockdown. SPRY2 knockdown also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation, EGFR expression, and EGFR recycling. In a cell line expressing mutant EGFR, this effect on ERK led to a marked increase in cell death response to EGFR inhibition. The effects of SPRY2 knockdown on EGFR endocytosis and recycling were primarily the result of the concomitant change in EGFR expression, but this was not true for the observed changes in ERK phosphorylation. Thus, our study demonstrates that SPRY2 and MIG6 are important regulators of wild-type and mutant EGFR trafficking and points to an EGFR expression-independent function of SPRY2 in the regulation of ERK activity that may impact cellular sensitivity to EGFR inhibitors, especially in the context of EGFR mutation.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endocitose/fisiologia , Receptores ErbB/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Endocitose/genética , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Masculino , Proteínas de Membrana/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genéticaRESUMO
Background: Tumor mutational burden (TMB) has been proposed as a predictive biomarker of response to immunotherapy. Efforts to standardize TMB scores for use in the clinic and to identify the factors that could impact TMB scores are of high importance. However, the biopsy collection site has not been assessed as a factor that may influence TMB scores. Methods: We examine a real-world cohort comprising 137,771 specimens across 47 tissues in 12 indications profiled by the FoundationOne assay (Foundation Medicine, Cambridge, MA) to assess the prevalence of biopsy sites for each indication and their TMB scores distribution. Results: We observe a wide variety of biopsy sites from which specimens are sent for genomic testing and show that TMB scores differ in a cancer- and tissue-specific manner. For example, brain or adrenal gland specimens from NSCLC patients show higher TMB scores than local lung specimens (mean difference 3.31 mut/Mb; p < 0.01, 3.90 mut/Mb; p < 0.01, respectively), whereas bone specimens show no difference. Conclusions: Our data shed light on the biopsied tissue as a driver of TMB measurement variability in clinical practice.
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BACKGROUND: Nivolumab is an immune checkpoint inhibitor targeting the programmed death-1 receptor that improves survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). In contrast to other tumor types that respond to immunotherapy, factors such as programmed death ligand-1 (PD-L1) status and tumor mutational burden show limited predictive utility in ccRCC. To address this gap, we report here the first molecular characterization of nivolumab response using paired index lesions, before and during treatment of metastatic ccRCC. METHODS: We analyzed gene expression and T-cell receptor (TCR) clonality using lesion-paired biopsies provided in the CheckMate 009 trial and integrated the results with their PD-L1/CD4/CD8 status, genomic mutation status and serum cytokine assays. Statistical tests included linear mixed models, logistic regression models, Fisher's exact test, and Kruskal-Wallis rank-sum test. RESULTS: We identified transcripts related to response, both at baseline and on therapy, including several that are amenable to peripheral bioassays or to therapeutic intervention. At both timepoints, response was positively associated with T-cell infiltration but not associated with TCR clonality, and some non-Responders were highly infiltrated. Lower baseline T-cell infiltration correlated with elevated transcription of Wnt/ß-catenin signaling components and hypoxia-regulated genes, including the Treg chemoattractant CCL28. On treatment, analysis of the non-responding patients whose tumors were highly T-cell infiltrated suggests association of the RIG-I-MDA5 pathway in their nivolumab resistance. We also analyzed our data using previous transcriptional classifications of ccRCC and found they concordantly identified a molecular subtype that has enhanced nivolumab response but is sunitinib-resistant. CONCLUSION: Our study describes molecular characteristics of response and resistance to nivolumab in patients with metastatic ccRCC, potentially impacting patient selection and first-line treatment decisions. TRIAL REGISTRATION NUMBER: NCT01358721.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Nivolumabe/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Antígeno B7-H1/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Antígenos CD4/genética , Antígenos CD8/genética , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Citocinas/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Neoplasias Renais/sangue , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Mutação , Nivolumabe/efeitos adversos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
INTRODUCTION: Somatic mutations in STK11 and KEAP1, frequently comutated in non-squamous non-small cell lung cancer (NSQ NSCLC), have been associated with poor response to immune checkpoint blockade (ICB). However, previous reports lack non-ICB controls needed to properly ascertain the predictive nature of those biomarkers. The objective of this study was to evaluate the predictive versus prognostic effect of STK11 or KEAP1 mutations in NSQ NSCLC. METHODS: Patients diagnosed with stage IIIB, IIIC, IVA or IVB NSQ NSCLC from a real-world data cohort from the Flatiron Health Network linked with genetic testing from Foundation Medicine were retrospectively assessed. Real-world, progression-free survival (rwPFS) and overall survival (OS) were calculated from time of initiation of first-line treatment. RESULTS: We analysed clinical and mutational data for 2276 patients including patients treated with anti-programmed death-1 (PD-1)/anti-programmed death ligand 1 (PD-L1) inhibitors at first line (n=574). Mutations in STK11 or KEAP1 were associated with poor outcomes across multiple therapeutic classes and were not specifically associated with poor outcomes in ICB cohorts. There was no observable interaction between STK11 mutations and anti-PD-1/anti-PD-L1 treatment on rwPFS (HR, 1.05; 95% CI 0.76 to 1.44; p=0.785) or OS (HR, 1.13; 95% CI 0.76 to 1.67; p=0.540). Similarly, there was no observable interaction between KEAP1 mutations and treatment on rwPFS (HR, 0.93; 95% CI 0.67 to 1.28; p=0.653) or OS (HR, 0.98; 95% CI 0.66 to 1.45; p=0.913). CONCLUSION: Our results show that STK11-KEAP1 mutations are prognostic, not predictive, biomarkers for anti-PD-1/anti-PD-L1 therapy.
