trabectedin inhibits ews-fli1 and evicts swi/snf from ...contrast, the phase ii study in ewing...

Translational Cancer Mechanisms and Therapy

Trabectedin Inhibits EWS-FLI1 and EvictsSWI/SNF from Chromatin in a Schedule-dependent MannerMatt L. Harlow1, Maggie H. Chasse2, Elissa A. Boguslawski2, Katie M. Sorensen2,Jenna M. Gedminas2,3,4, Susan M. Kitchen-Goosen2, Scott B. Rothbart2, Cenny Taslim5,Stephen L. Lessnick5,6, Anderson S. Peck2, Zachary B. Madaj2, Megan J. Bowman2, andPatrick J. Grohar2,3,4

Abstract

Purpose: The successful clinical translation of compoundsthat target specific oncogenic transcription factors will requirean understanding of the mechanism of target suppression tooptimize the dose and schedule of administration. We havepreviously shown trabectedin reverses the gene signature ofthe EWS-FLI1 transcription factor. In this report, we establishthe mechanism of suppression and use it to justify the reeval-uation of this drug in the clinic in patients with Ewing sarcoma.

Experimental Design:We demonstrate a novel epigeneticmechanism of trabectedin using biochemical fractionationand chromatin immunoprecipitation sequencing. We linkthe effect to drug schedule and EWS-FLI1 downstream targetexpression using confocal microscopy, qPCR, Westernblot analysis, and cell viability assays. Finally, we quantitatetarget suppression within the three-dimensional architec-ture of the tumor in vivo using 18F-FLT imaging.

Results: Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributesEWS-FLI1 in the nucleus leading to a marked increasein H3K27me3 and H3K9me3 at EWS-FLI1 target genes.These effects only occur at high concentrations of trabec-tedin leading to suppression of EWS-FLI1 target genesand a loss of cell viability. In vivo, low-dose irinotecanis required to improve the magnitude, penetrance, andduration of target suppression in the three-dimensionalarchitecture of the tumor leading to differentiation ofthe Ewing sarcoma xenograft into benign mesenchymaltissue.

Conclusions: These data provide the justification to eval-uate trabectedin in the clinic on a short infusion schedule incombination with low-dose irinotecan with 18F-FLT PETimaging in patients with Ewing sarcoma.

IntroductionOncogenic transcription factors are dominant oncogenes for

a large number of leukemias and solid tumors in both thepediatric and adult populations (1–3). These proteins arechallenging drug targets because the active site lacks a tradi-

tional druggable domain and most transcription factors interactwith complex networks of proteins. Nevertheless, compoundsthat have successfully targeted specific transcription, such asATRA and arsenic trioxide in acute promyelocytic (APL), areeffective in the clinic (4–6).

Ewing sarcoma is a bone and soft-tissue sarcoma that isabsolutely dependent on the EWS-FLI1 transcription factor forcell survival (7). This fusion transcription factor, formed by thet(11;22)(q24;12) chromosomal translocation, both drivesproliferation and blocks differentiation (8, 9). EWS-FLI1 actsas a pioneer transcription factor and binds to repetitive regionsof the genome called GGAA microsatellites (10–13). Oncebound, the protein exhibits phase-transition properties toestablish these microsatellites as enhancers to drive geneexpression (14). This requires a complex network of proteininteractions and relies heavily on the ATP-dependent chroma-tin-remodeling complex, SWI/SNF to maintain chromatin inan open state (14, 15). Therefore, it is likely that reversal ofEWS-FLI1 activity would lead to widespread changes in chro-matin structure and restore the differentiation program. How-ever, it is not clear whether the effective targeting of EWS-FLI1requires a blockade of SWI/SNF activity or whether the pioneertranscription factor activity of EWS-FLI1 is reversible genome-wide.

We have previously shown that the natural product trabectedininterferes with the activity of the EWS-FLI1 transcription fac-tor (16). We showed that trabectedin reverses expression of the

1Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.2Van Andel Research Institute, Grand Rapids, Michigan. 3Department of Pedi-atrics, Michigan State University, East Lansing, Michigan. 4Division of PediatricHematology/Oncology, Helen DeVos Children's Hospital, Grand Rapids, Michi-gan. 5Center for Childhood Cancer and Blood Diseases, Nationwide Children'sHospital Research Institute, Columbus, Ohio. 6Division of Pediatric Hematology/Oncology/BMT, The Ohio State University College of Medicine, Columbus, Ohio.

