Circulating biomarker analyses in a longitudinal cohort of patients with IPF.

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.

Neurodevelopmental dimensional assessment of young children at high genomic risk of neuropsychiatric conditions.

Background: Individuals with 22q11.2 deletion are at considerably increased risk of neurodevelopmental and psychiatric conditions. There have been very few studies investigating how this risk manifests in early childhood and what factors may underlie developmental variability. Insights into this can elucidate transdiagnostic markers of risk that may underlie later development of neuropsychiatric outcomes. Methods: Thirty two children with 22q11.2 Deletion Syndrome (22q11.2DS) (mean age = 4.1 [SD = 1.2] years) and 12 sibling controls (mean age = 4.1 [SD = 1.5] years) underwent in-depth dimensional phenotyping across several developmental domains selected as being potential early indicators of neurodevelopmental and psychiatric liability. Comparisons were conducted of the dimensional developmental phenotype of 22q11.2DS and sibling controls. For autistic traits, both parents and children were phenotyped using the Social Responsiveness Scale. Results: Young children with 22q11.2DS exhibited large impairments (Hedge's g ≥ 0.8) across a range of developmental domains relative to sibling controls, as well as high rates of transdiagnostic neurodevelopmental and psychiatric traits. Cluster analysis revealed a subgroup of children with 22q11.2DS (n = 16; 53%) in whom neurodevelopmental and psychiatric liability was particularly increased and who differed from other children with 22q11.2DS and non-carrier siblings. Exploratory analyses revealed that early motor and sleep impairments indexed liability for neurodevelopmental and psychiatric outcomes. Maternal autism trait scores were predictive of autism traits in children with 22q11.2DS (intraclass correlation coefficients = 0.47, p = 0.046, n = 31). Conclusions: Although psychiatric conditions typically emerge later in adolescence and adulthood in 22q11.2DS, our exploratory study was able to identify a range of early risk indicators. Furthermore, findings indicate the presence of a subgroup who appeared to have increased neurodevelopmental and psychiatric liability. Our findings highlight the scope for future studies of early risk mechanisms and early intervention within this high genetic risk patient group.

A multi-omic single cell sequencing approach to develop a CD8 T cell specific gene signature for anti-PD1 response in solid tumors.

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.

Plasma biomarkers associated with adverse outcomes in patients with calcific aortic stenosis.

AIMS: Enhanced risk stratification of patients with aortic stenosis (AS) is necessary to identify patients at high risk for adverse outcomes, and may allow for better management of patient subgroups at high risk of myocardial damage. The objective of this study was to identify plasma biomarkers and multimarker profiles associated with adverse outcomes in AS. METHODS AND RESULTS: We studied 708 patients with calcific AS and measured 49 biomarkers using a Luminex platform. We studied the correlation between biomarkers and the risk of (i) death and (ii) death or heart failure-related hospital admission (DHFA). We also utilized machine-learning methods (a tree-based pipeline optimizer platform) to develop multimarker models associated with the risk of death and DHFA. In this cohort with a median follow-up of 2.8 years, multiple biomarkers were significantly predictive of death in analyses adjusted for clinical confounders, including tumour necrosis factor (TNF)-α [hazard ratio (HR) 1.28, P < 0.0001], TNF receptor 1 (TNFRSF1A; HR 1.38, P < 0.0001), fibroblast growth factor (FGF)-23 (HR 1.22, P < 0.0001), N-terminal pro B-type natriuretic peptide (NT-proBNP) (HR 1.58, P < 0.0001), matrix metalloproteinase-7 (HR 1.24, P = 0.0002), syndecan-1 (HR 1.27, P = 0.0002), suppression of tumorigenicity-2 (ST2) (IL1RL1; HR 1.22, P = 0.0002), interleukin (IL)-8 (CXCL8; HR 1.22, P = 0.0005), pentraxin (PTX)-3 (HR 1.17, P = 0.001), neutrophil gelatinase-associated lipocalin (LCN2; HR 1.18, P < 0.0001), osteoprotegerin (OPG) (TNFRSF11B; HR 1.26, P = 0.0002), and endostatin (COL18A1; HR 1.28, P = 0.0012). Several biomarkers were also significantly predictive of DHFA in adjusted analyses including FGF-23 (HR 1.36, P < 0.0001), TNF-α (HR 1.26, P < 0.0001), TNFR1 (HR 1.34, P < 0.0001), angiopoietin-2 (HR 1.26, P < 0.0001), syndecan-1 (HR 1.23, P = 0.0006), ST2 (HR 1.27, P < 0.0001), IL-8 (HR 1.18, P = 0.0009), PTX-3 (HR 1.18, P = 0.0002), OPG (HR 1.20, P = 0.0013), and NT-proBNP (HR 1.63, P < 0.0001). Machine-learning multimarker models were strongly associated with adverse outcomes (mean 1-year probability of death of 0%, 2%, and 60%; mean 1-year probability of DHFA of 0%, 4%, 97%; P < 0.0001). In these models, IL-6 (a biomarker of inflammation) and FGF-23 (a biomarker of calcification) emerged as the biomarkers of highest importance. CONCLUSIONS: Plasma biomarkers are strongly associated with the risk of adverse outcomes in patients with AS. Biomarkers of inflammation and calcification were most strongly related to prognosis.