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Adenocarcinoma/genética , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Biomarcadores Tumorais , Estudos de Coortes , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Mutação , Prognóstico , Análise de SobrevidaRESUMO
Serum interleukin-8 (IL-8) levels and tumor neutrophil infiltration are associated with worse prognosis in advanced cancers. Here, using a large-scale retrospective analysis, we show that elevated baseline serum IL-8 levels are associated with poor outcome in patients (n = 1,344) with advanced cancers treated with nivolumab and/or ipilimumab, everolimus or docetaxel in phase 3 clinical trials, revealing the importance of assessing serum IL-8 levels in identifying unfavorable tumor immunobiology and as an independent biomarker in patients receiving immune-checkpoint inhibitors.
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Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Farmacológicos/sangue , Interleucina-8/sangue , Neoplasias/tratamento farmacológico , Neutrófilos/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Biomarcadores Tumorais/sangue , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Estudos de Coortes , Feminino , Humanos , Masculino , Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/mortalidade , Infiltração de Neutrófilos/efeitos dos fármacos , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Falha de Tratamento , Microambiente Tumoral/imunologia , Regulação para CimaRESUMO
CD141+ DC are implicated in antiviral and antitumor immunity. However, mechanistic studies in autoimmune disease are limited. This is the first study to our knowledge examining CD141+ DC in autoimmune disease, specifically inflammatory arthritis (IA). We identified significant enrichment of CD141+ DC in the inflamed synovial joint, which were transcriptionally distinct from IA and healthy control (HC) blood CD141+ DC and significantly more activated, and they exhibited increased responsiveness to TLR3. Synovial CD141+ DC represent a bone fide CD141+ DC population that is distinct from CD1c+ DC. Synovial CD141+ DC induced higher levels of CD4+ and CD8+ T cell activation compared with their peripheral blood counterparts, as made evident by expression of IFN-γ, TNF-α, and granulocyte-macrophage CSF (GMCSF). Autologous synovial CD141+ DC cocultures also induce higher levels of these cytokines, further highlighting their contribution to synovial inflammation. Synovial CD141+ DC-T cell interactions had the ability to further activate synovial fibroblasts, inducing adhesive and invasive pathogenic mechanisms. Furthermore, we identify a mechanism in which synovial CD141+ DC are activated, via ligation of the hypoxia-inducible immune-amplification receptor TREM-1, which increased synovial CD141+ DC activation, migratory capacity, and proinflammatory cytokines. Thus, synovial CD141+ DC display unique mechanistic and transcriptomic signatures, which are distinguishable from blood CD141+ DC and can contribute to synovial joint inflammation.
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Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Artropatias/imunologia , Adulto , Antígenos CD1 , Antígenos de Superfície/sangue , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Citocinas/metabolismo , Feminino , Glicoproteínas , Humanos , Inflamação , Interferon gama/metabolismo , Ativação Linfocitária , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores Imunológicos , Membrana Sinovial , Trombomodulina , Receptor 3 Toll-Like/metabolismo , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Immune checkpoint blockade with therapeutic anti-cytotoxic T lymphocyte-associated antigen (CTLA)-4 (Ipilimumab) and anti-programmed death (PD)-1 (Nivolumab and Pembrolizumab) antibodies alone or in combination has shown remarkable efficacy in multiple cancer types, concomitant with immune-related adverse events, including arthralgia and inflammatory arthritis (IA) in some patients. Herein, using Nivolumab (anti-PD-1 antagonist)-responsive genes along with transcriptomics of synovial tissue from multiple stages of rheumatoid arthritis (RA) disease progression, we have interrogated the activity status of PD-1 pathway during RA development. We demonstrate that the expression of PD-1 was increased in early and established RA synovial tissue compared to normal and OA synovium, whereas that of its ligands, programmed death ligand-1 (PD-L1) and PD-L2, was increased at all the stages of RA disease progression, namely arthralgia, IA/undifferentiated arthritis, early RA and established RA. Further, we show that RA patients expressed PD-1 on a majority of synovial tissue infiltrating CD4+ and CD8+ T cells. Moreover, enrichment of Nivolumab gene signature was observed in IA and RA, indicating that the PD-1 pathway was downregulated during RA disease progression. Furthermore, serum soluble (s) PD-1 levels were increased in autoantibody positive early RA patients. Interestingly, most of the early RA synovium tissue sections showed negative PD-L1 staining by immunohistochemistry. Therefore, downregulation in PD-1 inhibitory signaling in RA could be attributed to increased serum sPD-1 and decreased synovial tissue PD-L1 levels. Taken together, these data suggest that agonistic PD1 antibody-based therapeutics may show efficacy in RA treatment and interception.