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

M.L. Harlow and M.H. Chasse contributed equally to this article.

Current address for M.L. Harlow: Dana-Farber Cancer Institute, Boston, Massa-chusetts; current address for A.S. Peck, Bamf Health, Grand Rapids, Michigan;and current address for M.J. Bowman, Ball Horticultural Company, West Chi-cago, Illinois.

Corresponding Author: Patrick J. Grohar, Van Andel Research Institute, 333Bostwick Ave NE, Grand Rapids, MI 49503. Phone: 616-234-5000; Fax: 616-234-5309; E-mail: [email protected]

Clin Cancer Res 2019;25:3417–29

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EWS-FLI1 gene signature. In addition, we cloned EWS-FLI1 intoanother cellular context, induced an EWS-FLI1–driven promot-er luciferase construct, and then rescued this induction withtrabectedin (16). These findings were consistent with earlypreclinical and clinical experience with the drug that suggesteda heightened sensitivity of Ewing sarcoma to trabecte-din (17, 18). Most notably, a patient with treatment-refractoryEwing sarcoma achieved a durable complete response withsingle-agent trabectedin treatment in the phase I study. Incontrast, the phase II study in Ewing sarcoma was negativeand only 1 of 10 patients responded to the drug (19). However,the drug was administered on a different schedule in thenegative phase II study. Therefore, it is possible that a detailedunderstanding of the mechanism of EWS-FLI1 suppression bytrabectedin would allow us to optimize the schedule of admin-istration and achieve the therapeutic suppression of EWS-FLI1in the clinic.

Like many natural products, trabectedin has a complicatedmechanism of action (20, 21). The compound is known togenerate DNA damage and poison various repair pathways, blockspecific transcription factors such as the FUS-CHOP transcriptionfactor, and exert cytotoxicity with preference for specific cell typessuch as tumor-associated macrophages (TAM), myxoid liposar-coma cells, and Ewing sarcoma cells (22–24)

In this study, we define the mechanism of EWS-FLI1 sup-pression to establish trabectedin as a bona fide EWS-FLI1inhibitor. We show that the drug redistributes EWS-FLI1 withinthe nucleus and at the same time evicts the SWI/SNF chroma-tin-remodeling complex to trigger an epigenetic switch, leadingto global increases in H3K27me3 and H3K9me3 with prefer-ence for GGAA microsatellites and EWS-FLI1 target genes.Importantly, these effects are concentration dependent, andlead to sustained target suppression only if a threshold con-centration of drug is exceeded. This mechanistic insight isconsistent with the clinical experience with the drug wherethis threshold was exceeded in the phase I and patientsresponded, but not the negative phase II study. Finally, targetsuppression is amplified and sustained in vivo in combinationwith the topoisomerase inhibitor irinotecan to cause a com-plete histologic change in the tumor and differentiation intobenign mesenchymal tissues.

Materials and MethodsCell culture

TC32, A673 cells were obtained from Dr. Lee Helman andTC252, SK-N-MC, EW8 from Dr. Tim Triche (both at Children'sHospital of Los Angeles, Los Angeles, CA). Cell identity wasconfirmed by short tandem repeat profiling (DDC Medical; lasttest October 24, 2018). They were cultured at 37�C pathogen freewith 5% CO2 in RPMI-1640 (Gibco) with 10% FBS (Gemini Bio-Products), 2 mmol/L L-glutamine, and 100 U/mL and 100 mg/mLpenicillin and streptomycin (Gibco).

