Biochemical mechanisms of beta cell protection through bromodomain inhibition

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PROJECT SUMMARY Autoimmune diabetes is characterized by an inflammatory reaction in and around pancreatic islets followed by selective destruction of insulin producing β-cells. Low concordance rates of autoimmune diabetes in monozygotic twins indicate an important but poorly understood role for epigenetic factors in diabetes initiation and progression. Bromodomains are epigenetic “readers” of lysine acetylation on histones and transcription factors; bromodomain binding to acetylated histones/proteins regulates transcription in a cell-type dependent manner. Early treatment of non-obese diabetic (NOD) mice with an inhibitor of the bromodomain and extraterminal (BET) family (Brd2-4) was recently shown to suppress development of autoimmune diabetes. The protective effects of BET inhibition correlated with anti-inflammatory and pro-proliferative phenotypes in macrophages and β-cells, respectively; however, the mechanisms are poorly understood. We hypothesize that Brd4 regulates β-cell proliferation and macrophage inflammation in islets, and that inhibition of Brd2 and Brd3 are liabilities of pan-BET inhibitors in autoimmune diseases. Consistent with this hypothesis, pan-BET inhibition is associated with impaired learning and memory as well as reduced immune system function. As epigenetic intervention in autoimmune diabetes represents a novel therapeutic target in a primarily pediatric population, off-target effects must be minimized. Brd4 inhibition as a therapeutic strategy in autoimmune diabetes will be examined in three specific aims: Aim 1) Test the hypothesis that Brd4 inhibition prevents macrophage activation and production of inflammatory mediators known to damage β-cells. Studies will build on our preliminary data showing that pan-BET inhibitors attenuate macrophage production of inflammatory mediators such as IL-1β and nitric oxide. Aim 2) Test the hypothesis that Brd4 inhibition protects β-cells from cytokine-mediated damage by stimulating DNA damage repair pathways and protecting mitochondria from damage. Studies will use insulinoma cells deficient in Brd2, Brd3, and Brd4 and chemical inhibitors to explore the role of BET proteins in the regulation of β-cell responses to inflammatory mediators and the activation of defense pathways that facilitate β-cell recovery from oxidative stress. Aim 3) Develop selective Brd4 inhibitors for effective treatment of autoimmune diabetes. We provide evidence to support our innovative approach to selectively inhibit Brd4 using covalent targeting of a specific amino acid residue unique to Brd4. Biochemical, molecular, immunological, cell biological, genetic, and chemical biological approaches will be used to investigate the molecular and cellular pathways through which BET inhibition protects β-cells and to develop novel tools and reagents to selectively target the BET family of transcriptional regulators. Our long-term goals are to elucidate the cell-type specific mechanisms of transcriptional regulation by BET bromodomains and develop novel therapeutics to selectively inhibit the activity of individual BET proteins as an initial step in the design of strategies to halt the development and progression of autoimmune diabetes.