Assuntos
Artrite Reumatoide/patologia , Regulação para Baixo , Receptor de Morte Celular Programada 1/metabolismo , Membrana Sinovial/patologia , Progressão da Doença , Feminino , Humanos , Masculino , Membrana Sinovial/metabolismoRESUMO
OBJECTIVES: This study sought to investigate the genome-wide transcriptional effects of a combination of disease modifying anti-rheumatic drugs (tDMARD; methotrexate, sulfasalazine and hydroxychloroquine) in synovial tissues obtained from early rheumatoid arthritis (RA) patients. While combination DMARD strategies have been investigated for clinical efficacy, very little data exists on the potential molecular mechanism of action. We hypothesized that tDMARD would impact multiple biological pathways, but the specific pathways were unknown. METHODS: Paired synovial biopsy samples from early RA patients before and after 6 months of tDMARD therapy were collected by arthroscopy (n = 19). These biopsies as well as those from subjects with normal synovium (n = 28) were profiled by total RNA sequencing. RESULTS: Large differences in gene expression between RA and control biopsies (over 5000 genes) were identified. Despite clinical efficacy, the expression of a restricted set of less than 300 genes was reversed after 6 months of treatment. Many genes remained elevated, even in patients who achieved low disease activity. Interestingly, tDMARD downregulated genes included those involved in T cell activation and signaling and plasmablast/plasma cell differentiation and function. CONCLUSIONS: We have identified transcriptomic signatures that characterize synovial tissue from RA patients with early disease. Analysis after 6 months of tDMARD treatment highlight consistent alterations in expression of genes related to T cell activation and plasmablast/plasma cell differentiation. These results provide novel insight into the biology of early RA and the mechanism of tDMARD action and may help identify novel drug targets to improve rates of treatment-induced disease remission.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Membrana Sinovial/efeitos dos fármacos , Linfócitos T/citologia , Adulto , Idoso , Artroscopia , Biópsia , Estudos de Casos e Controles , Diferenciação Celular , Regulação para Baixo , Quimioterapia Combinada , Feminino , Humanos , Hidroxicloroquina/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Plasmócitos/citologia , Análise de Componente Principal , Indução de Remissão , Análise de Sequência de RNA , Sulfassalazina/uso terapêutico , Membrana Sinovial/metabolismo , Linfócitos T/efeitos dos fármacos , TranscriptomaRESUMO
BACKGROUND: Although genome-wide association studies (GWAS) have identified over 100 genetic loci associated with rheumatoid arthritis (RA), our ability to translate these results into disease understanding and novel therapeutics is limited. Most RA GWAS loci reside outside of protein-coding regions and likely affect distal transcriptional enhancers. Furthermore, GWAS do not identify the cell types where the associated causal gene functions. Thus, mapping the transcriptional regulatory roles of GWAS hits and the relevant cell types will lead to better understanding of RA pathogenesis. RESULTS: We combine the whole-genome sequences and blood transcription profiles of 377 RA patients and identify over 6000 unique genes with expression quantitative trait loci (eQTLs). We demonstrate the quality of the identified eQTLs through comparison to non-RA individuals. We integrate the eQTLs with immune cell epigenome maps, RA GWAS risk loci, and adjustment for linkage disequilibrium to propose target genes of immune cell enhancers that overlap RA risk loci. We examine 20 immune cell epigenomes and perform a focused analysis on primary monocytes, B cells, and T cells. CONCLUSIONS: We highlight cell-specific gene associations with relevance to RA pathogenesis including the identification of FCGR2B in B cells as possessing both intragenic and enhancer regulatory GWAS hits. We show that our RA patient cohort derived eQTL network is more informative for studying RA than that from a healthy cohort. While not experimentally validated here, the reported eQTLs and cell type-specific RA risk associations can prioritize future experiments with the goal of elucidating the regulatory mechanisms behind genetic risk associations.