Western blotting1.5 million cells (TC32, A673) or 3 million cells (TC252,

EW8, SK-N-MC) were incubated with drug, washed in PBS, andlysed in 4% lithium dodecyl sulfate (LDS) buffer. Thirty micro-grams of total protein were resolved on a NuPage 4–12% Bis-Tris gradient gel (Invitrogen) in 1� NuPage MOPS SDS Run-ning Buffer (Invitrogen) after diluting detergent and quantitat-ing by bicinchoninic acid (BCA) assay (Pierce, Thermo Scien-tific). The protein was transferred overnight to nitrocellulose at20 V in 1� Tris-Glycine-SDS Buffer (Bio-Rad) with 20% meth-anol. The membranes were blocked in 5% milk in TBS-T, andprobed with WRN, NR0B1, GAPDH (Abcam), or EZH2 (CellSignaling Technology) antibodies.

Quantitative RT-PCRRNAwas collected using the RNeasy Kit (Qiagen), immediately

reverse-transcribed using a High-Capacity Reverse TranscriptaseKit (Life Technologies) at 25�C for 10 minutes, 37�C for 120minutes, and 85�C for 10minutes. The products were quantitatedusing qPCR, SYBR green (Bio-Rad), and the following program:95�C for 10 minutes, 95�C for 15 seconds, 55�C for 15 seconds,and 72�C for 1 minute, for 40 cycles. Expression was determinedfrom three independent experiments relative to GAPDH andsolvent control using standard DDCt methods.

Luciferase assaysStable cell lines containing an EWS-FLI1–driven NR0B1 lucif-

erase or constitutively active CMV control (25) were incubatedwith drug in white, flat-bottom 96-well plates (Costar) for 8hours. Cells were lysed in 100 mL of Steady-Glo (Promega) andbioluminescence was measured on a BioTek plate reader.

Cell proliferation assaysIC50s were determined by nonlinear regression (GraphPad

Prism) as the average of three independent experiments usingstandard MTS assay CellTiter 96 (Promega). The results wereconfirmed with real-time proliferation assays on the IncucyteZoom as described previously (26).

Confocal microscopyTC32 cells were incubatedwithDMSOor trabectedin in aNunc

Lab-Tek II Chamber Slide (Thermo Fisher Scientific), fixed in 4%paraformaldehyde in PBS, washed, lysed in 1% Triton-X100, andblocked in 5% goat serum. Cells were incubated with primaryantibody (18 hours), secondary antibody (1 hour), and DAPI(10 minutes), mounted in VectaShield mounting media (VectorLaboratories; primary antibodies: nucleolin, Abcam, 1:1,000;HA-tag, Abcam, 1:500; FLI1, Abcam, 1:100; N-terminal EWSR1,Cell Signaling Technology, 1:1,000; secondary antibodies:

Translational Relevance

This article provides the basis for a clinical trial to evaluatetrabectedin in combination with low-dose irinotecan as anEWS-FLI1–targeted therapy. The clinical suppression of EWS-FLI1 has not been achieved despite a known dependence onthis target for more than 20 years. In addition, trabectedin hasfailed in the disease in a phase II study. These data provide anexplanation for the failed phase II, a schedule change that willimprove the therapeutic suppression of EWS-FLI1 and evi-dence that low-dose irinotecan improves the magnitude, pen-etrance, and duration of EWS-FLI1 suppression in vivo. Wedemonstrate the utility of 18F-FLT to serve as a biomarker ofEWS-FLI1 suppression in patients. In addition, we establisha novel mechanism of trabectedin as an inhibitor of theSWI/SNF chromatin-remodeling complex that is mutated inapproximately 25% of all human cancers.

Harlow et al.

Clin Cancer Res; 25(11) June 1, 2019 Clinical Cancer Research3418




Cy5-conjugated anti-mouse IgG, Vector Laboratories, 1:400,FITC-conjugated anti-rabbit IgG, Millipore, 1:200; DAPI SigmaAldrich, 1:10,000). All images were obtained with standardizedsettings on a Zeiss 510 confocal microscope.

