P-252 YI New Targets of Interest: Inhibition of PAK-1 Improves Inflammation in a Mouse Model of Colitis. (February 2017)

• Record Type: Journal Article
• Authors: Baldwin, Katherine; Lyons, Jesse; Ghazi, Phaedra; Kissil, Joseph; Haas, Wilhelm; Yajnik, Vijay; Haigis, Kevin
• Is Part Of: Inflammatory bowel diseases. Volume 23(2017)Supplement 1
• Publication Date: 2017-02
• Subjects: Inflammatory bowel diseases -- Periodicals; Colitis, Ulcerative -- Periodicals; Crohn Disease -- Periodicals; Inflammatory Bowel Diseases -- Periodicals; 616.344
• DOI: 10.1097/01.MIB.0000512795.37521.f2
• Languages: English
• ISSNs: 1078-0998

Abstract

Abstract : Background: Our lab seeks to identify signaling pathways that are dysregulated during the onset and progression of IBD and, in doing so, identify potential therapeutic targets. We performed TMT-labeled, multiplexed mass spectrometry (MS) to measure total and phosphoprotein levels in mice with colitis. While defective phosphorylation cascades represent excellent targets for therapy because they are readily targetable by small molecule inhibitors, their potential remains untapped in IBD. Using a novel computational modeling approach (PSSEA, for phosphorylation substrate set enrichment analysis), we identified within our phosphoproteomic dataset potential activation of a targetable kinase, PAK1. We then tested whether PAK1 contributed to colitis by treating our mouse model with FRAX597, a small molecule inhibitor of this kinase. Methods: We utilized a T cell transfer model of colitis where Rag1 null mice are injected with naive T cells to induce chronic inflammation. For the initial experiment, a cohort of 8 animals (5 naive T inflamed animals and 3 T-regulatory noninflamed animals) was subjected to TMT-labeled mass spectrometry for both proteins and phosphorylated proteins. We then applied PSSEA, a method of inferring kinase activity based on phosphorylation events to this data set. Up-regulated targets were confirmed by western blotting. We then generated a new cohort of T cell transfer mice that were monitored for development of colitis with regular weight measurements. Once mice developed signs of colitis they underwent rigid endoscopy to confirm evidence of inflammation. Once inflammation was demonstrated, mice were treated with either FRAX597 or vehicle by oral gavage for 7 days. A subset of mice underwent endoscopy post-treatment and then all were sacrificed at 7 days. The colons were resected for histology and a portion of the colon was utilized for flow cytometry to determine the percent of neutrophils, macrophages and T cells in the tissue as a measure of inflammatory infiltrate. Results: In our hypothesis generating experiment, PSSEA on global MS data from animals with colitis, we demonstrated that PAK-1 was activated in inflamed animals relative to controls. In the subsequent experiment, 3 mice were vehicle treated and 4 were treated with FRAX597 to inhibit PAK-1. The mean neutrophil count for the FRAX treated mice was 5.47% of cells, whereas the vehicle treated mice had a mean neutrophil count of 21.47% ( P = 0.03 using an unpaired t test). This observation demonstrates decreased immune infiltrate in the mice treated with FRAX597, one of the first markers for reversal of colitis. Several of the animals treated with FRAX597 also underwent follow-up endoscopy after treatment and all demonstrated significant improvment on endoscopy as demonstrated by wider lumen, decreased wall thickeness, decreased friability and improved vascular markings. Conclusions: PAK-1 kinase activity is up-regulated during colitis and treatment with FRAX597 led to attenuation of colitis in our mouse model, as demonstrated by both improved endoscopic findings as well as decreased neutrophilic infiltrate. This demonstrates that inhibition of PAK-1 may be a potential future therapeutic target in colitis and also underscores the potential for identifying novel therapeutic targets based on unbiased, global MS analysis.