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BACKGROUND: Lupus nephritis (LN) occurs in

Type I interferon (IFN) drives pathology in systemic lupus erythematosus (SLE) and can be tracked via IFN-inducible transcripts in blood. Here, we examined whether measurement of circulating proteins, which enter the bloodstream from inflamed tissues, also offers insight into global IFN activity. Using a novel protocol we generated 1,132 aptamer-based protein measurements from anti-dsDNApos SLE blood samples and derived an IFN protein signature (IFNPS) that approximates the IFN 21-gene signature (IFNGS). Of 82 patients with SLE, IFNPS was elevated for 89% of IFNGS-high patients (49/55) and 26% of IFNGS-low patients (7/27). IFNGS-high/IFNPS-high patients exhibited activated NK, CD4, and CD8 T cells, while IFNPS-high only patients did not. IFNPS correlated with global disease activity in lymphopenic and non-lymphopenic patients and decreased following type I IFN neutralisation with anifrolumab in the SLE phase IIb study, MUSE. In summary, we developed a protein signature that reflects IFNGS and identifies a new subset of patients with SLE who have IFN activity.

OBJECTIVE Neutrophil dysregulation and the type I interferon (IFN) axis have been proposed to contribute to premature cardiovascular disease, a leading cause of mortality in patients with systemic lupus erythematosus (SLE). In the present study, we evaluated the ability of anifrolumab, a type I IFN receptor-blocking antibody, to reduce neutrophil extracellular trap (NET) formation and modulate cardiometabolic disease markers in comparison to placebo. METHODS Study subjects comprised patients with moderate-to-severe SLE who were enrolled in phase IIb of the MUSE trial (A Phase II, Randomized Study to Evaluate the Efficacy and Safety of MEDI-546 in Subjects with Systemic Lupus Erythematosus), with healthy individuals as controls. Blood samples were collected from SLE patients (n = 305) and healthy controls (n = 10-20) before the initiation of treatment (baseline) and from SLE patients after they had been treated with 300 mg of anifrolumab (n = 99) or placebo (n = 102). Baseline IFN gene signature test status was determined, and the IFN gene signature (21-gene panel) was monitored over time. Serum proteins were measured by multiplex immunoassay or ultrasensitive Simoa assay. NET complexes, cholesterol efflux capacity (CEC), and glycoprotein acetylation (GlycA) and other lipid parameters were assessed in plasma. RESULTS Formation of NET complexes and levels of tumor necrosis factor (TNF) and interleukin-10 (IL-10) were correlated with extent of type I IFN pathway activity. NET complexes and IL-10 levels were up-regulated in SLE patients compared to healthy controls (P < 0.008). The cardiometabolic disease markers CEC and GlycA were also found to be dysregulated in patients with SLE (P < 0.001 versus healthy controls). Type I IFN receptor inhibition with anifrolumab significantly reduced NET complexes and GlycA and improved CEC compared to baseline (P < 0.05) whereas no improvements were seen with placebo. Levels of TNF and IL-10 were reduced with anifrolumab compared to placebo (P < 0.05). CONCLUSION These data support a key role for type I IFNs in modulating factors contributing to SLE vasculopathy and suggest that inhibition of this pathway could decrease cardiovascular risk in individuals with SLE.

Systemic lupus erythematosus (SLE) impacts multiple organ systems, although the causes of many individual SLE pathologies are poorly understood. This study was designed to elucidate organ-specific inflammation by identifying proteins that correlate with SLE organ involvement and to evaluate established biomarkers of disease activity across a diverse patient cohort. Plasma proteins and autoantibodies were measured across seven SLE manifestations. Comparative analyses between pathologies and correlation with the SLE Disease Activity Index (SLEDAI) were used to identify proteins associated with organ-specific and composite disease activity. Established biomarkers of composite disease activity, SLE-associated antibodies, type I interferon (IFN), and complement C3, correlated with composite SLEDAI, but did not significantly associate with many individual SLE pathologies. Two clusters of proteins were associated with renal disease in lupus nephritis samples. One cluster included markers of infiltrating leukocytes and the second cluster included markers of tissue remodelling. In patients with discoid lupus, a distinct signature consisting of elevated immunoglobulin A autoantibodies and interleukin-23 was observed. Our findings indicate that proteins from blood samples can be used to identify protein signatures that are distinct from established SLE biomarkers and SLEDAI and could be used to conveniently monitor multiple inflammatory pathways present in different organ systems.