Chromatin immunoprecipitation10 million TC32 cells were incubated with trabectedin or

DMSO for the indicated time, washed, cross-linked in 1% form-aldehyde for 10 minutes, and quenched with 0.2 mol/L glycine.The cells were collected in cold PBS with 1� protease inhibitor(SigmaAldrich), lysed in 20mmol/L Tri-HCl (pH7.5), 85mmol/LKCl, and 0.5% NP-40 for 15 minutes on ice with dounce homog-enizing. Chromatin was sheared with the E220 evolution focusedsonicator (Covaris) for 10 minutes. Ten micrograms solubilizedchromatin was immunoprecipitated with 1 mgmouse IgG (Abcam#18394), or H3K27me3 (Abcam #6002), 1 mg rabbit IgG (CellSignaling Technology #2729S) or 1 mgH3K9me3 (Abcam#8898),and 2 mg rabbit IgG or 1 mg SMARCC1/BAF155 (Cell SignalingTechnology #11956S). Antibody–chromatin complexes wereimmunoprecipitated with Magna ChIP Protein AþG magneticbeads (EMD Millipore) and washed. DNA was eluted with 100mmol/L NaHCO3, 1% SDS, and 1� proteinase K for 2 hours at65�C followed by 10-minute incubation at 95�C. Chromatinimmunoprecipitation (ChIP) DNA was purified with QiaQuickPurification Kit (Qiagen). Purified SMARCC1 ChIP DNA wasanalyzed with ChIP-qPCR, described below. Purified H3K27me3andH3K9me3ChIPDNAwas submitted for 2� 75bp sequencingand analyzed as described below.

ChIP with quantitative PCRSolubilized chromatin was treated with RNAse A at 37�C for

30 minutes followed by Proteinase K at 65�C for 2 hours, puri-fied with the QiaQuick purification kit (Qiagen), and quantifiedusing SYBR Green relative to a standard curve of DNA generatedwith input DNA from the respective sample independently foreach primer set. qPCR was performed with the following primersets (MYT1, NR0B1, SOX2, CCND1) using published primersequences (14).

ChIP SequencingLibraries for input and immunoprecipitated samples were

prepared by the Van Andel Genomics Core from 10 ng of inputmaterial and either 10 ng or all available IP material using theKAPA Hyper Prep Kit (v5.16; Kapa Biosystems). Prior to PCRamplification, end-repaired and A-tailed DNA fragments wereligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific).Quality and quantity of the libraries were by Agilent DNA HighSensitivity ChIP (Agilent Technologies, Inc.),QuantiFluor dsDNASystem (Promega Corp.), and Kapa Illumina Library Quantifica-tion qPCR assays (Kapa Biosystems). 50 bp, paired-end sequenc-ing was performed on an Illumina NovaSeq sequencer using a100-bpS1 sequencing kit (Illumina Inc.). Base callingwas donebyIllumina RTA v3.0 software and output of RTAwas demultiplexedand converted to FastQ format with Illumina Bcl2fastq2 v2.20.0.

ChIP-Seq bioinformatic analysisH3K27me3 and H3K9me3 ChIP sequencing (ChIP-seq) and

input reads were aligned to human genome (hg19) usingBWA-MEM v 0.7.15 and peaks were called using MACS2(v 2.1.1.20160309) compared with input using the broad param-eter and a q-value of 0.01 (27). Known ENCODE blacklist regions

were removed from called peaks using BEDtools intersect(v 2.27.1; refs. 28, 29). Peak intersections were also determinedusing BEDtools. SMARCC1 (BAF155) ChIP-seq data were down-loaded from NCBI-GEO (GSE94278; ref. 6) and processed usingthe same software and parameters. Peak annotation was com-pleted using the ChIPseeker package in R (v 1.14.2; ref. 30).Additional figures were generated using deepTools (v 2.3.6) andIntervene (v 0.6.2; refs. 31, 32).

Nuclear fractionation2.5 million TC32 cells were incubated with DMSO control for

the indicated times and collected or replacedwith drug-freemediafor 8 hours (9 hours total) or 15 hours (16 hours total). Cells werewashed in PBS and incubated in CSK buffer (100 mmol/L NaCl,300mmol/L sucrose, 3mmol/LMgCl2, 0.1%Triton X-100, RocheCOmplete EDTA-free tablet, 10 nmol/L Pipes, pH 7.0 withNaOH) for 20minutes on ice (33). The total fractionwas collectedand the soluble fraction was collected by centrifugation at1,300 � g for 5 minutes at 4�C. The nuclear insoluble pelletswere resuspended with CSK buffer, incubated on ice for 10minutes, and then the chromatin fraction was collected by cen-trifugation at 1,300 � g for 5 minutes at 4�C (33). Total proteinwas quantitated using Bradford assay (Bio-Rad Protein Assay DyeReagent Concentrate). Chromatin protein and soluble proteinquantitation were calculated from total protein quantitation.Total protein and chromatin protein were incubated with CSKbuffer plus Pierce Universal Nuclease (Thermo Fisher Scientific)for 20 minutes on ice. Ten micrograms of each protein samplewere resolved as described above (see Western blotting).

