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AB0111 Extracellular 14-3-3 ETA represents a novel rheumatology biomarker and drug target for personalized medicine
  1. A. Marotta1,
  2. R. Kilani2,
  3. A. Ghahary2,
  4. W.P. Maksymowych3
  1. 1Augurex Life Sciences Corp, North Vancouver
  2. 2Department of Surgery, University of British Columbia, Vancouver
  3. 3Department of Medicine, University of Alberta, Edmonton, Canada

Abstract

Background 14-3-3η is a novel therapeutic target that stimulates specific cell lines (THP-1), activates disease-specific pathways (MAPK) and potentiates pathogenic factors (TNFα, IL-6). Clinically, 14-3-3η serum expression is higher in both erosive RA and PsA, correlates strongly with both synovial and serum levels of metalloproteinases (MMP-1 & -3), and predicts likelihood of ACR50 response in PsA and Good EULAR response in RA patients receiving TNFα therapy. Extracellular 14-3-3η’s involvement in disease-specific processes together with its clinical expression underscores its potential as a personalized medicine drug target.

Objectives To further characterize 14-3-3η’s role in disease processes by examining its effects on signaling pathways targeted by small molecule compounds, its potency versus TNFα on inflammatory factors, and the effectiveness of 14-3-3η antibodies at blocking its effects.

Methods Phosphoproteomic profiling was conducted using THP-1 cells stimulated with either 14-3-3η or TNFα, at the same dose, to examine activation of intracellular pathways. The evaluation included 25 distinct phosphorylation sites on 20 different targets. Staying within the 14-3-3η clinical serum expression range (median 1.12ng/ml and range of 0.12 to 20ng/ml), 3 downstream targets (IL-8, IL-6 and TNFα) were selected to evaluate if increases in mRNA reflected increased protein expression. 14-3-3η’s potency on inflammatory factors (IL-6, IL-8, MCP-1) was compared to TNFα at an 18- and 48-hour end-point in human macrophages. Responsiveness of PBMC and monocyte donor cells to 14-3-3η stimulation was also evaluated. Dose escalation studies with 14-3-3η-specific monoclonal antibodies were performed using primary human monocyte and macrophage donor cells.

Results The table below summarizes the findings of the phosphoproteomic analysis. 76% of the targets had similar changes in their phosphorylation status when stimulated with either TNFα and 14-3-3η, whereas 24% exhibited clear differences.

Table 1. Summary of phosphoproteomic analysis comparing TNFα to 14-3-3η

Human PBMCs, monocytes and macrophages were responsive to 14-3-3η and its potency in inducing IL-6, IL-8 and TNFα as measured by mRNA analysis corresponded with changes in protein expression. In a comparison of 14-3-3η’s inducing effects to those of TNFα, similar degrees of induction were observed for levels of IL-8 and MCP-1. In contrast, 14-3-3η was a more potent inducer of IL-6. Antibody targeting studies demonstrate that the potentiating effects of 14-3-3η can be blocked in a dose-dependent manner with as little as 80ng/ml in vitro using both human monocyte and macrophage donors.

Conclusions 14-3-3η activates key intracellular signaling pathways that are targeted by small molecule compounds. It potently induces inflammatory pathways, especially IL-6, and its effects can be blocked with 14-3-3η-specific monoclonal antibodies. These findings, together with its association with disease expression in RA and PsA may guide in vivo evaluation and underscores 14-3-3η as a personalized medicine target.

Disclosure of Interest A. Marotta Shareholder of: Co-founder, R. Kilani: None Declared, A. Ghahary: None Declared, W. Maksymowych: None Declared

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