targeting mycn-driven transcription by bet-bromodomain … · 2021. 8. 2. · cancer therapy:...

Cancer Therapy: Preclinical

Targeting MYCN-Driven Transcription ByBET-Bromodomain InhibitionAnton Henssen1,2, Kristina Althoff2,3, Andrea Odersky2, Anneleen Beckers4,Richard Koche5, Frank Speleman4, Simon Sch€afers2,3, Emma Bell6, Maike Nortmeyer6,Frank Westermann6, Katleen De Preter4, Alexandra Florin7, Lukas Heukamp8,9, AnnikaSpruessel2, KathyAstrahanseff10, SvenLindner2, Natalie Sadowski2, Alexander Schramm2,Lucile Astorgues-Xerri11, Maria E. Riveiro11, Angelika Eggert10, Esteban Cvitkovic11,12, andJohannes H. Schulte2,3,6,13,14

Abstract

Purpose: Targeting BET proteins was previously shown tohave specific antitumoral efficacy against MYCN-amplifiedneuroblastoma. We here assess the therapeutic efficacy of theBET inhibitor, OTX015, in preclinical neuroblastoma modelsand extend the knowledge on the role of BRD4 in MYCN-drivenneuroblastoma.

Experimental Design: The efficacy of OTX015 was assessed inin vitro and in vivo models of human and murine MYCN-drivenneuroblastoma. To study the effects of BET inhibition in thecontext of high MYCN levels, MYCN was ectopically expressedin human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4and H3K27Ac chromatin immunoprecipitation coupled withDNA sequencing (ChIP-Seq) and gene expression analysis inneuroblastoma cells treated with OTX015 compared with vehiclecontrol.

Results: OTX015 showed therapeutic efficacy against preclin-ical MYCN-driven neuroblastoma models. Similar to previouslydescribed BET inhibitors, concurrent MYCN repression wasobserved in OTX015-treated samples. Ectopic MYCN expression,however, did not abrogate effects of OTX015, indicating thatMYCN repression is not the only target of BET proteins inneuroblastoma. When MYCN was ectopically expressed, BETinhibition still disruptedMYCN target gene transcription withoutaffecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specificallydisrupts BRD4 binding and transcription of these genes.

Conclusions:We show that OTX015 is effective against mouseand human MYCN-driven tumor models and that BRD4 notonly targets MYCN, but specifically occupies MYCN target geneenhancers as well as other genes associated with super-enhancers.Clin Cancer Res; 22(10); 2470–81. �2015 AACR.

IntroductionTargetingwhat has been recently coined as super-enhancers has

emerged as a powerful therapeutic strategy in cancer treatment(1). Super-enhancers have been shown to epigenetically regulatethe transcription of specific gene sets, and are most abundantupstream of oncogenes such as MYC (1). Epigenetic readersrecognize and bind to covalent chromatin modifications insuper-enhancer regions, influencing expression of associatedgenes (1). These regions are enriched with acetylated histoneH3 at lysine K27 (H3K27Ac; ref. 2). The bromodomain andextra-terminal domain (BET) protein family, composed of BRD2,BRD3, BRD4, andBRDT recognize andbind histone acetylation atsuper-enhancer sites (1, 3, 4). BRD4 interacts with the positivetranscription elongation factor b (P-TEFb; ref. 5). Through itsinteraction with BRD4, P-TEFb is recruited to promoters tophosphorylate RNA polymerase II. BRD4, thus, plays an impor-tant role in regulating gene expression by retaining P-TEFb at thepromoters of genes associated with super-enhancer regions (6, 7).

Neuroblastomas are embryonal childhood tumors of neuroec-todermal origin. They are the most common extracranial solidtumors in children, accounting for more than 15% of all child-hood cancer-related deaths (8). Despite multimodal therapy, therelapse rate of patientswithhigh-risk neuroblastoma exceeds 50%and is almost always fatal (9). Targeted therapies with low toxicity

1Molecular Pharmacology&Chemistry Program, Sloan Kettering Insti-tute, Memorial Sloan Kettering Cancer Center, New York, New York.2Department of Pediatric Oncology and Hematology, University Chil-dren's Hospital Essen, Essen, Germany. 3German Consortium forTranslational Cancer Research (DKTK), Partner Site Essen/Duessel-dorf, Essen, Germany. 4Center of Medical Genetics Ghent (CMGG),Ghent University Hospital, Ghent, Belgium. 5Human Oncology andPathogenesis Program, Memorial Sloan Kettering Cancer Center, NewYork, New York. 6Neuroblastoma Genomics, B087, German CancerResearch Center (DKFZ), Heidelberg, Germany. 7Institute of Patholo-gy, University Hospital Cologne, Cologne, Germany. 8New Oncology,K€oln, Germany. 9Institut f€ur H€amatopathologie Hamburg, Hamburg,Germany. 10Department of Pediatric Oncology/Hematology, Charit�e-Universit€atsmedizin Berlin, Berlin, Germany. 11Oncology TherapeuticDevelopment, Clichy, France. 12Oncoethix, Lausanne, Switzerland.13Translational Neuro-Oncology,West German Cancer Center (WTZ),University Hospital Essen, University Duisburg-Essen, Essen, Ger-many. 14Centre forMedical Biotechnology, University Duisburg-Essen,Essen, Germany.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A. Henssen and K. Althoff contributed equally to this article.

Corresponding Author: Anton Henssen, Memorial Sloan Kettering CancerCenter, 1275 York Avenue, New York, NY 10021. Phone: 917-283-0458; Fax:646-888-3557; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-1449

�2015 American Association for Cancer Research.

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would be a clear improvement over the aggressive cytotoxic agentscurrently used to treat children with high-risk neuroblastomas.The MYCN transcription factor is a central oncogenic driver inneuroblastoma (8).MYCN amplification is an adverse prognosticfactor used for patient stratification inmost neuroblastoma trials.As MYCN-driven neuroblastomas are addicted to high levels ofMYCN expression, development of targeted therapies function-ally disrupting MYCN is expected to be of therapeutic value forpatients with these tumors.