Xenograft experimentsTwo million TC32 cells were injected intramuscularly in the

gastrocnemius of female 8- to 10-week-old female homozygousnudemice (Crl; Nu-Foxn1Nu; Van Andel Research Institute, GrandRapids, MI) and established to a minimum diameter of 0.5 cm.Five cohorts of mice were treated with vehicle (n¼ 6), trabectedin(n¼9; 0.18mg/kg i.v. ondays 1 and8), irinotecan (n¼7; 5mg/kgintraperitoneal on days 2 and 4), the combination trabectedinplus irinotecan (n¼ 7; samedose route and schedule as the single-agent treatments). Tumor volume was measured daily and deter-mined using the equation (D � d2)/6 � 3.12 (where D is themaximum diameter and d is the minimum diameter). All experi-ments were performed in accordance with the guidelines andregulation of, and approved by the Van Andel Institute (VAI)Institutional Animal Care and Use Committee (IACUC). Inves-tigators were not blinded to the treatment groups.

18F-FLT PET imagingMice were anesthetized with 2% isoflurane in oxygen, injection

with approximately 25 mCi 18F-FLT (18F-30-Deoxy-30-Fluorothy-midine; Spectron MRC) and given 1-hour uptake time whileconscious before 10-minute imaging on a GENISYS4 pet scanner(Sofie Biosciences) and a 6-minute NanoSPECT/CT (BioscanInc.). PET reconstruction was performed using 3D maximum-likelihood expectation-maximization algorithm for 60 iterationsand CT reconstruction utilized filtered back-projection with aShepp–Loganfilter.Data visualization andanalysis utilizedOsirixMD (Pixmeo SARL) and the R statistical programming language.Reconstructed images were normalized for exact uptake time,actual injected dose, and residual dose remaining in the tail whenapplicable. Tumor uptake changes over time were assessed using

Trabectedin Inhibits EWS-FLI1

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percentage injected dose per mL (%ID/mL) and mean and max-imum standardized uptake value.

Tissue staining and IHCFive-micron sections of formalin-fixed, paraffin-embedded tis-

sue were mounted on charged slides and stained with hematox-ylin and eosin (H&E; Ventana Symphony). For IHC, antigenretrieval was performed on the PT Link platform on the DakoAutostainer Plus instrument or manually using Dako TargetRetrieval System citrate buffer. Following blocking, tissue sectionswere incubated with either SP7 Osterix antibody (Abcam,1:2,000) or MTCO2 antibody (Abcam, 1:800), washed, thenincubated in secondary antibody (polyclonal goat anti-rabbitHRP or EnvisionþSystem HRP-labeled polymer anti-rabbit,Dako, 1:100) and developed with Dako Liquid DABþ SubstrateChromogen System. Collagen staining was performed via PicroSirius Red Stain Kit (Connective Tissue Stain, Abcam).

Project statisticsAll qPCR data are normalized to solvent (expression data) or

input (CHIP data) as fold change from three independentexperiments. The P value was determined by two-sided Studentt test or one-way ANOVA using the Dunnett test for multiplecomparisons. For PET imaging, the signal above backgroundwas determined by a mixed-effects Poisson regression withrandom intercepts for each animal and false-discovery rateadjusted. Background signal was defined as the average signalfrom a similar sized region in the contralateral limb. Treatmentgroup differences were determined by a log-transformed linearmixed-effects regression with random intercepts for each ani-mal and false-discovery rate adjusted. All hypotheses were two-sided, significance level set at 0.05, and performed using Rv3.4.4. Data are plotted with signal broken out into "high,""medium," and "low," which are the tertiles of the vehicle'ssignal above background at hour 1.

