Effect and mechanism of BET bromodomain inhibition in macrophage transcriptional programming

Transcription

1 REVIEW Effect and mechanism of BET bromodomain inhibition in macrophage transcriptional programming Yu Qiao 1, Lionel B. Ivashkiv 1,2 1 Arthritis and Tissue Degeneration Program and David Z. Rosensweig Center for Genomics Research, Hospital for Special Surgery, New York, NY, USA 2 Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA Correspondence: Lionel B. Ivashkiv IvashkivL@hss.edu Received: February 03, 2015 Published online: March 11, 2015 Epigenetic regulation is at the center of gene transcriptional activity, and the epigenetic mechanisms in immune responses have gained increasing attention because of their potential as therapeutic targets. Bromodomain and extra terminal (BET) proteins are recognized as important players in transcriptional elongation; blockade of the recruitment of BET proteins to acetylated histones suppresses BET-mediated transcription. BET inhibitors have been tested in multiple mouse models as a promising approach to treat various diseases. Recently, a study reported the effect of a BET inhibitor I-BET151 on human monocyte and macrophage responses. The study focused on the interference of I-BET151 with cytokine-stimulated JAK-STAT pathways that are important for monocyte polarization and inflammatory responses. In both pro-inflammatory and alternative macrophage responses, I-BET151 exhibited differential repression of cytokine target genes and the repression was independent of protein synthesis. The study also found that I-BET151 repressed TLR- and pro-inflammatory cytokine-induced interferon responses by blocking both IFN- production and IFN- -induced immune response. Further investigation of interferon responses showed that I-BET151 administration functions downstream of JAK-STAT activation or STAT1 recruitment to target promoters, but instead blocked RNA polymerase II recruitment to gene proximal promoters as well as distal regulatory regions. These findings expand the understanding of the effect and therapeutic potential of BET protein inhibition in inflammatory diseases. To cite this article: Yu Qiao, et al. Effect and mechanism of BET bromodomain inhibition in macrophage transcriptional programming. Inflamm Cell Signal 2015; 2: e600. doi: /ics.600. Chromatin regulation and BET proteins in transcriptional activity Cell lineage and response-specific transcription is essential for the biology of the organism. Transcriptional specificity is achieved and regulated mainly by the collaboration of cell autonomous and environmental factors. Terminally differentiated immune cells such as monocytes and macrophages are specialized to quickly respond to external stimuli and to deploy inducible gene expression programs in accordance with the nature of the immunological process. The precision of the transcriptional response to corresponding environmental cues is critical for an effective immune response [1]. Inappropriate gene activation programs result in ineffective immune responses and even exaggeration of autoimmune or inflammatory diseases. A common theme of a specific cellular immune response starts from the engagement of cell surface receptors by their ligands, which triggers a series of intracellular signaling cascades. One consequence of the integrated signaling events is the activation of signal-specific transcriptional regulatory factors that translocate to the nucleus, recognize binding motifs Page 1 of 5

2 around their target genes and facilitate gene expression, thereby achieving stimulation-specific cellular responses. However, in the nucleus not all target genes are easily accessible because of the complex chromatin structure, which forms another layer of transcriptional regulation by exposing or blocking genes and DNA regulatory elements from the transcriptional machinery. Major mechanisms involved in chromatin structure modulation include DNA methylation, histone modification and ATP-dependent nucleosome remodeling [2]. In addition to the regulation of the proximal promoters, the interaction of gene promoters with distal enhancers is also known as an important cell-type and stimulation-specific mechanism of epigenetic regulation [3]. Signaling regulation and mechanisms in inflammatory responses have been extensively studied for decades, and more recently research has started to reveal the importance of epigenetic changes in inflammation. One of the first observations was that TLR stimulation results in gene-specific histone modification, which regulates gene expression long after the resolution of infection [4]. Later, in an attempt to find epigenetic signatures to address persisting inflammations, a study on rheumatoid arthritis synovial fibroblasts reported the correlation between decreased DNA methylation and elevated pro-inflammatory chemokine CXCL12 [5]. More recently, the modulation of histone acetylation by the micro-environment has been shown to contribute to prolonged and elevated TLR responses in human monocytes [6]. More comprehensive characterization has confirmed stimulation-specific epigenetic modulation in various types of human macrophage responses [7]. The implication of epigenetic regulation in gene expression has led to the targeting of transcriptional regulators that function on chromatin levels in the treatment