We and others have recently discovered that targeting BRD4with JQ1 disrupts MYCN transcription leading to significantantitumoral effects in neuroblastoma models (10–12). Mostcompelling are previous observations thatMYCN amplificationappeared to be the strongest positive predictor of sensitivity toBRD4 inhibition. However, the reason why MYCN-driven neu-roblastoma models were more susceptible to BRD4 inhibitionis still unclear. Considering recent observations describing theimportance of BRD4 binding to super-enhancers, we sought touncover if and how super-enhancer disruption contributes tothe antitumoral effects of BRD4 inhibition in MYCN-drivenneuroblastoma. OTX015 (Oncoethix) is a small molecule thatprevents BRD2/3/4 from binding to acetylated histones (13).Recently, OTX015 was shown to be active in the preclinicalmodels of acute leukemia and B-cell lymphoma (14, 15).Cellular uptake of OTX015 was shown to be rapid (<5 minutes)in both sensitive and resistant cell lines (16). Extracellularlevels of OTX015 remain stable up to 6 hours after exposure,and downstream transcriptional changes are observed as soonas 2 hours after exposure (16). OTX015 is orally bioavailableand clinical phase I/II trials in adults have shown promisingpharmacologic properties (refs. 17, 18; NCT01713582,NCT02259114, NCT02296476). No dose-limiting toxicity wasobserved for up to 80 mg OTX015 once daily or 40 mg twicedaily. These doses resulted in plasma trough concentrations>250 nmol/L, thus, well in excess of previously reported in vitroIC50 concentrations. Adverse events were mainly hematologicand gastrointestinal, including aggravation of pre-existent dia-betes, thrombocytopenia, neutropenia, diarrhea, and elevatedtransaminases. No cumulative toxicity was observed (16).Encouraged by the promising antitumoral activity of BET inhi-bitors, we here evaluated whether OTX015 is suited for furtherclinical testing in pediatric patients with neuroblastoma.

Materials and MethodsCell culture and reagents

Reagents were obtained from Sigma-Aldrich Chemie GmbH,and materials from Carl Roth GmbH & Co.KG unless otherwisespecified. OTX015 (Oncoethix SA) was provided as a powder. Forin vitro experiments, OTX015 was dissolved in DMSO. The Chp-212, Chp-134, GI-M-EN, IMR-5, IMR-32, NB69, SK-N-AS, SK-N-BE, and SK-N-BE (2) human neuroblastoma cell lines wereobtained from the German Collection of Microorganisms andCell Cultures (DSMZ) or the ATCCwithin the last 3 years. Humanforeskin fibroblasts were obtained from the ATCC (CRL-2522) in2013. The mNB-A1 murine cell line was described previously(12). The identity of all cell lines was verified at the DSMZ usingShort Tandem Repeats (STR) genotyping in 2014.Mycoplasma sp.contamination was excluded as described previously (13). Celllines were cultured under standard conditions in RPMI1640supplemented with 10% FCS and antibiotics.

Cell viability, death, proliferation, and cycle analysesCell lines were treated with 0.1 to 6,000 nmol/L OTX015 for

72hours andcell viabilitywasassessedusing theMTTassay(Roche)as previously described (13). GraphPad Prism 5.0 (GraphPadSoftware Inc.) was used to calculate 50% inhibition of growth(IC50) andmaximal effect of the highest OTX015 dose (6 mmol/L).Apoptosis and cell proliferation were assessed after 72-hour treat-ment with 250 and 500 nmol/L OTX015 using the Cell DeathDetection or BrdU ELISA (Roche) assays performed according tothe manufacturer's protocols, respectively. For cell-cycle analysis,cell lines were treated for 72 hours with 500 nmol/L OTX015 orDMSO. Cells were harvested and incubated with propidiumiodide (100 mg/mL) to stain DNA and RNAse (1 mg/mL). CellularDNA content was analyzed on a FC500 flow cytometer (BeckmanCoulter). Cell survival after treatment with 250, 500, and 1,000nmol/L OTX015 or 0.2% DMSO was also evaluated in real-timeusing the xCELLigence system (ACEABiosciences Inc.) as describedbefore (13). Adherence to the culture plates was continuouslymonitored for 120 hours (or until cell growth reached a plateau).Proliferative capacity was calculated by means of the slope usingRTCA Software version 2.0 (ACEA).

Affymetrix microarray analysisIMR-5 and mNB-A1 cells were plated at 1 � 105 cells/well in

6-well plates, cultured overnight, then treated in triplicate with0.2% DMSO (control), 500 nmol/L OTX015 or 500 nmol/L JQ1for 24 hours. Total RNA was extracted using the RNeasyMini kit(Qiagen) according to the manufacturer's instructions. Human-derived samples were profiled using the HG-U133 Plus 2.0Human Gene Expression Array (Affymetrix), and murine-derivedsamples using the MG-MM4302 Murine Gene Expression Array(Affymetrix), using established protocols (12). Microarray CELfiles were normalized and summarized to gene level using theBioconductor R statistical language repository for gcRMAnormal-ization (19). Probes forwhich the log2 expressionwas<4 in>10%of samples were defined as "not expressed" and were not used inanalyses. The strength of similarity between different samples wasevaluated usingWard–Manhattan clustering performed using the50 genes with the highest SD over all analyzed samples. Differ-ential expression analysis was performed using Rank ProductAnalysis in R (v 2.13, RankProd package; ref. 20). Hierarchicalclustering of the Manhattan distance of log2 expression values of

Translational Relevance

We provide preclinical data about the BET inhibitor,OTX015, and mechanistic insight into BRD4 functions inMYCN-amplified neuroblastoma. We show that BRD4 specif-ically occupies MYCN target genes as well as other genesassociated with super-enhancers. OTX015 specifically disruptsBRD4 binding to chromatin and mouse models of MYCN-driven neuroblastoma treated with OTX015 show significantsurvival advantage compared with untreated controls.OTX015 therefore has the potential to generate a measurableresponse in patients with high-risk MYCN-driven neuroblas-toma. This new insight should provide a translational frame-work for clinical trial development of OTX015 for pediatricpatients with MYCN-amplified neuroblastoma.

Targeting MYCN in Neuroblastoma

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the 50most differentially expressed geneswas applied to visualizedifferential gene expression after treatment. Gene set enrichmentanalysis (GSEA) was conducted using GSEA version 2.0 (www.broadinstitute.org/gsea) and the c2.cgp.v4.0.symbols.gmt and c6.all.v4.0.symbols.gmt gene set collections (21). Genes were rankedusing a signal-to-noise ranking metric calculated using the differ-ence of the comparator population means scaled by SD. MYCNgene signature scores were calculated using a previously reportedalgorithm(22). The following gene signatureswere used to analyzeMYCN activity: MYCN-regulated genes as previously defined byWestermann and colleagues (23), JQ1-responsive genes as previ-ously defined by Puissant and colleagues (11) and MYCN signa-tures retrieved from curated gene sets (c2) in MSigDB, version 4.0(24). Microarray data are available in the ArrayExpress database(www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3672and are in a format conforming to theMinimumInformationAbout a Microarray Gene Experiment (MIAME) guidelines of theMicroarray Gene Expression Data society (MGED).