ObjectivesDespite the therapeutic value of current rheumatoid arthritis (RA) treatments, agents with alternative modes of action are required. Mavrilimumab, a fully human monoclonal antibody targeting the granulocyte–macrophage colony-stimulating factor receptor-α, was evaluated in patients with moderate-to-severe RA.MethodsIn a phase IIb study (NCT01706926), patients with inadequate response to ≥1 synthetic disease-modifying antirheumatic drug(s), Disease Activity Score 28 (DAS28)−C reactive protein (CRP)/erythrocyte sedimentation rate ≥3.2, ≥4 swollen joints despite methotrexate (MTX) were randomised 1:1:1:1 to subcutaneous mavrilimumab (150, 100, 30 mg), or placebo every other week (eow), plus MTX for 24 weeks. Coprimary outcomes were DAS28−CRP change from baseline to week 12 and American College of Rheumatology (ACR) 20 response rate (week 24).Results326 patients were randomised (150 mg, n=79; 100 mg, n=85; 30 mg, n=81; placebo, n=81); 305 completed the study (September 2012–June 2013). Mavrilimumab treatment significantly reduced DAS28−CRP scores from baseline compared with placebo (change from baseline (SE); 150 mg: −1.90 (0.14), 100 mg: −1.64 (0.13), 30 mg: −1.37 (0.14), placebo: −0.68 (0.14); pSignificantly more mavrilimumab-treated patients achieved ACR20 compared with placebo (week 24: 73.4%, 61.2%, 50.6% vs 24.7%, respectively (pConclusionsMavrilimumab significantly decreased RA disease activity, with clinically meaningful responses observed 1 week after treatment initiation, representing a novel mechanism of action with persuasive therapeutic potential.Trial registration numberNCT01706926; results.

Non-receptor and receptor tyrosine kinases, such as Src and EGF receptor (EGFR), are major inducers of vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis. While tyrosine kinases signal through multiple pathways, signal transducer and activation of transcription 3 (Stat3) is a point of convergence for many of these and is constitutively activated with high frequency in a wide range of cancer cells. Here, we show that VEGF expression correlates with Stat3 activity in diverse human cancer cell lines. An activated Stat3 mutant (Stat3C) up-regulates VEGF expression and stimulates tumor angiogenesis. Stat3C-induced VEGF up-regulation is abrogated when a Stat3-binding site in the VEGF promoter is mutated. Furthermore, interrupting Stat3 signaling with dominant-negative Stat3 protein or Stat3 antisense oligonucleotide in tumor cells down-regulates VEGF expression. Consistent with an important role of Stat3 in VEGF up-regulation induced by various oncogenic tyrosine kinases, v-Src-mediated VEGF expression is inhibited when Stat3 signaling is blocked. Moreover, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in vivo and mutation of a Stat3-binding site in the VEGF promoter abrogates v-Src-induced VEGF promoter activity. These studies provide evidence that the VEGF gene is regulated directly by Stat3 protein, and indicate that Stat3 represents a common molecular target for blocking angiogenesis induced by multiple signaling pathways in human cancers.

A popular commercially available oligonucleotide microarray technology employs sets of 25 base pair oligonucleotide probes for measurement of gene expression levels. A mathematical algorithm is required to compute an estimate of gene expression from the multiple probes. Previously proposed methods for summarizing gene expression data have either been substantially ad hoc or have relied on model assumptions that may be easily violated. Here we present a new algorithm for calculating gene expression from probe sets. Our approach is functionally related to leave-one-out cross-validation, a non-parametric statistical technique that is often applied in limited data situations. We illustrate this approach using data from our study seeking a molecular fingerprint of STAT3 regulated genes for early detection of human cancer.

Type I interferon (IFN) signaling drives pathology in systemic lupus erythematosus (SLE) and can be tracked via IFN-inducible transcripts present in whole blood as described by several IFN gene signatures. As SLE is a complex disease affecting diverse organ systems, we examined whether measurement of circulating proteins, which can infiltrate the bloodstream from afflicted tissues, might also offer insight into global IFN activity. The presence of anti-DNA autoantibodies in patient serum has prevented effective use of SOMAmers for the evaluation of circulating proteins in SLE. Here, we adapted protocols to mitigate for those autoantibodies and report high reproducibility and accuracy with 100% QC pass rate and improved correlation with previously validated multi-analyte platform results. Using SOMAmers together with the IFN 21-gene signature1 (IFNGS), we derived an IFN protein signature that can approximate the IFNGS score. In a cohort of 82 SLE patients and 48 healthy donors, the protein signature was found elevated above healthy donors for most of IFNGS-high patients (49/55, 89%) and also for a subgroup of IFNGS-low patients (7/27, 26%). The protein signature correlated with global disease activity (median SLEDAI score of 4 for the cohort) in both lymphopenic and non-lymphopenic patients. Significant associations with skin involvement, low complement, anti DNA auto-antibodies, and thrombocytopenia were also observed. In sum, our results suggest blood derived protein measurements may complement validated gene signatures to monitor IFN activity.

