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PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain

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PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Published OnlineFirst April 26, 2017; DOI: 10.1158/2159-8290.CD-16-1297 RESEARCH ARTICLE PAX3–FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability Berkley E. Gryder 1 , Marielle E. Yohe 1 , 2 , Hsien-Chao Chou 1 , Xiaohu Zhang 3 , Joana Marques 4 , Marco Wachtel4 , Beat Schaefer 4 , Nirmalya Sen 1 , Young Song 1 , Alberto Gualtieri 5 , Silvia Pomella 5 , Rossella Rota5 , Abigail Cleveland 1 , Xinyu Wen 1 , Sivasish Sindiri 1 , Jun S. Wei 1 , Frederic G. Barr 6 , Sudipto Das7 , Thorkell Andresson 7 , Rajarshi Guha 3 , Madhu Lal-Nag 3 , Marc Ferrer 3 , Jack F. Shern 1 , 2 , Keji Zhao8 , Craig J. Thomas 3 , and Javed Khan 1 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. 16-CD-16-1297_p884-899.indd 884 7/20/17 2:21 PM Published OnlineFirst April 26, 2017; DOI: 10.1158/2159-8290.CD-16-1297 ABSTRACT Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and ado- lescent young adults, driven primarily by the chimeric transcription factor PAX3–FOXO1. The mechanisms by which PAX3–FOXO1 dysregulates chromatin are unknown. We fi nd PAX3–FOXO1 repro- grams the cis -regulatory landscape by inducing de novo super enhancers. PAX3–FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3–FOXO1-occupied super enhancers. Furthermore, PAX3–FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a signifi cant susceptibility to BRD inhibition. These results yield insights into the epigenetic func- tions of PAX3–FOXO1 and reveal a specifi c vulnerability that can be exploited for precision therapy. SIGNIFICANCE: PAX3–FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin- level functions are critical to understanding its oncogenic activity. We fi nd that PAX3–FOXO1 estab- lishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3–FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. Cancer Discov; 7(8); 884–99. ©2017 AACR. INTRODUCTION oncogenic driver. Many other transcription/chromatin factor fusion gene–driven sarcomas have similar low mutational Transcription factors (TF) recognize specifi c noncoding burdens ( 5–7 ). Importantly, patients with RMS who harbor a sequences across the genome, recruiting epigenetic machin- PAX3 fusion are more likely to be metastatic at presentation, ery to regulate key cell identity genes, and are sequentially relapse despite aggressive therapy, and have very poor survival exchanged during development and differentiation ( 1 ). Onco- ( 8 ), underscoring the critical need to develop therapeutic genic fusion genes involving TFs are predicted to profoundly strategies for this subset of patients. alter normal developmental progression and cell identity in Early (PAX family TFs) and late (MYOG) regulators of many malignancies ( 2 ). normal myogenesis are temporally mutually exclusive in nor- Rhabdomyosarcoma (RMS) is a cancer of childhood and mal muscle development, yet FP-RMS tumors concurrently adolescence characterized by its inability to exit the pro- express high levels of PAX3–FOXO1 and the myogenic MYOD, liferative myoblast-like state. Genomic and transcriptomic MYOG, as well as MYCN ( 9, 10 ). Although the transcriptional characterization implicates either chromosomal transloca- perturbation caused by the PAX fusions has been previously tion resulting in the oncogenic fusion transcription factor reported ( 11, 12 ), the chromatin mechanisms by which PAX PAX3/7–FOXO1 (fusion-positive alveolar subtype, FP-RMS) fusions dysregulate the myogenic program are unknown. In or mutations in receptor tyrosine kinase/RAS pathways this work, we interrogated the underlying epigenetics that (fusion-negative embryonal subtype, FN-RMS; refs. 3, 4 ). enforce the myogenic and oncogenic transcriptional program FP-RMS is characterized by a strikingly low somatic muta- of cell lines and clinical tumor samples with PAX3 fusions. In tional burden indicating that the fusion gene is the primary charting the genome-wide landscape of histone modifi cations, we discovered that PAX3–FOXO1 drives expression of its target 1 Genetics Branch, NCI, NIH, Bethesda, Maryland . 2 Pediatric Oncology oncogenes by creating large deposits of active histone marks 3 Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. Divi- exclusively at enhancers, collaboratively with myogenic TFs, sion of Preclinical Innovation, National Center for Advancing Translational Sciences, NIH, Rockville, Maryland. 4 University Children’s Hospital, Zurich, and by recruiting chromatin reader bromodomain-containing Switzerland. 