Chromatin immunoprecipitation-sequencing and PCR(ChIP-PCR) of H3K27Ac and BRD4

IMR-5 cells were seeded at 1 � 107 cells/175 cm2 cell cultureflask, and harvested either after adherence (ChIP-seq forH3K27Ac) or after 24-hour treatment with 500 nmol/L OTX015or DMSO control medium (ChIP-PCR and ChIP-seq for Brd4).After harvesting, 5 � 106 cells were cross-linked with 1% form-aldehyde in 1 mL PBS for 7 minutes at room temperature. Cross-linking was stopped by adding 50 mL of 2.5 mol/L glycine for 5minutes and cell pelletswerewashed twicewith PBS then stored at�80�C. ChIP pull-downs were prepared by Zymo Research usingfrozen cross-linked cells and the following antibodies: anti-BRD4(A301-985A50, Bethyl Laboratories), anti-H3K27ac (ab4729, lot#GR144577-2; Abcam) and normal rabbit IgG (12-370, lot#2295402; Millipore). H3K27Ac ChIP DNA enriched in threeindependent ChIP assays was verified by qPCR using positivecontrol primers for the human RPL10 promoter. ChIP-seq librar-ies were sequenced on the HiSeq next-generation sequencingplatform. Samples had >40 million raw reads that were trimmedof low-quality and adapter sequences. Read alignment to thehuman hg19 genome assembly was performed using bowtie2with default settings, which achieved >93% sequence mapping.Genome browser maps were created by extending reads to 200 bpand computing the density using BEDTools (25). All ChIP-seqand input maps (H3K27Ac and BRD4) were normalized forsequencing depth by scaling to 10 million reads. Previouslypublished primers for the MYCN promoter were used for BRD4ChIP-PCR afterOTX015or JQ1 treatment (11, 26). Real-time PCRwas used to analyze BRD4 abundance at MYCNpromoter regionsbefore and after treatment with OTX015, JQ1, or the DMSOcontrol as previously described (11). GSEA was performed usingthe preranked option, whereby each promoter region (�2 kb ofstart site) was ranked by the log2 ratio of the normalized readcounts after OTX015 versus DMSO treatment. MYCN target genesets defined in the studies by Valentijn and colleagues andWestermann and colleagues were tested for enrichment in genesdifferentially bound by BRD4 (23, 27). For composite plots, theChIP-seq signal from cells treated with OTX015, JQ1 or DMSOwas averagedover 25bpbins inpromoters andputative enhancerscontaining BRD4 peaks in the DMSO-treated sample. MACS v1.4with default parameters was used to identify genome-wide enrich-ment of the ChIP signal relative to input in chromosomal regions

(28). Putative enhancers were defined as enriched segmentslocated at least 5 kb from a transcriptional start site documentedin RefSeq (http://www.ncbi.nlm.nih.gov/refseq/). Putative super-enhancers were identified as previously described (29). Briefly,enriched segments within 12.5 kbweremerged, and a normalizedChIP signal calculated and plotted against signal rank. Thethreshold for putative super-enhancers was defined by the pointat which the y¼ x line was tangent to the signal-rank curve. Geneswere assigned to super-enhancers if theyoccurredwithin100kbofthe enhancer.

Real-time reverse transcriptase PCRTotal RNA was isolated from cells using the RNeasyMini Kit

(Qiagen) and cDNA synthesiswas generatedusing the SuperScriptreverse transcription kit (Invitrogen). Quantitative mRNA expres-sion levels were monitored using Assays-on-Demand (AppliedBiosystems). Alternatively, fluorescence-based kinetic RT-PCRwas performed using the StepOnePlus PCR platform (AppliedBiosystems) in combination with the SYBR Green I intercalatingfluorescent dye according to the standard protocol. Expressionwas calculated using the DCt method and GAPDH expression asinternal reference. Data analysis was performed using theqbasePLUS software, version 1.5 (http://www.biogazelle.com).

Western blot analysisProtein lysate preparation and blotting has been described

previously (13). Primary antibodies against the following pro-teins were used: BRD4 (1:500; ab75898, Abcam), MYC (1:1,000;#9402, Cell Signaling Technology), Cyclin D1 (1:200; sc-753,Santa Cruz Biotechnology), E2F1 (1:500; AF4825, R&D Systems),and MYCN (1:1,000; #9405, Cell Signaling Technology). b-Actin(1:2,000; A3853, Sigma-Aldrich) or GAPDH (1:2,000; MA3374,Millipore) were used as loading controls. Horseradish peroxidase(HRP)-conjugated secondary antibodies, anti-mouse IgG(#NA9310V; GE Healthcare), anti-rabbit IgG (#NA9340V; GEHealthcare) or anti-sheep IgG (#HAF016; R&D Systems), diluted1:2,000 in 5% dry milk were used. Proteins were visualized usingthe ECLplus Western Blotting Detection Kit (GE Healthcare) andanalyzed on the FusionFX7 detection device (Peqlab).

OTX015 treatment of IMR-5 neuroblastoma xenograft tumorsin nude mice

IMR-5 cells (2 � 107) were suspended in 200 mL Matrigel (BDBiosciences) and subcutaneously inoculated into the left flank of6-week-old female athymic (nu/nu) mice. Mice were randomlyassigned to vehicle control (100mLwater) orOTX015 (n¼9mice/group) after tumors reached 150–200 mm3 in size. OTX015 wasadministered by oral gavage at 25 or 50mg/kg body weight daily,or 25 mg/kg body weight twice daily. Tumor growth was mon-itored using calipers, and tumor volume was calculated using theformula (width� length�height)/2.Micewere sacrificed after 42days of treatment or when tumor size exceeded 2,500 mm3. Sixdoses of 100mgOTX015/kg bodyweightwere administered twicedaily over 3 days to mice whose xenograft tumors were to beexamined for apoptosis (cleaved caspase-3) and proliferation(Ki-67). Mice were sacrificed by cervical dislocation 4 hours afterthe final OTX015 dose. Xenograft tumors were excised, anddivided in half. One half was snap-frozen in liquid nitrogen thenstored at �80�C and the other half was formalin-fixed andparaffin-embedded for immunohistochemical analyses. All ani-mal experiments were performed in accordance with the Council

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of Europe guidelines for accommodation and care of laboratoryanimals. Protocols were approved by the Ethical Commission forAnimal Experimentation at the University Hospital Essen.

Ectopic MYCN expression and OTX015 treatment of murinecells and tumors grown as grafts in nude mice

Murine tumors (�1,000 mm3) derived from the geneticallyengineered mouse model LSL-MYCN;Dbh-iCre (12) were mincedmanually with scissors and digested with 2 mg/mL collagenase inPBS for 30 minutes at 37�C. Tumor pieces were passed through asieve (400 mm pore size) to obtain a cell suspension. Cells werewashed with PBS and suspended in 2.5 ml Matrigel� for sub-cutaneous inoculation (200 ml per mouse) into the left flank of6-week-old female athymic (nu/nu) mice. OTX015 treatmentwas the same as described for xenografts, as was grafted tumorexcision and processing. M-NB-A1 cells were used as a murinemodel of cells ectopically expressing MYCN (12). To modelectopic expression of MYCN in human neuroblastoma cells, wetransfected IMR-5 cells with pMSCV-puro plasmids (#21654,Addgene) containing MYCN, GFP, or empty negative control.Cells were selected with 2 mg/mL puromycin for two days. Afterselection, cells were expanded and treated with OTX015 asdescribed above. Cells treated with 500 nmol/L OTX015 wereharvested for RT-PCR and Western blot analysis of MYCNexpression.