Background Mavrilimumab is a fully human monoclonal antibody targeting granulocyte-macrophage colony-stimulating factor receptor α (GM–CSFR-α). It is the first therapeutic to target this pathway in the treatment of rheumatoid arthritis (RA). A Phase IIb clinical trial (EARTH EXPLORER 1; [NCT01706926][1])1 of patients with moderate to severe RA has demonstrated mavrilimumab to be an efficacious and well-tolerated treatment option for RA. Objectives We assessed peripheral biomarkers and pathophysiologic pathways modulated by mavrilimumab in association with clinical response through GM–CSFR-α blockade in RA patients. Methods In a 24-week, randomized controlled study, RA patients received subcutaneous mavrilimumab (30, 100, or 150 mg every other week [eow]) or placebo, both in combination with methotrexate. Serum concentrations of 19 RA-associated proteins, as well as 6 protease-derived protein fragments, from 326 RA patients were measured at baseline and at 5 time points post-administration. Affymetrix HGU133Plus2 arrays were used to measure gene expression changes in whole blood of RA patients receiving mavrilimumab 100 or 150 mg eow, or placebo. Results Several proteins demonstrated significant down-regulation at Days 8, 15, 29, 85, and 169 after mavrilimumab 100 mg and/or 150 mg eow treatment, compared with placebo, including CRP, SAA, IL-6, macrophage-derived chemokine (CCL22), IL-2RA, MMP3, and VEGF (Mann-Whitney U test, p

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Background Mavrilimumab is a human monoclonal antibody targeting the alpha subunit of GM-CSF receptor and was recently evaluated in RA subjects in a phase 2a study (EARTH). Results reported from the European (EU) cohort demonstrated that mavrilimumab shows activity in subjects with moderate to severe RA (Burmester et al. 2013). Here, we describe final data from EARTH comparing results in the EU and Japanese (JA) cohorts. Objectives Biomarker (BM) assessments were performed to elucidate mechanistic aspects of mavrilimumab. Methods Mavrilimumab (10, 30, 50 or 100 mg) or placebo was administered SC every other week to subjects with moderate/severe RA (DAS28 >3.2) on stable methotrexate for 12 weeks followed by a 12 week drug free follow up period. A multi-biomarker disease activity (MBDA) score was calculated using the validated Vectra ®DA algorithm and used to track the effect of the drug on disease activity over time. An additional multi-BM-based algorithm (MBSD) was used to assess the impact of mavrilimumab on markers known to be associated with progressive joint damage. The relative ability of different mavrilimumab doses, over time, to saturate GM-CSF receptors in whole blood was examined by flow cytometry using receptor occupancy assay (ROA) in the EU cohorts. Results In the EARTH study, the primary endpoint (DAS28-CRP reduction ≥1.2 at Week 12) was met. Improvements were seen as early as week 2 and persisted through the 12 week follow up. The MBDA score decreased significantly as early as day 8 (p Conclusions Promising results of mavrilimumab support further clinical development at doses greater than 10 mg. Mechanistically, the drug suppressed both acute phase and inflammatory blood markers. Tracking of disease activity by MBDA showed a clear biomarker-based dose-response relationship. The association of MBSD decline with radiographic damage will be assessed in an on-going phase 2b study. References Burmester GR, et al. 2013, ARD, 72 (9):1445-52. Disclosure of Interest W. White Shareholder of: AstraZeneca, Employee of: MedImmune, P. Ryan Shareholder of: AstraZeneca, Employee of: MedImmune, X. Guo Shareholder of: AstraZeneca, Employee of: MedImmune, D. Sinibaldi Shareholder of: AstraZeneca, Employee of: MedImmune, G. Ranganna Shareholder of: AstraZeneca, Employee of: MedImmune, A. Godwood Shareholder of: AstraZeneca, Employee of: MedImmune, D. Saurigny Shareholder of: AstraZeneca, Employee of: MedImmune, D. Close Shareholder of: AstraZeneca, Employee of: MedImmune, S. Eck Shareholder of: AstraZeneca, Employee of: MedImmune, D. Wilkins Shareholder of: AstraZeneca, Employee of: MedImmune, L. Roskos Shareholder of: AstraZeneca, Employee of: MedImmune, M. Sleeman Shareholder of: AstraZeneca, Employee of: MedImmune, W. Li Shareholder of: Crescendo Bioscience, Employee of: Crescendo Bioscience, G. Cavet Shareholder of: Crescendo Bioscience, Employee of: Crescendo Bioscience, N. Defranoux Shareholder of: Crescendo Bioscience, Employee of: Crescendo Bioscience DOI 10.1136/annrheumdis-2014-eular.5731

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c -myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully defined. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse fibroblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the findings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These findings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse fibroblasts.