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Artrite Reumatoide/genética , Epigênese Genética , Genoma Humano , Linfócitos/metabolismo , Locos de Características Quantitativas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Estudo de Associação Genômica Ampla , Humanos , Linfócitos/classificação , Masculino , Pessoa de Meia-Idade , Receptores de IgG/genéticaRESUMO
Stratifying patients on the basis of molecular signatures could facilitate development of therapeutics that target pathways specific to a particular disease or tissue location. Previous studies suggest that pathogenesis of rheumatoid arthritis (RA) is similar in all affected joints. Here we show that distinct DNA methylation and transcriptome signatures not only discriminate RA fibroblast-like synoviocytes (FLS) from osteoarthritis FLS, but also distinguish RA FLS isolated from knees and hips. Using genome-wide methods, we show differences between RA knee and hip FLS in the methylation of genes encoding biological pathways, such as IL-6 signalling via JAK-STAT pathway. Furthermore, differentially expressed genes are identified between knee and hip FLS using RNA-sequencing. Double-evidenced genes that are both differentially methylated and expressed include multiple HOX genes. Joint-specific DNA signatures suggest that RA disease mechanisms might vary from joint to joint, thus potentially explaining some of the diversity of drug responses in RA patients.
Assuntos
Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Metilação de DNA/genética , Perfilação da Expressão Gênica , Articulações/metabolismo , Articulações/patologia , Transcriptoma/genética , Análise por Conglomerados , Bases de Dados como Assunto , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Quadril/patologia , Humanos , Interleucina-6/metabolismo , Joelho/patologia , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Osteoartrite/genética , Osteoartrite/patologia , Análise de Componente Principal , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Sinoviócitos/metabolismo , Sinoviócitos/patologiaRESUMO
UNLABELLED: Glioblastoma multiforme (GBM) is notoriously resistant to therapy, and the development of a durable cure will require the identification of broadly relevant regulators of GBM cell tumorigenicity and survival. Here, we identify Sprouty2 (SPRY2), a known regulator of receptor tyrosine kinases (RTK), as one such regulator. SPRY2 knockdown reduced proliferation and anchorage-independent growth in GBM cells and slowed xenograft tumor growth in mice. SPRY2 knockdown also promoted cell death in response to coinhibition of the epidermal growth factor receptor (EGFR) and the c-MET receptor in GBM cells, an effect that involved regulation of the ability of the p38 mitogen-activated protein kinase (MAPK) to drive cell death in response to inhibitors. Analysis of data from clinical tumor specimens further demonstrated that SPRY2 protein is definitively expressed in GBM tissue, that SPRY2 expression is elevated in GBM tumors expressing EGFR variant III (EGFRvIII), and that elevated SPRY2 mRNA expression portends reduced GBM patient survival. Overall, these results identify SPRY2 and the pathways it regulates as novel candidate biomarkers and therapeutic targets in GBM. IMPLICATIONS: SPRY2, counter to its roles in other cancer settings, promotes glioma cell and tumor growth and cellular resistance to targeted inhibitors of oncogenic RTKs, thus making SPRY2 and the cell signaling processes it regulates potential novel therapeutic targets in glioma.
Assuntos
Neoplasias Encefálicas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Adulto , Animais , Biomarcadores Tumorais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Receptores ErbB/metabolismo , Feminino , Glioblastoma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Transplante de Neoplasias , Fosforilação , Proteínas Serina-Treonina Quinases , RNA Mensageiro/metabolismoRESUMO
Due to the existence of parallel pathways for receptor endocytosis and their complexities, a quantitative understanding of receptor endocytosis in normal and pathological settings requires computational analysis. Here, we develop a mechanistic model of epidermal growth factor receptor (EGFR) endocytosis to determine the relative contributions of three parallel pathways: clathrin-dependent internalization mediated by mitogen-inducible gene 6 (MIG6), an endogenous EGFR kinase inhibitor that links EGFR to endocytic proteins; clathrin-dependent internalization mediated by the ubiquitin ligase CBL, which can be sequestered by the regulatory protein Sprouty2; or alternative pathways that may be non-clathrin mediated. We applied the model to interpret our previous measurements of EGFR endocytosis in lung cancer cells. Interestingly, our results suggest that MIG6 is responsible for at least as much wild-type EGFR internalization as CBL, indicating that a significant fraction of internalizing EGFR may be incapable of driving signaling. Model results also suggest that MIG6's endocytic function is reduced for the kinase-activated and internalization-impaired EGFR mutants found in some lung cancers. Analysis of Sprouty2 knockdown data indicates that Sprouty2 regulates EGFR endocytosis primarily by controlling EGFR expression, rather than by sequestering CBL, and supports the notion that CBL-mediated internalization is impaired for EGFR mutants. We further demonstrate that differences in internalization between wild-type and mutant EGFR cannot explain differences in EGF-mediated EGFR degradation without concomitant changes in EGFR recycling, which we previously quantified. This work provides new quantitative insights into EGFR trafficking in lung cancer and provides a framework for studying parallel endocytosis pathways for other receptors.