Tumor tissue immunohistochemistrySamples of xenograft andmurine tumors treatedwith vehicle or

OTX015 were fixed embedded in paraffin and stained as previ-ously described (30). Primary antibodies against Ki-67 (1:50, pH6; #275R-16, Cell Marque) and cleaved caspase-3 (1:100, pH 6;#9661, Cell Signaling Technology) were used with correspondingsecondary antibody detection kits for reduced background (His-tofine Simple Stain MAX PO, Medac) and the automated LabVi-sion Autostainer 480S (Thermo Scientific). Slides were scannedwith a Pannoramic 250 slide scanning program (3D Histech.com).

Statistical analysisExpression data from IMR-5 cells treated with DMSO, JQ1, or

OTX015 were analyzed with the R2 visualization and analysisplatform (http://hgserver1.amc.nl/cgi-bin/r2/main.cgi). SPSS,version 18.0 (IBM) and R, version 2.13, were used for furtherstatistical analyses. Interval variables were compared using theStudent two-sided t test and categorical variables compared by c2

test. GraphPad Prism 5.0 was used to perform Kaplan–Meiersurvival analysis with log-rank statistics on OTX015-treated andcontrol mouse cohorts.

ResultsOTX015 is a BET protein inhibitor with no significant effect onnormal human cells

OTX015 is a synthetic small molecule targeting the BET bro-modomain proteins, BRD2/3/4 (Fig. 1A; ref. 13). To assess puta-tive effects of OTX015 on nonmalignant primary human cells, wemeasured the cell viability of human fibroblasts after 72 hours ofOTX015 treatment. Although OTX015 concentrations above 1mmol/L slightly reduced fibroblast viability (maximal reduction<50% at 4 mmol/L), this was not significant (Fig. 1B and Supple-mentary Fig. S1A and S1B).

OTX015-induced reductions in neuroblastoma cell viabilitycorrelate with MYCN status and expression

OTX015 significantly reduced viability of neuroblastoma cells(Fig. 1C and Supplementary Fig. S2A and S2B) with IC50 valuesranging from 37 nmol/L to >1 mmol/L (Supplementary Fig. S2A).Maximal reduction of cell viability was almost 100% in IMR-5,CHP-134, CHP-212, NB69, and GI-M-EN cell lines (Supplemen-tary Fig. S2B). MYCN-amplified neuroblastoma cell lines (redcurves, Fig. 1C)weremore sensitive toOTX015 treatment than celllines lacking MYCN amplification (P ¼ 0.003, Fig. 1D). Theseresults are in line with those previously reported for the BETinhibitor, JQ1 (11). The cell lines IMR-5, IMR-32, CHP-134, andCHP-212, which express higher levels of MYCN at both mRNAand protein levels, were more sensitive than the SK-N-AS, GI-M-EN and NB-69 cells lines with low-level expression (Supplemen-tary Figs. S2C–S2E); however, there was a poor correlationbetween IC50 values and both MYCN mRNA and protein levels.Notably, SK-N-AS cells did not respond to OTX015 even thoughexpressing highMYC levels. Taken together, OTX015 treatment invitro strongly reduces viability of neuroblastoma cell lines withpredominant effects on MYCN-amplified cells.

Oral OTX015 shows therapeutic efficacy against humanMYCN-amplified neuroblastoma xenografts

To further investigate antitumoral effects of OTX015 in vivo, weutilized a mouse xenograft model of MYCN-amplified humanneuroblastoma, with the human IMR-5 cell line. Average tumorvolumewas significantly smaller in theOTX015 groups comparedwith control mice after day 9 of treatment (P < 0.05, Fig. 1E).Kaplan–Meier analysis showed that tumor-free survival wasdelayed significantly in all treatment groups compared with thecontrol group (P < 0.02, Fig. 1F). Thus, OTX015 treatmentsignificantly improved survival in mice harboring MYCN-ampli-fied neuroblastoma xenografts. Two of 9 mice from the grouptreated with 25 mg OTX015/kg body weight twice a day diedbecause of treatment misapplication on days 8 and 9. No con-siderable side-effects were observed inmice treated withOTX015.Oral administration of OTX015 achieved antitumoral effects in aMYCN-driven neuroblastoma xenograft model.

OTX015 reduces proliferation and induces apoptosis and cell-cycle arrest in human neuroblastoma cell lines and in vivoneuroblastoma models

When investigating the impact of OTX015 on typical tumor-igenic properties, MYCN-amplified cells most predominantlyshowed significantly reduced cell growth over time (Supplemen-tary Fig. S3B and S3C). The percentage ofMYCN-amplified cells inG1 increased after OTX015 treatment compared with DMSO-treated control cells. This was accompanied by fewer cells in theS-phase. The percentage of cells in sub-G1 also increased in theIMR-5 cell line after treatment (Supplementary Fig. S3D).OTX015 treatment of SK-N-AS cells, lackingMYCN amplification,did not alter the distribution of cells in the cell cycle. These datasuggest that OTX015 can cause a shift towards either G1 arrest orapoptosis in MYCN-amplified cells.

The fraction of apoptotic cells was significantly higher inIMR-5, Chp-134, and Chp-212 MYCN-amplified cell lines afterOTX015 treatment compared with the DMSO control, but therewas no detectable induction of apoptosis in SK-N-BE (MYCN-amplified) and SK-N-AS (single-copy MYCN) cells (Supple-mentary Fig. S3E). BrdUrd incorporation showed significantly






reduced proliferative capacity in all cell lines except Chp-212 afterOTX015 compared with the DMSO control (SupplementaryFig. S3F).

Tumors from OTX015-treated IMR-5 xenografts also showeda significant increase in the proportion of apoptotic cells(Supplementary Fig. S4), and the proportion of cells stainingpositively for Ki-67 was significantly reduced (SupplementaryFig. S4B and S4C). Taken together, OTX015 induced cell-cyclearrest and apoptosis with a range of sensitivity across a panel ofneuroblastoma cells, most strongly dependent on cellularMYCN status.

