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OP0289 Integration of chromatin conformation, transcriptome and genome-wide landscape of brd2 and brd4 binding motifs identifies mechanisms of bet inhibitor action in rheumatoid arthritis synovial fibroblasts

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by infiltration of immune cells into the synovium and hyperplasia of the synovial lining, resulting in the formation of pannus that degrades cartilage and bone. Fibroblast-like synoviocytes (FLS) are the main cell types of the rheumatoid synovium and possess phenotypic and molecular characteristics of transformed cells. JQ1, an inhibitor of the bromodomain and extra terminal domain (BET) family that includes BRD2, BRD3, BRD4 and BRDt has shown efficacy in vitro on RA-FLS proliferation and in vivo in a murine model of arthritis. Objectives We sought to elucidate the mechanism of action of BET proteins in FLS biology and determine the potential therapeutic utility of targeting BRD2/BRD4 for RA disease treatment and interception. Methods To understand the mechanism of JQ1 action, we subjected JQ1-treated RA-FLS to transcriptional profiling by RNA-Seq and determined BRD2 and BRD4 cistromes by identifying global BRD2/BRD4 chromatin binding sites by ChIP-Seq. In addition, Assay for Transposable Accessible Chromatin by high throughput Sequencing (ATAC-Seq) was employed to identify open and closed regions of chromatin in JQ1-treated RA-FLS. Results We demonstrate that the active isomer of JQ1 but not its inactive isomer inhibits IL-1b-induced RA-FLS activation and proliferation. Through an integrated analysis of RNA-Seq, ATAC-Seq to profile changes in chromatin accessibility and ChIP-Seq of BRD2/4 and Pol2 proteins, we found that JQ1 inhibited multiple key inflammatory pathways, and altered the genome wide occupancy of crucial transcription factors involved in inflammatory signalling. Specifically, JQ1 treatment resulted in reduced occupancy of both BRD2 and BRD4 in approximately 2000 regions genome-wide, and a loss of Pol2 occupancy in approximately 600 genomic regions. Collectively we found that 105 genes had altered occupancy in all three proteins (BRD2, BRD4 and Pol2) and were also differentially expressed. Most prominently, JQ1 resulted in down regulation of IL6, IL8, p38 MAP kinase and HMGB1/TLR4 signalling pathways. In addition, we have identified BRD2/BRD4 super-enhancer genes and demonstrate that JQ1 altered BRD2/BRD4 occupancy in the IL6 and IL8 super-enhancer regions, and significantly down-regulated IL6, IL8, TLR4 and IL1b expression. Conclusions Our results suggest pleiotropic effects of JQ1 on pathways that have been individually targeted and shown to be efficacious for the treatment of RA. These studies provide a strong rationale for targeting of BRD2/BRD4 in RA. Disclosure of Interest S. Nagpal Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, V. Krishna Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, X. Yin Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, D. Pocalyko Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, A. Walsh Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, K. Bachman Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, I. Anderson Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson, L. Madakamutil Shareholder of: Johnson and Johnson, Grant/research support from: Johnson and Johnson, Employee of: Johnson and Johnson