6 NOVEL BRD4 INHIBITORS BLOCK THE PATHOLOGICAL ACTIVATION OF BRD4-NFκB SIGNALING AND SUPPRESS COLONIC INFLAMMATION IN IBD MOUSE MODELS

Abstract Ulcerative Colitis (UC) and Crohn’s Disease (CD) are two major types of inflammatory bowel disease (IBD), with recurrent symptoms and significant morbidity. Long-term persistence of chronic inflammation in IBD is among the major factors contributing to neoplastic transformation and development of colitis-associated colorectal cancer. There is a lack of efficient medications for IBD, due to either limited efficacy or side effects. Antibodies against TNFα are effective therapies; however, they are expensive, and up to 40% of patients are non-responders. Thus, improved therapy with higher efficacy, enhanced safety and better patient affordability is urgently needed. The master regulator of innate immune response NFκB plays a critical role in the initiation and chronicity of IBD mucosal inflammation. We demonstrated that bromodomain-containing protein 4 (BRD4), an epigenetic reader, is required for stabilization of NFκB binding on the promoters of inflammatory genes, activation of RNA polymerase II, and histone H3 Lys122 acetylation (H3K122ac) to permit high levels of inflammatory gene expressions by sentinel epithelial cells and stromal fibroblasts. Our data using human IBD patient samples suggest that BRD4 activation is critical to the initiation of colonic inflammation. We have successfully identified highly potent and specific BRD4 inhibitors, demonstrating that inhibition of BRD4 suppresses expression of inflammatory cytokines TNFα, IL-6, IL-17A, and IL-8. Administration of our lead inhibitors (ZL0454 & ZL0420) significantly reduces initiation of the mucosal inflammation in both dextran sulfate sodium (DSS)-induced and Cbir1 T cell transfer colitis models in vivo, but with low toxicity and much safer than the generic NFκB/IKK inhibitor BMS345541. The levels of NFkB and BRD4 activation were also compared using immunofluorescence staining of NFkB/RelA translocation, phospho-Ser276 RelA formation, and induction of the BRD4 marker H3K122Ac. The BRD4 inhibitors reduced DSS-induced NFkB-BRD4 activation in colon tissue, demonstrating the target specificity in vivo. Our data suggest that BRD4 activation is critical to the initiation of the colonic inflammation during IBD. BRD4 inhibition disrupts its interactions with acetylated histone lysine residues, thereby blocking the pathological activation of the BRD4-NFκB signaling and suppressing colonic inflammation. Therefore, inhibition of the BRD4 activation is an attractive target for the development of novel IBD therapy and in prevention of CAC. Using our novel BRD4 inhibitors, we will further seek to evaluate the effect of long-term BRD4 inhibition on UC-induced neoplastic transformation and development of CAC. Funding support: Crohn’s & Colitis Foundation Entrepreneurial Investing (EI) Initiative award and a research fellowship award from the Crohn’s & Colitis Foundation of America.