OTX015 disrupts the BRD4–chromatin interaction andrepresses MYCN expression in neuroblastoma cell lines

BRD4 inhibition has been shown to suppress transcription ofMYCN in neuroblastoma, thus, the phenotypic effects were pre-viously primarily attributed to this observation (11). Consistentwith this previous observation, treatment with OTX015 or JQ1significantly reduced BRD4 binding to theMYCN promoter (Fig.2A). We next examined the effect of OTX015 treatment onMYCNexpression in several genetic backgrounds relevant for humanneuroblastoma in vitro and in vivo. OTX015 treatment reducedMYCN transcript levels in all MYCN-amplified cell lines after 4

Figure 1.The BET inhibitor, OTX015, displays antitumoral activity against MYCN-amplified neuroblastoma in vitro and in vivo. A, the molecular structure of OTX015, with theformula (6S)-4-(4-chlorophenyl)-N-(4-hydroxyphenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetamide. B, OTX015 had nosignificant effect on human fibroblast viability, as shown in a dose–response curve of fibroblasts treated with increasing doses of OTX015 for 72 hours andmeasuredwith the MTT cell viability assay. C, cell viability dose–response curve for the IMR-5, Chp-134, Chp-212, SK-N-BE(2), IMR-32 SK-N-BE, NB69, SK-N-AS,and GI-M-EN neuroblastoma cell lines treatedwith increasing OTX015 concentrations for 72 hours. Red curves indicate cells harboringMYCN amplifications and bluecurves are cell lines lacking amplification. D, the IC50 values for the OTX015 impact on cell viability are plotted for cell lines harboring MYCN amplifications(red) and cell lines lacking MYCN amplification (blue). The difference between the two groups was analyzed using the Student t test, supporting a significantcorrelation betweenMYCN status andOTX015 efficacy against neuroblastoma cell lines in vitro. E, nudemice harboring IMR—5 xenograft tumorswith volumes of 200mm3 were treated with OTX015 by oral gavage at the concentrations, 25 mg/kg body weight/day (light green, n ¼ 9), 2 � 25 mg/kg body weight/day(green, n¼ 9), and 50mg/kg bodyweight/day (dark green, n¼ 9), orwith 200 mLH2O/day (black, n¼ 9) as a negative control. Tumor volumewasmeasured using acaliper every 2 days during treatment, and the average tumor volume in each treatment group is plotted for the course of treatment. Statistical differencebetween the treatment and control groups was analyzed by Student t test (all treatment groups have P < 0.05 from day 9 to the end of treatment). F, Kaplan–Meieranalysis was conducted for the xenograft mouse model treated in E, showing that OTX015 treatment significantly improved survival of mice with established IMR-5xenograft tumors (log-rank test, P < 0.02). BID, twice daily.

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hours and up to 72 hours of treatment (Fig. 2B). This reduction ingene expression in OTX015-treated cell lines was recapitulated atthe protein level for MYCN and other BRD4 targets (Fig. 2C). Invivo OTX015 treatment of IMR-5 xenografts reduced MYCNexpression at both the mRNA and protein levels as well asE2F1 and cyclin D1 protein expression (Fig. 2D and E). Together,these observations support the previous observations of MYCNbeing a target of BRD4 inMYCN-driven neuroblastoma cells, andprovide evidence for this activity with OTX015.

EctopicMYCNexpression does not abrogate the effects of BRD4inhibition

The effect of BRD4 inhibition in neuroblastoma cells haspreviously been attributed to MYCN repression (11). We set outto test the existing hypothesis that BET protein inhibition mainlyacts through transcriptional suppression ofMYCN. To do this, weectopically expressed MYCN in IMR-5 cells to stabilize its highexpression level (Fig. 3A) then treated cells with OTX015. Inter-estingly, there was no difference in OTX015 dose responsebetween IMR-5 cells ectopically expressingMYCN or control cells(Fig. 3a). As an orthogonal model, we used the geneticallyengineered mouse model (GEMM), LSL-MYCN;Dbh-iCre, andthemNB-A1 cell line derived froma neuroblastoma tumor arisingin this GEMM (12). In this murine system,MYCN transcription is

strongly driven by a chicken actin promotor. We hypothesizedthat MYCN would not be under the epigenetic control of endog-enous elements such as enhancers in this model. It follows thatMYCN expression should not be affected by BRD4 inhibition ifour hypothesis is correct, and that MYCN expression from thetransgene shouldnot be affected to the sameextent as endogenousMYCN. Any effects of BET bromodomain inhibition observed inthis context would not be due toMYCN transcriptional silencing.Indeed, in LSL-MYCN;Dbh-iCre–derived mNB-A1 cells, MYCNprotein levels remained stable even after treatment with OTX015(Fig. 3D). Interestingly, in vitro treatment of the mNB-A1 cell linewith OTX015 significantly reduced cell viability as did JQ1 treat-ment (Fig. 3C). Mice harboring regrafted LSL-MYCN;Dbh-iCretumors were orally treated with OTX015 or control (n ¼ 6).Consistentwith in vitro treatment of LSL-MYCN;Dbh-iCre–derivedmNB-A1 cells, we observed a delay in tumor progression inOTX015-treated mice harboring regrafted LSL-MYCN;Dbh-iCretumors, resulting in significantly prolonged mouse survival (Fig.3E and F). Again, MYCN expression was not affected by OTX015treatment (Fig. 3G). Concurrently, the fraction of apoptotic cellswas significantly increased in the LSL-MYCN;Dbh-iCre tumors,while the fraction of proliferating cells was reduced (Fig. 3H andI). Together, OTX015 showed therapeutic efficacy in both murineneuroblastoma GEMM models and human cells ectopically

Figure 2.OTX015 treatment reduces BRD4 binding to chromatin and leads to decreased BRD4 target gene expression in vitro and in vivo. A, schematic H3K27Ac track(adapted from genome browser, http://genome.ucsc.edu/) showing PCR primer localization (region 1 and 2) relative to MYCN gene body (left). Quantitativechromatin immunoprecipitation (ChIP) and PCR (ChIP-PCR) of BRD4 from IMR-5 cells treated with OTX015, JQ1 or control, at the MYCN promotor regions1 and 2. B, time course ofMYCNmRNAexpression (measured using real-timeRT-PCR) after treatment of neuroblastoma cell lineswith 500nmol/LOTX015 relative toDMSO control. Expression was normalized to GAPDH expression. MYCN expression was not assessed in SK-N-AS cells, as they did not express significantendogenous levels ofMYCN. C, whole-cell protein lysates collected after 72 hours of 500 nmol/L OTX015 (þ) or control DMSO (�) treatment of neuroblastoma celllines in vitro, and known BRD4 targets were analyzed in Western blots. b-Actin was used as loading control. D, real-time RT-PCR was used to assess MYCNexpression in IMR-5 xenograft tumors. Expression was normalized to GAPDH expression, and statistical difference between groups was assessed by Student t test.E, protein lysates were prepared from IMR-5 xenograft tumors, and BRD4 targets were analyzed in Western blots. b-Actin was used as loading control.