5 Department of Oncohematology, Ospedale Pediatrico protein 4 (BRD4), which function at looped enhancer–pro- Bambino Gesù Research Institute, Rome, Italy. 6 Laboratory of Pathology, moter pairs within topological domain boundaries. Integrat- NCI, NIH, Bethesda, Maryland. 7 Laboratory of Proteomics and Analytical ing epigenetic and mechanistic drug screening data exposed Technologies, Advanced Technologies Center, NCI, Frederick, Maryland. multiple biological nodes of chemical vulnerability, including 8 Systems Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland. BET bromodomains. The BRD4 inhibitor JQ1 has recently Note: Supplementary data for this article are available at Cancer Discovery shown effi cacy in RMS ( 13 ), but no mechanistic connection Online (http://cancerdiscovery.aacrjournals.org/). has been shown between BRD4 and PAX3–FOXO1. Here, we Corresponding Author: Javed Khan , NIH, 37 Convent Drive, Building 37, report that BRD4 inhibition disrupts a hitherto undiscovered Room 2016B, Bethesda, MD 20892. Phone: 240-760-6135; Fax: 301-480- PAX3–FOXO1 interaction with BRD4, causes a rapid degrada- 0314; E-mail: [email protected] tion of the fusion gene, and ablates its transcriptional output, doi: 10.1158/2159-8290.CD-16-1297 thus revealing a subtype-selective therapeutic vulnerability to © 2017 American Association for Cancer Research. BRD4 inhibition. AUGUST 2017 CANCER DISCOVERY | 885 Downloaded from cancerdiscovery.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. 16-CD-16-1297_p884-899.indd 885 7/20/17 2:21 PM Published OnlineFirst April 26, 2017; DOI: 10.1158/2159-8290.CD-16-1297 RESEARCH ARTICLE Gryder et al. A PAX3–FOXO1 peak distribution P3F H3K27ac H3K4me1H3K4me2 B Mean = 18 kb H3K27me3 PAX3–FOXO1 Nearest genes regions 106 4 5 3 PAX3–FOXO1 PAX3–FOXO1 PAX3–FOXO1 H3K27ac H3K27ac H3K27ac 105 H3K27me3 H3K27me3 H3K4me3 H3K4me3 H3K4me3 104 Signal (RPM) Signal (RPM) Signal (RPM) 103 Exon n = 17 0 0 0 2 10 Intergenic n = 1,488 –4 kb 0 +4 kb −2.5 kb TSS +2.5 kb –4 kb 0 +4 kb Intron n = 1,383 Distance to PAX3–FOXO1 Distance to H3K27me3 peak Distance to closest TSS (bp) Promoter n = 32 10 40 80 120 160 0 Count 400 –4 kb 0 +4 kb Peak signal -log10(q-value) C D % 1 2 3 H3K27ac signal at P3F sites = 334 1 7ac me me me 6me3 7me3 P3F n FP-RMS FN-RMS Myogenesis e PAX3–FOXO1 nome K2 K4 K4 K4 K3 K2 at occupancy St H3 H3 H3 H3 H3 CTCF RAD2 H3 Ge 1 10 24 82 41 11 51122 0.6 Poised promoter 2 83 14 90 100 4426140.6 Active promoter 3 95 95 100 49 49 52 70 7 0.7 Strong enhancer 4 73 76 73 215611 3 1.4 Enhancer 5 783020103.4 Weak enhancer 6 70 81 78 687921 4 0.4 Genic enhancer 7 21 10 16 586142271.3 Transcript.Transition = 446 n 8 100072 1203.3 Transcript. Elongation 9 000010 00012 Weak transcribed Recurrent enhancers 10 0000000053 Heterochromatin 11 312273783.2 Low signal 12 102212481 5.7 Polycomb repressed 13 000000010 14 Weak polycomb rep. = 1107 n 14 51017310 89 69 18 0.7 5 2 Domain insulator 4 3 4 5 RD 206 216 008 238 0100 200300 209 RH RH RH RH Frequency in state (%) CTR Adult 007 008 S S S S S I I Peaks per Gb of state SCMC SCMC otubes oblasts Muscle Skeletal RM RM RM RM RM NC NC My My Figure 1. Chromatin state mapping pinpoints PAX3–FOXO1 (P3F) in active enhancers. A, PAX3–FOXO1 peak distribution and heat maps of PAX3– FOXO1, H3K27ac, H3K4me1, and H3K4me2 at distal regulatory elements in PAX3–FOXO1-bearing cell line (RH4). The scatter plot is accompanied by a histogram showing the amounts of PAX3–FOXO1 in intronic, exonic, intergenic, or promoter-proximal sites. TSS, transcription start site. Rows are cen- tered around PAX3–FOXO1 peaks and extended 4 kb in each direction, sorted by PAX3–FOXO1 signal strength. B, PAX3–FOXO1, H3K27ac, H3K27me3, and H3K4me3 signal at PAX3–FOXO1 peaks (left), genes nearest to PAX3–FOXO1 peaks (center), and Polycomb-repressed chromatin (right). Mean dis- tance of PAX3–FOXO1 to its nearest genes (18 kb) is indicated. C, Chromatin states in FP-RMS cells (left) and abundance of PAX3–FOXO1 peaks per Gb of each state (right). States were discovered de novo using ChIP-seq data for all histone marks (plus, CTCF and RAD21) with the hidden Markov modeling algorithm chromHMM, which bins the genome into states by recurring patterns. Frequency corresponds to the probability of each mark being present in a given state. D, High-confidence PAX3–FOXO1 sites bound to enhancers recurrent n( = 1,107) in FP-RMS cell lines and tumors, some of which are shared with FN-RMS (n = 334) and/or myogenic cells and tissue (n = 446). RESULTS enhancer marks (Fig. 1A), including acetylation at histone 3 lysine 27 (H3K27ac) and H3 lysine 4 mono/dimethylation PAX3–FOXO1 Establishes Active Chromatin at (H3K4me1 and H3K4me2), but not the active promoter- Distal Enhancers associated mark H3K4me3 (Fig.
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