Published OnlineFirst April 20, 2016; DOI: 10.1158/2159-8290.CD-16-0217 RESEARCH ARTICLE BRD4 Connects Enhancer Remodeling to Senescence Immune Surveillance Nilgun Tasdemir 1 , 2 , 3 , Ana Banito 1 , Jae-Seok Roe 3 , Direna Alonso-Curbelo 1 , Matthew Camiolo 4 , Darjus F. Tschaharganeh 1 , Chun-Hao Huang 1 , 5 , Ozlem Aksoy 1 , 2 , 3 , Jessica E. Bolden 1 , Chi-Chao Chen 1 , 5 , Myles Fennell 1 , Vishal Thapar 1 , Agustin Chicas 1 , Christopher R. Vakoc 3 , and Scott W. Lowe 1 , 6 Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst April 20, 2016; DOI: 10.1158/2159-8290.CD-16-0217 A B S T R A C T Oncogene-induced senescence is a potent barrier to tumorigenesis that limits cellular expansion following certain oncogenic events. Senescent cells display a repressive chromatin confi guration thought to stably silence proliferation-promoting genes while simul- taneously activating an unusual form of immune surveillance involving a secretory program referred to as the senescence-associated secretory phenotype (SASP). Here, we demonstrate that senescence also involves a global remodeling of the enhancer landscape with recruitment of the chromatin reader BRD4 to newly activated super-enhancers adjacent to key SASP genes. Transcriptional profi ling and functional studies indicate that BRD4 is required for the SASP and downstream paracrine signaling. Consequently, BRD4 inhibition disrupts immune cell–mediated targeting and elimination of premalignant senescent cells in vitro and in vivo . Our results identify a critical role for BRD4-bound super-enhancers in senes- cence immune surveillance and in the proper execution of a tumor-suppressive program. SIGNIFICANCE: This study reveals how cells undergoing oncogene-induced senescence acquire a dis- tinctive enhancer landscape that includes formation of super-enhancers adjacent to immune-modula- tory genes required for paracrine immune activation. This process links BRD4 and super-enhancers to a tumor-suppressive immune surveillance program that can be disrupted by small molecule inhibitors of the bromo and extra terminal domain family of proteins. Cancer Discov; 6(6); 612–29. ©2016 AACR. See related commentary by Vizioli and Adams, p. 576. INTRODUCTION Beyond the cell-cycle arrest phenotype, senescent cells dis- play marked changes in cell morphology, show increased Cellular senescence is a cell-cycle arrest program that lim- senescence-associated β -galactosidase (SA- β -gal) activity, and its proliferation of damaged cells and can be triggered in undergo dramatic changes in higher-order chromatin organi- response to diverse forms of cellular stress ( 1, 2 ). Although zation that are associated with a distinct transcriptional initially defi ned as a phenotype of cultured fi broblasts ( 3 ), profi le ( 2 ). Mechanistically, the best-understood feature of cellular senescence appears to have a broad impact on cellular senescence is its stable cell-cycle arrest program, human health and disease. For example, cellular senescence which renders senescent cells insensitive to mitogenic stimuli can restrict tumorigenesis ( 4, 5 ) and modulate chemotherapy and undoubtedly contributes to key senescence phenotypes responses ( 6–8 ), and may exert a primordial role in wound ( 2, 19 ). This arrest program involves a complex interplay healing, tissue remodeling, and repair ( 9–13 ). Moreover, between the RB and p53 tumor suppressor pathways ( 19 ), senescent cells have been observed in aged or damaged tis- and is accompanied by marked changes in the chromatin sues ( 14 ) and can contribute to the decline of tissue regen- landscape involving the redistribution of cellular heterochro- eration capacity with age ( 15, 16 ). Accordingly, disruption of matin and leading to stable repression of cell-cycle–regula- senescence regulators can promote cancer or impair wound tory genes ( 20, 21 ). Such a potently repressive chromatin healing, and the elimination of senescent cells from tissues environment presumably buffers against the activation of can enhance lifespan in mice ( 17, 18 ). proliferation genes in response to mitogenic cues. Conversely, senescent cells activate the expression of genes known to infl uence the tissue microenvironment, including 1 Memorial Sloan Kettering Cancer Center, New York, New York. 2 Watson matrix remodeling enzymes, growth factors, and immune- 3 School of Biological Sciences, Cold Spring Harbor, New York. Cold Spring modulatory cytokines ( 22, 23 ). As a consequence, senescent Harbor Laboratory, Cold Spring Harbor, New York. 4 Medical Scientist Training Program, Stony Brook University, Stony Brook, New York. 5 Weill cells display qualitative and quantitative changes in their Graduate School of Medical Sciences, Cornell University, New York, New secretome, a feature collectively referred to as the senescence- York. 6 Howard Hughes Medical Institute , New York, New York. associated secretory phenotype (SASP; refs. 24, 25 ). This Note: Supplementary data for this article are available at Cancer Discovery gene activation program is controlled in part by factors such Online (http://cancerdiscovery.aacrjournals.org/). as NF κ B ( 26 ), C/EBP β ( 27 ), mTOR ( 28, 29 ), MacroH2A.1 N. Tasdemir, A. Banito, J.