Figure 3.Ectopic MYCN expression does notattenuate effects of BET inhibition. A,Western blot analysis of MYCN and V5in IMR-5 cells transfected withpMSCV-MYCN, V5-tagged MYCN,EGFP, or empty control after 48 hoursof treatment with 500 nmol/L OTX015and DMSO control. B, viability(measured by MTT assay) of IMR-5cells ectopically expressing MYCN,V5-tagged MYCN, GFP, or emptycontrol. C, viability (measured by MTTassay) of the murine neuroblastomacell line mNB-A1 ectopicallyexpressing MYCN, over 96 hours aftertreatment with either OTX015 (darkgray), JQ1 (light gray) or DMSOcontrol (black). D, MYCN proteinexpression in LSL-MYCN;Dbh-iCremurine neuroblastoma cell line mNB-A1 after treatment with OTX015. E,tumor volumes of LSL-MYCN;Dbh-iCre murine neuroblastomatumors grown as grafts in nude miceandorally treatedwith 25mg/kg bodyweight/day OTX015 or H2O alone(control). The average tumor volumewas smaller in the OTX015-treatedgroups compared with the controlgroup (Student t test: P ¼ 0.07 ontreatment day 13). F, Kaplan–Meieranalysis showing the survival of micebearing tumors described in C treatedwith OTX015 or H2O alone. Log-ranktests were used to calculatesignificance. G, relative MYCN mRNAexpression in LSL-MYCN;Dbh-iCretumors after treatment with OTX015or vehicle control. H, representativepictures of sections of graftedLSL-MYCN;Dbh-iCre tumor stainedwith hematoxylin/eosin andimmunohistochemical staining forapoptotic cells (cleaved caspase-3)and proliferating cells (Mib-1/Ki-67).Scale bar ¼ 250 mm. I, the relativefraction of positively stained cells forcleaved caspase-3 (cl. cas3) and Mib-1were calculated from threerepresentative images from eachgrafted LSL-MYCN;Dbh-iCre tumorand are shown as box plots. Statisticaldifference between groups wasassessed by Student t test.

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expressing MYCN without repressingMYCN transcription. Theseresults suggest that effects of BET protein inhibition are at leastpartly independent of MYCN repression.

Inhibiting BRD4 by OTX015 disrupts transcription of MYCNtarget genes independently of MYCN expression levels

Considering the interesting observation that OTX015 showedantitumoral effects on cells ectopically expressing MYCNwithoutrepressing MYCN expression, we performed gene expressionprofiling to explore the underlying mechanisms and alternativetargets of the BET proteins. OTX015 or JQ1 treatment of IMR-5cells significantly altered gene expression patterns compared withDMSO-treated control cells (Fig. 4A). Genes that were differen-tially expressed in OTX015- or JQ1-treated cells showed signifi-cant overlap. Treatment with either OTX015 or JQ1 commonlyupregulated 63.7% (2,534/3,976) and downregulated 66.5%(3,051/4,590) of genes in IMR-5 cells. This similar action isconsistent with the similar specificity of JQ1 and OTX015 forBET proteins. Gene sets enriched among the genes downregulatedby OTX015 represented known oncogenic pathways, such as theKRAS, ALK, andMYC pathways (Fig. 4B). The same gene sets wereenriched among the genes downregulated by JQ1, supporting the

high overlap in differentially expressed genes between the twoBET inhibitors.

Interestingly, treatment of both IMR-5 and the LSL-MYCN;Dbh-iCre–derived mNB-A1 cell line with either BET inhibitor signif-icantly decreased MYCN target gene signature scores (Fig. 4C andD). This indicates that MYCN target genes are repressed by BETinhibition even at unchangedMYCN levels (seeMYCNexpressioninmNB-A1 in Fig. 3D). As this effect onMYCN target genes occurswithoutMYCN transcriptional suppression in mNB-A1 cells, thisraises the question whether MYCN target genes are direct BRD4targets. Our data suggests that BRD4 inhibition in nonengineeredhuman neuroblastoma cells directly suppresses both MYCN andMYCN target genes.

OTX015 selectively disrupts BRD4 binding at MYCN targetgenes and represses super-enhancer–associated genetranscription

Our observation that the therapeutic efficacy of BET inhibitorsagainst MYCN-driven tumors was partly independent of MYCNrepression raised the question about other therapeutically impor-tant BRD4 targets in neuroblastoma. Our current understandingof BRD4 function supports BRD4binding to acetylatedhistones at

Figure 4.BET inhibition causes distinct gene expression changes and affects MYCN target genes even at stable MYCN levels. A, heatmap of the top 50 differentiallyexpressed genes after OTX015 treatment in IMR-5 cells compared with DMSO-treated control cells (left) and a heatmap of the top 50 commonly differentiallyexpressed genes after OTX015 and JQ1 treatment (right). B, GSEA (version 2.0, Broad Institute) of differentially expressed genes in IMR-5 cells aftertreatment with OTX015 or JQ1 using C6 gene sets representing signatures of cellular pathways often dysregulated in cancer. C, enrichment of the published geneexpression signature for MYCN target genes (24) in differentially expressed genes in IMR-5 cells after treatment with OTX015, JQ1, or DMSO control. D,enrichment of the published gene expression signature for MYCN target genes (24) in differentially expressed genes in LSL-MYCN;Dbh-iCre murineneuroblastoma cell line mNB-A1 cells after treatment with OTX015, JQ1, or DMSO control (� , P < 0.05; ��, P < 0.01; ��, P < 0.001).






what is referred to as super-enhancer sites that regulate transcrip-tional pause release of oncogenes such as MYC. We hypothesizedthat super-enhancers associated with genes other than MYCNmight be important to mediate the effects of BET inhibition inneuroblastoma. Using H3K27Ac ChIP-seq we detected 1335super-enhancers in the IMR-5 genome using an algorithm pro-posed by Whyte and colleagues (Fig. 5a) (29). Among the genesassociated with the top 200 super-enhancers (SE-genes), wedetected a large number of genes of known importance in cancer,including MYCN and GLI2 (Fig. 5A and Supplementary Fig. S5).We hypothesized that super-enhancer–associated genes would bemost affected by BRD4 inhibition. Indeed, genes downregulatedin IMR-5 cells after treatment with OTX015 or JQ1 were signif-icantly enriched for super-enhancer–associated genes (Supple-mentary. Fig. S5C). The cumulative distribution of gene expres-sion changes was also significantly shifted towards a strongerrepression of genes associated with super-enhancers (Fig. 5b).Expression of genes associated with super-enhancers was signif-icantly lower than genes not associatedwith super-enhancers aftertreatment with either OTX015 or JQ1 (Fig. 5C). Consistent withthis, ChIP-Seq detected a significant decrease in BRD4 binding tochromatin, particularly at super-enhancer regions (Fig. 5D–F). AsOTX015 significantly affected MYCN target gene expression, wesearched for MYCN target genes in the BRD4 ChIP-seq data.MYCN target genes were significantly enriched in chromatinareas where BRD4 binding was strongly reduced. This was observ-ed for both the Valentijn and colleagues and Westermann andcolleagues independently developed MYCN target gene sets(Fig. 5G; refs. 23, 27). This suggests a functional importance forBRD4 in MYCN-driven gene expression, and may explain ourobservations that BET inhibition disrupts MYCN target genetranscription even when MYCN is ectopically expressed (Fig.5h). Our data show that pharmacologic BRD4 inhibition speci-fically disrupts BRD4 binding not only to MYCN but also toMYCN target genes and preferentially leads to transcriptionalrepression of super-enhancer–associated genes.