ResultsSuppression of EWS-FLI1 by trabectedin requires high serumconcentrations

To determine whether the schedule of administration maycorrelate with EWS-FLI1 suppression and clinical response inpatients with Ewing sarcoma, we modeled the effects of drugexposure on cell viability and EWS-FLI1 activity in vitro. In thepediatric phase I study, trabectedinwas administered over 3 hoursand accumulated to a high maximal serum concentration (Cmax)of either 6 ng/mL (7.8–12.2 nmol/L at 1.1 mg/m2 dose) or10.5 ng/mL [13.8–20.4 nmol/L) at 1.3 mg/m2 dose] but lowerAUCof 39 ng/mL/hr. In contrast, when administered as a 24-hourinfusion in the phase II study, a greater exposure of 112 ng/mL/hrwas achieved at the expense of a lower serum Cmax of only 2.5ng/mL (3.2 nmol/L). Interestingly, 2 of 3 patients with Ewingsarcoma responded to the drug in thephase I (highCmax) andonly1 of 10 patients with Ewing sarcoma responded with stabledisease in the phase II study despite a substantially higher expo-sure (AUC) of the tumor to the drug (17, 19). These data suggestthat tumor response correlates with concentration (Cmax), nottotal exposure (AUC). Because Ewing sarcoma is dependent onEWS-FLI1, it suggests that a threshold concentration is required toblock target and impact viability.

To test this hypothesis, we pulsed cells with compoundthen changed medium to evaluate the impact of brief exposuresto trabectedin on cell viability, EWS-FLI1 activity, and down-stream target expression. This is possible because trabectedin–DNA adducts are known to be repaired and cleared from treatedcells (34). We treated cells with the identical exposure ofdrug (AUC ¼ Concentration � time; 600 nmol/L/hr), but atvarying maximal concentrations, removed the drug from medi-um, and measured the effect on viability using real-timemicroscopy. We observed sustained suppression of cell viabilityover time with as little as 1 hour of exposure if a 10 nmol/Lconcentration threshold was exceeded (Fig. 1A). To see whetherthis threshold translates to suppression of EWS-FLI1 activity,we repeated the experiment and evaluated the effect on EWS-FLI1 activity 18 hours later using a NR0B1 promoter-drivenluciferase construct (16, 35). The activity of this NR0B1-pro-moter driven luciferase is highly specific for EWS-FLI1 becauseit contains an EWS-FLI1–responsive GGAA microsatellite in thepromoter that is the proper length to induce transcrip-tion (13, 36). CRISPR/Cas9 elimination of this microsatelliteeliminates NR0B1 expression and while both FLI1 and EWS-FLI1 can bind this region, only EWS-FLI1 can activate NR0B1expression (12, 37, 38). We found marked suppression ofNR0B1 luciferase 18 hours after drug removal with no impacton a constitutively active CMV-driven control again with a 10nmol/L threshold concentration that reflects the phase I, highCmax exposure observed in patients (Fig. 1B).

To directly compare the impact of exposure on mRNA expres-sion of target genes, we performed the same experiments andevaluated mRNA expression of three target genes, NR0B1, EZH2,andWRNat 24hours (35, 39,40).We treated the cells for 1nmol/Lfor 24 hours tomaximize the likelihood that lower dose over time(AUC) would block target expression as this is 2� the GI50 of thedrug that we have previously established (16). Target suppressionwas foundonlywith a highCmax (Cmax; 24 nmol/L for 1 hour), butnot with a sustained lower dose exposure (AUC; 1 nmol/L for24hours) despite the fact that the identical total exposurewas usedin both treatments (Fig. 1C). These effects extended to the proteinlevel where again only the Cmax exposure, but not the AUCexposure, led to a loss of expression of NR0B1, EZH2, and WRNin two different Ewing sarcoma cell lines (Fig. 1D). This wasa generalized effect on EWS-FLI1 activity and suppression ofNR0B1expressionwasobserved in three additional Ewing sarcomacell lines, SK-N-MC, EW8, and TC252 cells with a high Cmax

exposure (Cmax), but not with prolonged but identical exposure(AUC; Fig. 1E).