-S. Roe, and D. Alonso-Curbelo contributed equally ( 30 ), and GATA4 ( 31 ). Among their many functions, SASP to this article. proteins mobilize immune cells and modify their activities, Corresponding Authors: Scott W. Lowe, Memorial Sloan Kettering Cancer for example, by recruiting natural killer (NK) cells or alter- Center, 417 E 68th Street, New York, NY, 10065. Phone: 646-888-3342; ing macrophage polarization to kill and/or engulf senescent Fax: 646-888-3347; E-mail: [email protected] ; and Christopher R. Vakoc, Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, cells ( 9, 32–34 ). These immune-modulatory activities may NY 11724 . Phone: 516-367-5045; Fax: 516-367-8453; E-mail: vakoc@ be important for the resolution of certain wound-healing cshl.edu responses ( 9, 10, 13 ) and underlie a unique form of immune doi: 10.1158/2159-8290.CD-16-0217 surveillance that can eliminate premalignant or malignant ©2016 American Association for Cancer Research. cells undergoing senescence in vivo ( 33, 35, 36 ). JUNE 2016CANCER DISCOVERY | 613 Downloaded from cancerdiscovery.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst April 20, 2016; DOI: 10.1158/2159-8290.CD-16-0217 RESEARCH ARTICLE Tasdemir et al. As is clear from the above, the biological roles of senes- although such quiescent cells can be triggered to re-enter the cence rely on gene repression and activation programs that cell cycle upon serum addition, senescent cells cannot ( 2 ). substantially alter cell function and fate. In certain devel- Following lysis of cross-linked proliferating, senescent, and opmental and pathologic contexts, such dramatic gene quiescent IMR90 cells, the enhancer landscape was visualized expression changes rely on dynamic remodeling of enhancer using chromatin immunoprecipitation coupled with high- landscapes, regulatory regions that when activated induce throughput sequencing (ChIP-Seq) using the histone modi- gene expression and maintain downstream biological cir- fi cation H3K27Ac as a marker of active enhancers ( 45 ). This cuits. For example, in embryonic stem cells (ESC), excep- analysis identifi ed a total of approximately 33,000 H3K27Ac- tionally large stretches of H3K27Ac-marked regulatory detectable enhancers in proliferating, quiescent, and senes- elements, termed super-enhancers (SE), are associated with cent IMR90 cells (union enhancers; Fig. 1A ). Previous studies genes that play prominent roles in ESC self-renewal ( 37, have documented distinct classes of enhancers termed SEs, 38 ). In other cell types, SEs can contribute to lineage- and which accumulate disproportional levels of specifi c chroma- cellular state–specifi c transcription, for example, by sustain- tin modifi cations and are associated with key cell identity ing the expression of the oncogene MYC in leukemia and genes ( 38, 39 ). By implementing the previous defi nition of some solid tumors ( 37, 39 ) or driving proinfl ammatory SEs based on H3K27Ac ChIP-Seq profi ling ( 39 ), we identifi ed responses in endothelial cells ( 40 ). Importantly, transcrip- approximately 1,250 SEs present across the three cell condi- tion of SE-associated genes relies on dense accumulation tions and classifi ed the approximately 32,000 remaining as of cofactors such as the bromo and extra terminal domain typical enhancers (TE; Fig. 1B and C ). (BET) proteins, and is highly sensitive to perturbations in To reveal enhancer dynamics during OIS, we next com- cofactor binding ( 39, 40 ). BET proteins such as BRD4 bind pared the H3K27Ac levels at enhancers in senescent cells to chromatin in a manner that can be disrupted with small- to those in proliferating cells. Based on a cut-off of 2-fold molecule drugs, and, as such, BET inhibitors are currently in H3K27Ac enrichment over the proliferating condition, our development to suppress certain hyperinfl ammatory diseases analysis called approximately 6,500 senescence-activated and cancer ( 41–43 ). TEs and approximately 7,090 senescence-inactivated TEs Studies to date have explored enhancer dynamics in ESC ( Fig. 1B ; Supplementary Table S1A and S1B), as well as 198 self-renewal, cell lineage specifi cation, infl ammation, and senescence-activated SEs