Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition
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Published OnlineFirst February 21, 2013; DOI: 10.1158/2159-8290.CD-12-0418 RESEARCH ARTICLE Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition Alexandre Puissant1,3, Stacey M. Frumm1,3, Gabriela Alexe1,3,5,6, Christopher F. Bassil1,3, Jun Qi2, Yvan H. Chanthery8, Erin A. Nekritz8, Rhamy Zeid2, William Clay Gustafson8, Patricia Greninger7, Matthew J. Garnett10, Ultan McDermott10, Cyril H. Benes7, Andrew L. Kung1,3, William A. Weiss8,9, James E. Bradner2,4, and Kimberly Stegmaier1,3,6 Downloaded from cancerdiscovery.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. 15-CD-12-0418_p308-323.indd 1 22/02/13 12:15 AM Published OnlineFirst February 21, 2013; DOI: 10.1158/2159-8290.CD-12-0418 A BSTRACT Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bro- modomain inhibitors in patients with neuroblastoma. SIGNIFICANCE: Biomarkers of response to small-molecule inhibitors of BET bromodomains, a new com- pound class with promising anticancer activity, have been lacking. Here, we reveal MYCN amplification as a strong genetic predictor of sensitivity to BET bromodomain inhibitors, show a mechanistic rationale for this finding, and provide a translational framework for clinical trial development of BET bromodomain inhibitors for pediatric patients with MYCN-amplified neuroblastoma.Cancer Discov; 3(3); 1–16. ©2012 AACR. See related commentary by Schnepp and Maris, p. 255. INTRODUCTION Modulation of the epigenetic regulators known as “read- ers” has recently emerged as a therapeutic strategy in cancer The interplay between master regulatory transcription treatment. These epigenetic “readers” are structurally diverse factors and specific chromatin-associated coactivators is an proteins, which recognize and bind to covalent modifica- emerging hallmark of cancer. Cancer genomic discovery efforts tions of chromatin (2). One important modification associ- continue to reveal mutations in epigenetic modifiers, and labo- ated with open chromatin and transcriptional activation is ratory efforts to validate functional dependencies are ongo- the side-chain acetylation of lysine residues on histone tails ing (1). Moreover, notable examples of successful commercial (3). The dominant mode of recognition of acetylated lysine development of drugs targeting epigenetic modifiers have residues is by bromodomains present in 47 human proteins come to the fore, including the development of inhibitors of (4–6). We and others have described the therapeutic potential enzymatic “writers,” such as DNA methyltransferases for mye- of targeting one bromodomain-containing family impor- lodysplastic syndrome, and inhibitors of enzymatic “erasers,” tant in regulating transcription, epigenetic memory, and such as histone deacetylases for cutaneous T-cell lymphoma. cell growth: The bromodomain and extraterminal domain (BET) family is composed of BRD2, BRD3, BRD4, and BRDT. Numerous hematologic malignancies and the highly malig- Authors’ Affiliations: Departments of 1Pediatric Oncology and 2Medical nant solid tumor NUT midline carcinoma are responsive to Oncology, Dana-Farber Cancer Institute; 3Boston Children’s Hospital; BET inhibition in vitro and in mouse models (7–12). 4Department of Medicine, Harvard Medical School; 5Bioinformatics Graduate Although disease-specific indications for drugs modifying Program, Boston University, Boston; 6The Broad Institute of Harvard Univer- sity and Massachusetts Institute of Technology, Cambridge; 7Massachusetts epigenetic regulators have been uncovered, precise genomic General Hospital Cancer Center, Harvard Medical School, Charlestown, Mas- biomarkers predictive of treatment response remain elusive. sachusetts; 8Department of Pediatrics, Helen Diller Family Comprehensive To date, the best validated genetic predictor of response to Cancer Center; 9Departments of Neurology and Neurosurgery, Brain Tumor BET inhibitors is in a rare genetically defined subset of poorly Research Center, University of California, San Francisco, San Francisco, differentiated squamous cell carcinomas (NUT midline car- California; and 10Cancer