DiscussionThe importance of super-enhancers for transcriptional regu-

lation of oncogenes such as MYC family transcription factorshas recently been brought to light, opening up new opportu-nities for drug development and raising new questions abouttheir functions in cancer. Here, we demonstrate antitumoralpotency of the BET protein inhibitor, OTX015, against celland animal models of MYCN-driven neuroblastoma, and pro-vide mechanistic insights indicating that OTX015 selectivelydisrupts MYCN-driven transcription, preferentially affectingsuper-enhancer–associated genes.

Neuroblastoma is the most common extracranial tumor inchildren (31). The majority of children with high-risk diseasesuccumb to neuroblastoma and surviving patients often sufferfrom long-term sequelae due to cytotoxic chemotherapy (32).Novel targeted treatment approaches with lower toxicity areclearly needed for these patients. The MYCN transcription factoris a driving oncogene in neuroblastoma. Tumor cells are addictedto high levels of MYCN. The critical role of MYCN renders it acompelling target for drug development, and several strategiesindirectly targeting MYCN are emerging (refs. 33–37; reviewed inref. 38). BRD4 inhibition was the first successful approach used totarget MYCN transcription in neuroblastoma (10, 11, 39).

There has been widespread interest in developing drugs selec-tively targeting BET proteins that are well suited to clinical use. Inpast years, several BET inhibitors such as I-BET 762 (GSK525762),TEN-010, and CPI-0610 have been introduced into clinicalphase I trials (NCT02308761, NCT01987362, NCT01587703,NCT01949883, NCT02157636, NCT02158858), but to ourknowledge, none have yet entered pediatric clinical trials. Withdevelopment for oral bioavailability, OTX015 entered adult clin-ical phase I/II trials in 2012 (16, 17). Here, we explored thepossibility of selectively targeting BRD4 with OTX015 in preclin-ical neuroblastoma models. Similar to JQ1, OTX015 effectivelyreduced neuroblastoma cell viability in vitro and in vivo. OTX015potently inhibited growth in a MYCN-amplified neuroblastomaxenograft tumor model and significantly prolonged mouse sur-vival. Responsiveness to BRD4 inhibition has previously beenattributed to the extent of endogenousMYCNexpression (11).Weobserved similar correlations betweenMYCN status and sensitiv-ity to OTX015 treatment. High-level MYCN expression andMYCN amplification were significantly correlated with theresponse toOTX015 treatment. SK-N-AS cells, which express highlevels of MYC, were rather resistant to BET inhibition, indicatingthat sensitivity to BET inhibition might indeed be specific toMYCN status in the context of neuroblastoma biology. Together,our findings indicate OTX015 is a potent agent against preclinicalmodels of MYCN-driven neuroblastoma.

As neuroblastoma cells are addicted to high MYCN levels, theeffect of BRD4 inhibition on cell viability was previouslyattributed to MYCN repression (3, 39). Supporting these find-ings, we observe a similar reduction of MYCN expressionfollowing OTX015 treatment. Previous reports showed thatdisrupting BRD4-chromatin binding was the mechanistic causefor these transcriptional changes. OTX015 treatment inhibitedBRD4 binding to chromatin to a comparable or stronger extentas the known BRD4 inhibitor, JQ1, providing support thatOTX015 robustly reduces BRD4 binding to acetylated histonetails at the MYCN promoter. To further understand the role ofMYCN as a known mediator of antitumoral effects of BRD4inhibiton in neuroblastoma, we investigated the effects ofBRD4 inhibition on models ectopically expressing MYCN.MYCN expression was not affected by BET inhibition in ourmodels. Interestingly, ectopic MYCN expression did not atten-uate the antitumoral effects of OTX015 in this context. This is inline with our previous observations that JQ1 is effective againstLSL-MYCN;Dbh-iCre neuroblastoma (12), but was intriguing asMYCN amplification and high-level MYCN expression arestrong predictors of BRD4 inhibitor susceptibility. These find-ings suggested that mechanisms other than transcriptionalrepression ofMYCN contribute to the effect of BRD4 inhibitionin neuroblastoma. Understanding these mechanisms is essen-tial for the future clinical application of OTX015 and otherBRD4 inhibitors. We observed that genes transcriptionallyrepressed after OTX015 treatment were enriched for MYCNtarget genes, in bothMYCN-amplified IMR-5 cells and mNB-A1cells ectopically expressing MYCN. This suggested that BRD4inhibition suppresses MYCN target gene transcription even inthe presence of high MYCN levels. Gergano and colleaguesdescribed MYC recruitment of P-TEFB to its target genes (40)and recent findings indicate a similar recruitment of CDK7 tosuper-enhancer sites by MYCN (37). This recently publishedinsight indicates that MYCN-driven transcription is dependenton recruitment of P-TEFB and, thus, BRD4 to MYCN target gene

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Figure 5.BET protein inhibition specifically disrupts BRD4binding toMYCN target genes andpreferentially represses super-enhancer driven transcription. A, theH3K27Ac signalin MYCN-amplified IMR-5 cells is shown across enhancer regions for all enhancers. Super-enhancers were defined as surpassing the threshold defined bythepointatwhich the y¼ x linewas tangent to thesignal-rankcurve. In IMR-5cells, 4.6%of theenhancerswereclassedas super-enhancers, includingthosegenesnamedon the right. B, cumulative distribution plots of the top 200 super-enhancer–associated genes and their fold-change measured using genes which are differentiallyregulated in IMR-5 cells by OTX015 treatment. Plots show a shift towards negative logFC for super-enhancer associated genes (blue curves) compared withgenes not associated with super-enhancers (black curves). C, box plots of log2-fold changes in the top 200 genes associated with super-enhancers (SE) and regularenhancers (non-SE) in genes differentially regulated (compared with DMSO-treated controls) in IMR-5 cells treated with OTX015. Statistical significance wasdetermined using a two-sided Mann–Whitney test. D–F, the normalized BRD4 ChIP-Seq signal across the genome (D), at enhancers and promoters (E) as wellas at super-enhancer sites (F) in IMR-5 cells after treatment with OTX015 or DMSO control. G, GSEA of genes where BRD4 binding was most significantlyreduced show significant enrichment for MYCN target genes [Valentijn and colleagues, MYCN target genes (left), P < 0.002;Westermann and colleagues MYCN targetgenes, P < 0.001 (right)]. H, schematic view of proposed mechanism of BRD4 inhibition in neuroblastoma. BRD4 is recruited to super-enhancer complexes oftheMYCN gene (top). MYCN acts as a transcription factor at MYCN target gene promoter and enhancer siteswhere BRD4 is also recruited to (bottom). BRD4 inhibitionby OTX015 therefore not only indirectly leads to decreased expression of MYCN target genes by inhibiting MYCN expression, but also suppressed MYCNactivity at its target genes directly.