of various diseases [8-10]. One promising drug target is the bromodomain and extra terminal domain (BET) family proteins (BrdT, Brd2, Brd3 and Brd4), which play important roles in histone acetylation-dependent transcriptional regulation [11]. The tandem bromodomains on BET proteins bind to acetylated lysine on histone H3 and H4. The recruited BET proteins function as anchors for the positive transcription elongation factor b (p-tefb) [12], which in turn phosphorylates RNA polymerase II on Ser2 residue, which is necessary for transcription elongation [13]. In addition to recruitment and functioning around on gene promoters, Brd4 is also recruited to subsets of distal enhancers to promote gene transcription [14]. Various BET small molecule inhibitors successfully block BET protein recruitment and BET-mediated transcriptional activity [15-17], and the function of these inhibitors has been explored in various disease settings including cancer [4, 18-21], autoimmune response [4], osteoporosis [22, 23] and atherosclerosis [24]. Here we will focus our discussion on how BET inhibition affects inflammatory responses. BET inhibition in inflammation in mouse models A study in 2010 first revealed the role of BET in LPS response and evaluated the potency of a competitive BET protein inhibitor I-BET in LPS-induced gene expression [4]. Among hundreds of LPS-induced target genes in mouse bone-marrow derived macrophages (BMDMs), I-BET repressed the transcriptional regulation of a subgroup of these genes, including key LPS-inducible cytokines and chemokines such as IL-6, IFNβ, IL-1β, IL-12α, CXCL9, and CCL12, whereas some other important LPS-induced pro-inflammatory genes such as TNF-α were unaffected. Genome-wide analysis demonstrated LPS-induced BET occupancy around the promoters of I-BET repressible genes, which was diminished by I-BET administration. The potency of I-BET in repressing inflammation in vivo was demonstrated by the rescue of I-BET-treated mice from LPSor Salmonella-triggered systemic inflammation. The importance of BET proteins for mouse macrophage inflammatory responses was confirmed in another study using a different BET small-molecule inhibitor JQ1 [25]. The study showed that JQ1 interrupted the BRD proteins association with certain inflammatory cytokine gene promoters and blocked pro-inflammatory cytokine expression in vivo and in vitro. Although the earlier study discussed above did not observe an effect of I-BET on TNF expression, JQ1 treatment decreased TNF levels in endotoxemic mice. Another study investigated the effect of JQ1 on IFN- triggered interferon-stimulated gene (ISG) expression [26]. The study found that IFN- induced recruitment of BRD4 and p-tefb to ISGs in the murine 3T3 cell line, and that JQ1 treatment blocked ISG transcriptional elongation and repressed both inflammatory and osteoclastogenic gene transcription. The in vivo significance of JQ1 administration was implicated by the decrease of inflammation and bone absorption in a mouse model. BET inhibition in human inflammatory responses Although BET inhibitors have been tested in various mouse models, their efficacy in human conditions, outside of human cancer cell line xenografts in mouse models, is less explored [27]. Monocytes and macrophages are immune cells with highly versatile functionality and exhibit a broad spectrum of phenotypes in response to diverse signaling cues. Pro-inflammatory cytokines and TLR ligands drive the Page 2 of 5

3 classical inflammatory activation in human monocytes and macrophages to promote immune responses [28, 29], while regulatory cytokines such as IL-4/13 and IL-10 drive monocytes and macrophages to alternative immune-regulatory phenotypes [30-33]. Considering the pivotal roles of monocytes and macrophages in various physiological activities and the implications of their modulation in clinical environments, recently, Chan et al investigated the effects of I-BET151 administration in various types of activations of human monocytes. The study first assessed the effect of I-BET151 on the transcriptional activity of the inflammatory chemokines CXCL10 and CXCL11 upon the stimulation of pro-inflammatory cytokine TNF and the TLR ligand LPS. I-BET administration inhibited the induction of these chemokines in a dose dependent manner and resulted in decreased upstream STAT1 activity. In addition, the expression of IFN-, which is induced in TNF and LPS responses and forms an autocrine loop to activate STAT1 and an interferon response [28], was also blocked by I-BET151. Therefore the authors next investigated the role of I-BET151 in IFN- response and found that I-BET151 inhibited IFN- -induced CXCL10 and CXCL11 transcription. In contrast, the JAK-STAT activation remains intact in IFN- response, supporting that I-BET151 interferes with TNF and LPS responses at least in part through blocking IFN- response. I-BET displayed differential inhibition of various target genes in all pro-inflammatory responses investigated. IFN- -induced expression of the ISGs IFIT1, IFIT2, STAT1 and IRF1 was much less susceptible to inhibition by I-BET151 than the induction of CXCL10 and CXCL11. In comparison, LPS- and TNF-induced IFIT and IRF gene transcription was effectively repressed by I-BET151, consistent with the hypothesis that blocking IFN- results in global ISG repression. In regard to alternative