Finally, to firmly establish the schedule dependence of theseeffects,we evaluated the effect of drug treatment on cell viability asa function of AUC. Full dose–response curves were washed out atvariable time points and the effect on viability was determined 48hours later (Fig. 1F). As long as a threshold concentration wasachieved, as little as 6 minutes of drug exposure suppressed cellviability leading therefore minimizing the AUC needed to sup-press proliferation, leading to a shift in the curve to the left (Fig. 1F;ref. 41). These effects are specific for trabectedin as a similarrelationship with Cmax was not found with an alternative EWS-FLI1 inhibitor, mithramycin (25). Even at high concentrationsthat exceed what is required to suppress EWS-FLI1, the suppres-sion of viability by mithramycin exactly correlated with AUCregardless of concentration/time of exposure (Supplementary Fig.S1A and S1B).

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EWS-FLI1 redistributes in the nucleus to the nucleolus onlywith high serum concentrations

We have previously shown that treatment of Ewing sarcomacells with trabectedin and a second-generation analogue redis-tributes EWS-FLI1 within the nucleus to the nucleolus (26).Therefore, we investigated whether the Cmax exposure wasrequired for nucleolar redistribution and if it would be sustainedfollowing drug removal. A short 24 nmol/L 1-hour pulse of drugcaused EWS-FLI1 to redistribute within the nucleus to the nucle-

olus (Fig. 2A). This effect persisted following drug removalconsistent with the sustained suppression of targets describedabove. It is notable that the penetrance of the effect within thepopulation of cells decreases over time (data not shown). Theeffectwas concentration dependent and a similar redistribution ofEWS-FLI1 was not seen with 1 nmol/L treatment even after 24hours of exposure at this concentration consistent with therequirement for high concentrations to inhibit EWS-FLI1(Fig. 2B). The effect was not dependent on TP53 status as a similar

Figure 1.

The suppression of EWS-FLI1 by trabectedin is concentration dependent.A, Direct comparison of identical exposures of trabectedin for the indicated timefollowed by replacement with drug-free medium in TC32 cells (exposure¼ concentration� time). Greater suppression of cell viability (percent confluence)occurs above a 10 nmol/L threshold (10 nmol/L, 60minutes) relative to solvent control (solvent). B, Sustained suppression of EWS-FLI1 activity as measured byNR0B1-Luc (black bars) in comparison with CMV-driven (gray bars) reporter. Cells exposed to drug for 1 hour followed by a 17-hour incubation in drug-freemedium. C, Sustained suppression of EWS-FLI1 target genes (EZH2, WRN, NR0B1) favors high concentration (Cmax) exposure to drug. Data are direct comparisonof identical exposure of 24 nmol/L trabectedin for 1 hour followed by 23 hours in drug-free medium (Cmax) or 1 nmol/L trabectedin for 24 hours (AUC) exposureasmeasuredbyqPCRfoldchange relative toGAPDH(2DDCt). �� , P < 0.0001. D and E,Western blot analysis in 5 Ewing sarcoma cell lines comparing the effect ofSolvent (S) to Cmax or AUC exposure on the expression of the EWS-FLI1 downstream targets NR0B1, EZH2, WRN relative to the GAPDH loading control. F, Dose–response curves of cell number as a function of exposure (concentration � time ¼ logAUC) in TC32 Ewing sarcoma cells. Trabectedin was incubated at 10concentrations for the indicated time and then replaced with normal medium for a total of 48 hours. Concentrations tested were 25, 20, 15, 12.5, 10, 5, 2.5, 1.25, 0.625,and 0.3125 nmol/L. Above a threshold concentration, 6minutes of drug exposure leads to sustained effects on viability 48 hours after drug is removed as indicated bythe red curve.






redistribution of EWS-FLI1was seen onlywith high dose exposure(24 nmol/L for 1 hour) in the A673 cell line (Fig. 2C).