Molecular correlates of response to nivolumab at baseline and on treatment in patients with RCC.

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.

Quantitative Proteomic Analysis of Diabetes Mellitus in Heart Failure With Preserved Ejection Fraction.

Diabetes mellitus (DM) is associated with a higher risk of heart failure hospitalization and mortality in patients with heart failure with preserved ejection fraction (HFpEF). Using SomaScan assays and proteomics analysis of plasma from participants in the TOPCAT (Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist) trial and the Penn Heart Failure Study, this study identified 10 proteins with significantly different expression in patients with HFpEF and DM. Of these, apolipoprotein M was found to mediate 72% (95% CI: 36% to 100%; p < 0.001) of the association between DM and the risk of cardiovascular death, aborted cardiac arrest, and heart failure hospitalization.

Rapid single cell evaluation of human disease and disorder targets using REVEAL: SingleCell™.

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.

Pan-cancer analysis of the effect of biopsy site on tumor mutational burden observations.

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.

Integration of the Transcriptome and Genome-Wide Landscape of BRD2 and BRD4 Binding Motifs Identifies Key Superenhancer Genes and Reveals the Mechanism of Bet Inhibitor Action in Rheumatoid Arthritis Synovial Fibroblasts.

Fibroblast-like synoviocytes (FLS), one of the main cell types of the rheumatoid arthritis (RA) synovium, possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain family that includes BRD2, BRD3, BRD4, and BRDt, has shown efficacy in models of arthritis. We demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1ß-induced RA-FLS activation and proliferation. To understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling and determined BRD2 and BRD4 cistromes by identifying their global chromatin binding sites. In addition, assay for transposable accessible chromatin by high throughput sequencing was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS. Through an integrated analysis of expression profiling, Brd2/Brd4 cistrome data, and changes in chromatin accessibility, we found that JQ1 inhibited key BRD2/BRD4 superenhancer genes, downregulated multiple crucial inflammatory pathways, and altered the genome-wide occupancy of critical transcription factors involved in inflammatory signaling. Our results suggest a pleiotropic effect of JQ1 on pathways that have shown to be individually efficacious in RA (in vitro, in vivo, and/or in humans) and provide a strong rationale for targeting BRD2/BRD4 for disease treatment and interception.

Elevated serum interleukin-8 is associated with enhanced intratumor neutrophils and reduced clinical benefit of immune-checkpoint inhibitors.

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.

STK11 and KEAP1 mutations as prognostic biomarkers in an observational real-world lung adenocarcinoma cohort.

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.

Enriched Cd141+ DCs in the joint are transcriptionally distinct, activated, and contribute to joint pathogenesis.

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.

Integrative analysis reveals CD38 as a therapeutic target for plasma cell-rich pre-disease and established rheumatoid arthritis and systemic lupus erythematosus.

BACKGROUND: Plasmablasts and plasma cells play a key role in many autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study was undertaken to evaluate the potential of targeting CD38 as a plasma cell/plasmablast depletion mechanism by daratumumab in the treatment of patients with RA and SLE. METHODS: RNA-sequencing analysis of synovial biopsies from various stages of RA disease progression, flow cytometry analysis of peripheral blood mononuclear cells (PBMC) from patients with RA or SLE and healthy donors, immunohistochemistry assessment (IHC) of synovial biopsies from patients with early RA, and ex vivo immune cell depletion assays using daratumumab (an anti-CD38 monoclonal antibody) were used to assess CD38 as a therapeutic target. RESULTS: We demonstrated that the plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from patients with arthralgia, undifferentiated arthritis (UA), early RA and established RA as compared to healthy controls and control patients with osteoarthritis. In addition, the highest CD38 expression was observed on plasma cells and plasmablasts compared to natural killer (NK) cells, classical dendritic cells (DCs), plasmacytoid DCs (pDCs) and T cells, in blood from healthy controls and patients with SLE and RA. Furthermore, IHC showed CD38 staining in the same region as CD3 and CD138 staining in synovial tissue biopsies from patients with early RA. Most importantly, our data show for the first time that daratumumab effectively depletes plasma cells/plasmablasts in PBMC from patients with SLE and RA in a dose-dependent manner ex vivo. CONCLUSION: These results indicate that CD38 may be a potential target for RA disease interception and daratumumab should be evaluated clinically for the treatment of both RA and SLE.

Immune checkpoint inhibitor PD-1 pathway is down-regulated in synovium at various stages of rheumatoid arthritis disease progression.

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.

Triple DMARD treatment in early rheumatoid arthritis modulates synovial T cell activation and plasmablast/plasma cell differentiation pathways.

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.

CD40L-Dependent Pathway Is Active at Various Stages of Rheumatoid Arthritis Disease Progression.

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.

Joint-specific DNA methylation and transcriptome signatures in rheumatoid arthritis identify distinct pathogenic processes.

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.

Integrative genomic deconvolution of rheumatoid arthritis GWAS loci into gene and cell type associations.

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.

Sprouty2 Drives Drug Resistance and Proliferation in Glioblastoma.

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.