macrophage responses, I-BET151 inhibited IL-4 and IL-10 responses in gene-specific manners as well. Genes important in IL-10 response such as IL-7R, ABIN3, and ENPP2 were repressed by I-BET151. Interestingly, the expression of SOCS3, and critical regulator in the negative feedback of the JAK-STAT pathway, is unaffected by this inhibitor. In IL-4 response, I-BET151 also exhibited differential inhibition of gene transcription. The inhibited genes included PPARγ, ENPP2, and MS4A4A but not some other genes important for certain biological activities such as JMJD3. Together, these results showed that I-BET151 targeted the transcriptional activity of subgroups of genes in JAK-STAT mediated M1 and M2 activity. The authors further addressed the mechanism underlying I-BET151-mediated transcriptional inhibition. In previous tumor studies, it has been discovered that I-BET151 and other related inhibitors such as JQ-1 could repress Myc expression. Since Myc is an important transcriptional factor in tumorigenesis, Myc-dependent gene expression was repressed by BET inhibition [34]. However, human macrophages differ from tumor models and suppress target genes independent of Myc, as the blocking of Myc function with a Myc inhibitor did not mimic I-BET151 administration. Furthermore, the block of protein synthesis did not abolish the inhibitory effect of I-BET151. These results suggest that gene transcriptional repression by I-BET151 is not mediated by newly synthesized transcriptional repressors, nor through inhibiting transcriptional regulators that are essential in JAK-STAT mediated gene expression. Thus, I-BET151 is likely to act directly on the target gene loci to inhibit immediate transcriptional activity rather than through indirect mechanisms. Finally, the authors examined the mechanism by which I-BET151 represses IFN- -mediated gene induction at the chromatin level. In line with the earlier result showing that STAT activation was intact with I-BET151 administration, neither STAT2 recruitment nor histone acetylation levels around CXCL10 promoter was affected by I-BET151 in IFN- -stimulated monocytes. On the contrary, the recruitment of BRD4 as well as pol II was blocked by I-BET151. This suggests that BET proteins function downstream of STAT recruitment in the assembly and stability of pol II transcriptional complex. Therefore, I-BET151 not only blocks transcriptional elongation, but also transcriptional initiation in this case. In addition, I-BET151 also appeared to interfere with the pol II enhancer interaction upstream of CXCL10 promoter, thus adding another layer of transcriptional repression. Conclusions and future directions The recent study expands our knowledge on the mechanism and effect of BET inhibition in human conditions. The effect of I-BET151 in human macrophage responses suggests that BET inhibition dampens inflammation at least partially through blocking pro-inflammatory cytokine transcription. The study suggests that I-BET151 function downstream of STAT recruitment at the specific gene loci to interfere and repress JAK-STAT target gene activation in human monocyte cytokine responses. The study further demonstrated the transcriptional inhibition occurs in a gene-specific manner, and thus implies greater specificity of BET inhibitors compared to JAK inhibitors in JAK-STAT mediated cytokine responses. The Myc-independent mechanism of I-BET151 inhibition also indicates that this inhibitor functions through different Page 3 of 5

4 mechanisms depending on the cell type or response, which bears implications on different treatment strategies for different diseases. For future directions, the study focused on genes known to be important for macrophage functions. Although the regulation of these genes yields valuable information on BET inhibition, the knowledge of how this inhibitor affects cell biology globally in these responses could be more instrumental for therapeutic application. This could be achieved by genome-wide gene expressional profiling using RNAseq. Furthermore, knowledge of the change of BET occupancy by I-BET151 and/or the occupancy of this inhibitor across the whole genome would be valuable for further investigation of the mechanism of this inhibitor. The characterization of the BET and inhibitor genome-wide occupancy would help evaluate the global impact of BET inhibition and its differential effect on transcriptional initiation and transcriptional elongation. Combined with RNAseq data, it would help to further dissect the direct and indirect targets for further construction of regulatory network of the effect of BET inhibition in cytokine responses. Another interesting area to explore is how I-BET151 affects super-enhancer structure. Super-enhancers are large clusters of regulatory regions typically covered by extensive histone acetylation, and have been associated with elevated gene expression in cancer studies [35]. BET inhibitor administration has been shown to repression cancer genes by disturbing super enhancers. It would be interesting to see whether I-BET also disrupt super-enhancers in human monocyte and macrophage responses as a mechanism of gene repression. Conflicting interests The authors have declared that no competing interests exist. References 1. Xue J, Schmidt SV, Sander J, Draffehn A, Krebs W, Quester I, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 