Genome Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom cinoma), in which the presence of recurrent t(15;19) chro- Note: Supplementary data for this article are available at Cancer Discovery mosomal translocation results in the expression of the twin Online (http://cancerdiscovery.aacrjournals.org/). N-terminal bromodomains of BRD4 as an in-frame fusion A. Puissant and S.M. Frumm contributed equally to this work. with the NUT protein (13). High-throughput pharmacoge- Corresponding Authors: Kimberly Stegmaier and James E. Bradner, Dana- nomic profiling offers the opportunity to reveal new insights Farber Cancer Institute, Boston, MA 02215. Phone: 617-632-4438; Fax: into selective responses to drugs in defined cancer genotypes. 617-632-4850; E-mail: [email protected]; and James Initial efforts to connect drug response with genotype in the E. Bradner, [email protected] NCI60 cell line panel have since been expanded to screening doi: 10.1158/2159-8290.CD-12-0418 campaigns in large panels of genetically characterized cancer ©2012 American Association for Cancer Research. cell lines (14–17). These efforts have revealed both expected March 2013 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. 15-CD-12-0418_p308-323.indd 2 22/02/13 12:15 AM Published OnlineFirst February 21, 2013; DOI: 10.1158/2159-8290.CD-12-0418 RESEARCH ARTICLE Puissant et al. and unexpected connections. For example, the anticipated to JQ1 and a panel of structurally distinct BET bromodo- response to anaplastic lymphoma receptor kinase (ALK) main inhibitors, each of which conferred a dose-responsive, inhibitors in tumors with aberrant ALK activation, such as inhibitory effect on cell viability as well as growth over time lymphoma, non–small cell lung cancer, and neuroblastoma, (Fig. 1D–F and Supplementary Table S2). The only exception was shown in a screen of more than 600 tumor cell lines (15). in this cell line panel was NGP, which was comparatively More recently, the unexpected connections between response insensitive to all of the BET bromodomain inhibitors tested. to PARP inhibitors and expression of the EWS/FLI fusion Importantly, cell growth was not affected in any cell lines protein in Ewing sarcoma was elucidated in a screen of 130 by the (−)-JQ1 enantiomer, which lacks activity against BET drugs in more than 600 cancer cell lines (16). In an independ- bromodomains in biochemical and biologic assays, further ent study of 24 anticancer drugs in 479 human cancer cell supporting an on-target mechanism of action. Similarly, we lines, new connections were also observed between small- retested one of the least sensitive cell lines from the primary molecule sensitivities and cell lineage, gene expression, and screen, the MYCN–wild-type neuroblastoma cell line SK-N- genotype (17). AS and a second MYCN–wild-type cell line SH-SY5Y not in We conducted a high-throughput pharmacogenomic the primary screen. As predicted, SK-N-AS was insensitive screen to identify biomarkers of response to BET bromodo- to the effects of JQ1, and SH-SY5Y was less sensitive than main inhibitors. The prototype ligand JQ1, a novel thieno- the MYCN-amplified cell lines based on Emax (Supplementary triazolo-1,4-diazepine, which displaces BET bromodomains Fig. S2A and S2B and Supplementary Table S2). from chromatin by competitively binding to the acetyl lysine recognition pocket, has been validated in numerous mod- JQ1 Induces Cell-Cycle Arrest and Apoptosis els, nominating it as an excellent chemical probe for high- To further characterize the phenotypic consequences of throughput screening (7–10). In this study, we therefore JQ1 treatment on responsive MYCN-amplified neuroblas- queried a large compendium of genetically characterized toma cells, we determined its effects on cell-cycle arrest tumor cell lines to identify predictors of sensitivity to JQ1. We and apoptosis. JQ1 treatment induced a G0–G1 arrest and a identified MYCN amplification as a top predictive marker of decrease in S-phase at 24 hours by flow-cytometric evaluation response to JQ1 treatment and characterized the mechanistic of propidium iodide (PI) staining (Fig. 2A and Supplemen- and translational significance of this