promoters and enhancers. Indeed, our BRD4 ChIP-Seq analysisshowed that BRD4 binding was most significantly reduced atMYCN target genes. This further sustains our hypothesis thatinhibiting BRD4 with OTX015 blocks BRD4 and P-TEFb-dependent MYCN-driven transcription. Considering, however,that Chipumuro and colleagues described MYCN as a generaltranscriptional amplifier (37) similar to MYC, our gene expres-sion results might be underestimating the effect of BET inhi-bition on MYCN-driven transcription. Conducting geneexpression analysis using spike-in normalization, as recentlysuggested by Loven and colleagues (38, 39), should revealMYCN-induced changes on global expression levels. Consis-tent with previous reports, we conclude that MYCN-driventranscription might be dependent on BRD4-mediated tran-scriptional pause release, which would explain why MYCNtarget genes were more susceptible to BET inhibition. Thisconclusion is further supported by (i) the strong correlation ofMYCN amplification with susceptibility to OTX015 treatment,(ii) the widespread and robust inhibition of MYCN target genetranscription in cells ectopically expressing MYCN by OTX015and (iii) the fact that ectopic MYCN expression in a murineneuroblastoma model did not significantly attenuate OTX015effects.

Considering recent discoveries implicating BRD4 in super-enhancer regulation, we set out to understand the extent to whichinhibiting BRD4-chromatin binding would specifically disruptsuper-enhancer associated gene expression. Interestingly, wefound that H3K27Ac-enriched areas were most abundant neargenes of known importance for tumorigenesis. As expected, theexpression of genes associated with super-enhancers was mostsignificantly reduced after OTX015 treatment. MYCN was highlyranked in the list of super-enhancer-associated genes, indicatingits regulation by a super-enhancer and further supporting its roleas a BRD4 target. Building on published work implicating MYCNin BRD4 inhibition, we verified and extended understanding ofthe mechanism of BRD4 inhibition in neuroblastoma, reportingthat inhibiting BRD4 binding to the chromatin using OTX015preferentially disrupts transcription of super-enhancer–associat-ed genes, which include MYCN.

Previous studies have proposed MYCN amplification as abiomarker to predict tumor susceptibility to BET inhibitors(11). Considering our new findings, we propose that a MYCNactivity score be evaluated as an improved biomarker for tumorsusceptibility to BET protein inhibitors. A MYCN activity scorebased on the expression ofMYCN target geneswould also identifypatients with tumors lackingMYCN amplifications, but otherwiseactivated MYCN, as described previously (27). Neuroblastomaswith activatedMYCNbut lackingMYCN amplification are knownto be similarly aggressive and result in poor patient outcome (27).Remarkably, such signature scores are also capable of identifyingpoor outcome in patients with tumors expressing low MYCNtranscript levels but high nuclearMYCNprotein levels (27). Thesepatients would likely also benefit from BET protein inhibitortreatment and this could be applicable in other tumor types inwhich MYCN is the driving oncogenes.

The results we report here provide conclusive evidence that BETprotein inhibition disrupts super-enhancer functions and prefer-entially suppresses MYCN-driven transcription. We show thatOTX015 is active against both in vitro and in vivo preclinicalneuroblastoma models. On the basis of the survival advantageobserved in mouse models of MYCN-driven neuroblastomatreated with OTX015, this drug has the potential to generate ameasurable response in patients with high-risk MYCN-drivenneuroblastoma. To identify patients who would likely benefitfrom BET inhibitor treatment, we propose using the previouslyestablished MYCN activity score (27). Our data present a preclin-ical rationale that can feed into upcoming phase I/II trials ofOTX015 in pediatric patients.

Disclosure of Potential Conflicts of InterestE. Cvitkovic has ownership interest (including patents) in and is a consul-

tant/advisory boardmember forOncoethix SA.Nopotential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: A.G. Henssen K. Althoff, J.H. SchulteDevelopment of methodology: A.G. Henssen, E. Bell, L. Astorgues-Xerri,F. WestermannAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.G. Henssen, F. Speleman, S. Schaefer, K.D. Preter,L. Heukamp, A. Spruessel, N. Solomentsew, A. Schramm, A. Eggert, J.H. Schulte,F. WestermannAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.G. Henssen, K. Althoff, A. Beckers, R. Koche,F. Speleman, E. Bell, K.D. Preter, L. Heukamp, L. Astorgues-Xerri, J.H. Schulte,F. WestermannWriting, review, and/or revision of the manuscript: A.G. Henssen, K. Althoff,A. Beckers, F. Speleman, K.D. Preter, K. Astrahanseff, S. Lindner, L. Astorgues-Xerri, M.E. Riveiro, A. Eggert, E. Cvitkovic, J.H. SchulteAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):A.G.Henssen, K. Althoff, A.Odersky, S. Schaefer,A. Florin, N. Solomentsew, A. Eggert, J.H. SchulteStudy supervision: A. Schramm, M.E. Riveiro, A. EggertOther (specify): M. Nortmeyer

AcknowledgmentsThe authors thankAlex Kentsis, Gudrun Fleischhack, and Scott Armstrong for

critical discussions. The authors also thank the University of Essen PediatricOncology Research Program for their support.

Grant SupportThis work was supported by the German Cancer Aid (#111302; to J.H.

Schulte), by the German Ministry of Science and Education (BMBF) as part ofthe e:Med initiative (grant no. 01ZX1303B and 01ZX1307E; to J.H. Schulte), bythe GOA (#01G01910; to F. Speleman), by the EuropeanUnion FP7 (#259348-2, ASSET project, to F. Speleman, J.H. Schulte, and A. Eggert), by the fund forscientific research (FWO; grant number G.0530.12N; to F. Westermann and apost-doc grant to K.D. Preter), and by a PhD grant from the Agency forInnovation by Science and Technology (IWT; grant number IWT 101506; toA. Beckers). This study was also sponsored in part by Oncology TherapeuticDevelopment.

Received June 25, 2015; revised October 15, 2015; accepted November 3,2015; published OnlineFirst December 2, 2015.

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2016;22:2470-2481. Published OnlineFirst December 2, 2015.Clin Cancer Res Anton Henssen, Kristina Althoff, Andrea Odersky, et al. InhibitionTargeting MYCN-Driven Transcription By BET-Bromodomain

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