2014; 40: Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature genetics 2003; 33 Suppl: Ong CT, Corces VG. Enhancer function: new insights into the regulation of tissue-specific gene expression. Nature reviews Genetics 2011; 12: Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung CW, et al. Suppression of inflammation by a synthetic histone mimic. Nature 2010; 468: Karouzakis E, Rengel Y, Jungel A, Kolling C, Gay RE, Michel BA, et al. DNA methylation regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts. Genes and immunity 2011; 12: Qiao Y, Giannopoulou EG, Chan CH, Park SH, Gong S, Chen J, et al. Synergistic activation of inflammatory cytokine genes by interferon-gamma-induced chromatin remodeling and toll-like receptor signaling. Immunity 2013; 39: Ostuni R, Piccolo V, Barozzi I, Polletti S, Termanini A, Bonifacio S, et al. Latent enhancers activated by stimulation in differentiated cells. Cell 2013; 152: West AC, Johnstone RW. New and emerging HDAC inhibitors for cancer treatment. The Journal of clinical investigation 2014; 124: Crea F, Fornaro L, Bocci G, Sun L, Farrar WL, Falcone A, et al. EZH2 inhibition: targeting the crossroad of tumor invasion and angiogenesis. Cancer metastasis reviews 2012; 31: He Y, Korboukh I, Jin J, Huang J. Targeting protein lysine methylation and demethylation in cancers. Acta biochimica et biophysica Sinica 2012; 44: Hargreaves DC, Horng T, Medzhitov R. Control of inducible gene expression by signal-dependent transcriptional elongation. Cell 2009; 138: Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, et al. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Molecular cell 2005;19: Bres V, Yoh SM, Jones KA. The multi-tasking P-TEFb complex. Current opinion in cell biology 2008; 20: Vahedi G, Takahashi H, Nakayamada S, Sun HW, Sartorelli V, Kanno Y, et al. STATs shape the active enhancer landscape of T cell populations. Cell 2012; 151: Gosmini R, Nguyen VL, Toum J, Simon C, Brusq JM, Krysa G, et al. The discovery of I-BET726 (GSK A), a potent tetrahydroquinoline ApoA1 up-regulator and selective BET bromodomain inhibitor. Journal of medicinal chemistry 2014; 57: Pastori C, Daniel M, Penas C, Volmar CH, Johnstone AL, Brothers SP, et al. BET bromodomain proteins are required for glioblastoma cell proliferation. Epigenetics: official journal of the DNA Methylation Society 2014; 9: Picaud S, Wells C, Felletar I, Brotherton D, Martin S, Savitsky P, et al. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, Bantscheff M, Chan WI, et al. Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 2011; 478: Qi J. Bromodomain and Extraterminal Domain Inhibitors (BETi) for Cancer Therapy: Chemical Modulation of Chromatin Structure. Cold Spring Harbor perspectives in biology 2014; 6:a Ceribelli M, Kelly PN, Shaffer AL, Wright GW, Xiao W, Yang Y, et al. Blockade of oncogenic IkappaB kinase activity in diffuse large B-cell lymphoma by bromodomain and extraterminal domain protein inhibitors. Proceedings of the National Academy Page 4 of 5

5 of Sciences of the United States of America 2014; 111: Wyce A, Degenhardt Y, Bai Y, Le B, Korenchuk S, Crouthame MC, et al. Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer. Oncotarget 2013; 4: Lamoureux F, Baud'huin M, Rodriguez Calleja L, Jacques C, Berreur M, Redini F, et al. Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle. Nature communications 2014; 5: Park-Min KH, Lim E, Lee MJ, Park SH, Giannopoulou E, Yarilina A, et al. Inhibition of osteoclastogenesis and inflammatory bone resorption by targeting BET proteins and epigenetic regulation. Nature communications 2014; 5: Jahagirdar R, Zhang H, Azhar S, Tobin J, Attwell S, Yu R, et al. A novel BET bromodomain inhibitor, RVX-208, shows reduction of atherosclerosis in hyperlipidemic ApoE deficient mice. Atherosclerosis 2014; 236: Belkina AC, Nikolajczyk BS, Denis GV. BET protein function is required for inflammation: Brd2 genetic disruption and BET inhibitor JQ1 impair mouse macrophage inflammatory responses. J Immunol 2013; 190: Meng S, Zhang L, Tang Y, Tu Q, Zheng L, Yu L, et al. BET Inhibitor JQ1 Blocks Inflammation and Bone Destruction. Journal of dental research 2014; 93: Klein K, Kabala PA, Grabiec AM, Gay RE, Kolling C, Lin LL, et al. The bromodomain protein inhibitor I-BET151 suppresses expression of inflammatory genes and matrix degrading enzymes in rheumatoid arthritis synovial fibroblasts. Annals of the rheumatic diseases 2014: Yarilina A, Park-Min KH, Antoniv T, Hu X, Ivashkiv LB. TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes. Nature immunology 2008; 9: Lawrence T, Natoli G. Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nature reviews Immunology 2011; 11: Gordon S. Alternative activation of macrophages. Nature reviews Immunology 2003; 3: Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nature reviews Immunology 2011; 11: Murray PJ, Wynn TA. Obstacles and opportunities for understanding macrophage polarization. Journal of leukocyte biology 2011; 89: Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nature reviews Immunology 2008; 8: Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, et al. BET bromodomain inhibition as a therapeutic strategy to target c-myc. Cell 2011;146: Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR, et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153: Page 5 of 5