Expression of NFkB-regulated genes associates with RA disease activity and is altered by anti-TNFa treatment, but not by methotrexate 

Parker A1, Izmailova E1, Pickard M1, Bryant B1, Narang J1, Maier A2, Coblyn JS2, Shadick NA2, Roubenoff R1,  Weinblatt ME2. 1. Millennium Pharmaceuticals, Cambridge MA, 2. Brigham and Women’s Hospital, Boston MA

Purpose:
The two most commonly prescribed disease-modifying rheumatoid arthritis therapies are methotrexate (MTX) and TNFa inhibitors (antiTNF).  The two drugs have fundamentally different mechanisms of action (MOA): MTX inhibits multiple folate-dependent enzymes, and its pleiotropic effects on the RA disease process might occur via inhibition of either DNA or protein biosynthesis; antiTNF, in contrast, specifically targets a proinflammatory cytokine central to RA pathogenesis.  We sought to identify biomarkers predictive of treatment-specific response to these therapies, and to elucidate the molecular mechanisms underlying their disease-modifying activities.  

Methods:
RA patients beginning either MTX (N=28) or antiTNF (N=29) therapy provided blood at baseline and after 2, 6, and 12 wks and were evaluated for disease activity (ACR core set) at baseline, 6 and 12 weeks.  Whole blood RNA was prepared and analyzed using the Affymetrix U133 Gene Chip system.  To elucidate antiTNF-specific biomarkers, we selected probesets representing 100 genes known to be regulated by NFkB, which transduces signals from the TNF receptor.  Subsequently we analyzed the entire Affymetrix dataset, with the goal of identifying biomarkers reflective of the MOA of MTX.  Analyses used multivariate linear and logistic regression, controlling for clinical covariates including gender and duration of disease; p-values were FDR-adjusted. 

Results:
Robust associations with baseline disease severity were observed in the NFkB-focused data set; most significant were calgranulin B (P<10-5), MVP (P<10-4), and IL7R (P<0.0003).  Post-treatment changes in RNA expression also differed substantially between MTX and antiTNF groups, with the strongest signals centered on antiTNF suppression of NFkB-regulated genes like MCL1, SOD2, and calgranulins A, B and C (all P<0.001).  Within two weeks of treatment initiation, changes in regulation of AQP3, NFkBIA, TNFRSF9 and  TRAILR3 were predictive of ACRN outcome at week 12 (P<0.01), and at six weeks TRAILR1, TRAIL, COX2, caspases 8 and 9, and calgranulins A and C also predicted ACRN (P<0.02).  Analysis of the genome-wide Affymetrix data set revealed several genes whose expression was specifically altered by MTX exposure, including IL15 and CD49d, but none remained significant after adjustment for multiple testing.  

Conclusions:
Different genomic biomarker sets may be required for mechanistically different RA therapies, and targeted therapies, like antiTNF, may more readily yield transcriptional biomarker signatures.  Most importantly, our data indicate that calgranulin mRNA levels in peripheral blood serve as robust biomarkers of both RA disease activity and pathway-level impact of antiTNF, and may be useful biomarkers for future treatments directed at NFkB-mediated signaling.