Redistribution of EWS-FLI1 coincides with loss of SWI/SNFbinding to chromatin

A recent report has shown that the activity of EWS-FLI1 requiresthe recruitment of the ATP-dependent SWI/SNF chromatin-remo-deling complex to open chromatin and allow EWS-FLI1 to act as apioneer transcription factor (14). In addition, it is known thatboth trabectedin and SWI/SNF bind the minor groove ofDNA (42, 43). Therefore, to determine the impact of drug treat-ment on the chromatin binding of EWS-FLI1 and SWI/SNF, weagain pulsed the cells with drug and biochemically fractionatedthe cells into chromatinboundor soluble fractions.We found thatindeed, the redistribution of EWS-FLI1 led to less binding of EWS-FLI1 to chromatin. However, even more impressive was theimmediate eviction of SMARCC1 (BAF155) from chromatin thatoccurred within an hour of treatment with trabectedin (Fig. 3A).In both cases, this eviction was accompanied by accumulation ofSMARCC1 and EWS-FLI1 in the soluble fraction; an effect thatpersisted after drug removal (Fig. 3A). Importantly, this effect onlyoccurred at relatively high concentrations of trabectedin; theidentical concentration associated with target suppressionand nucleolar redistribution of EWS-FLI1. Neither SWI/SNF orEWS-FLI1 were evicted from chromatin at 1 nmol/L even with

prolonged exposure (Fig. 3B). To confirm that these effectsoccurred at EWS-FLI1 target genes and SWI/SNF–binding sites inthe genome, we used ChIP and qPCR to quantitate the impact ofdrug treatment on binding at previously identified EWS-FLI1 andSMARCC1-binding sites (from an independent study; ref. 14).Weconfirmed loss of binding of SMARCC1 to chromatin at severalkey loci (Fig. 3C). Importantly, SMARCC1 binds throughout thegenome, so as an additional control, we mapped and immuno-precipitated SMARCC1 at GAPDH. While GAPDH could beimmunoprecipitated, binding of SMARRC1 at this site wasnot impacted by drug treatment suggesting the importance ofEWS-FLI1 to this effect of trabectedin (Fig. 3D). It is notable thatidentical inputs were loaded into all immunoprecipitations (Sup-plementary Fig. S2A).

SWI/SNF eviction reverses the pioneering transcription factoractivity of EWS-FLI1

A link between SWI/SNF and EWS-FLI1 and the establishmentof GGAA microsatellites as enhancers has already been estab-lished (9, 14). Therefore, we were interested in determiningwhether the histonemodifications at EWS-FLI1 targeting changedfrom enhancer marks (K3K27ac, H3K4me1) to marks associatedwith epigenetically silenced chromatin (H3K27me3, H3K9me3).We treated cells with trabectedin (Cmax exposure), washed out thedrug, and then performedChIP ofH3K9me3 andH3K27me3 at 1

Figure 2.

Trabectedin redistributesEWS-FLI1 within the nucleus in aschedule-dependent manner.Redistribution of EWS-FLI1 withinthe nucleus in TC32 Ewingsarcoma cells with high-doseexposure (Cmax, 24 nmol/L for 1hour; A), drug removal, andincubation for the indicated time,but not with low-dose continuousexposure (AUC, 1 nmol/L for 24hours; B). C, Similar redistributionof EWS-FLI1 only with high Cmax

exposure (24 nmol/L for 1 hour) inTP53-mutant A673 cells. Confocalmicroscopy stained for nucleolin(NCL), EWS-FLI1.

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and 9 hours following drug removal. We chose these time pointsbecause they both featured the redistribution of EWS-FLI1 andloss of SMARCC1binding to chromatin (Figs. 2A, 3A, and 3B).Wefound that high-dose trabectedin led to themarked accumulationof both H3K27me3 and H3K9me3 epigenetic marks throughoutthe genome (Fig. 4A). This effect was most prominent withH3K9me3 as the number of peaks increased from 1,104 peaksin solvent to 28,901 by hour 1, with an additional 7,957 peaks byhour 9. In addition, we found an enrichment of both marks attranscriptional start sites (Fig. 4B andC). Therewas an enrichmentof H3K27me3 marks at transcriptional start sites with drug

treatment consistent with a known antagonism betweenSWI/SNF and the PRC2 complex (Fig. 4B; Supplementary Fig.S2B; refs. 44, 45). There was also a major increase in H3K9me3enrichment at transcriptional start sites (Fig. 4C). Indeed,pretreatment, there was little association between H3K9me3and transcriptional start sites consistent with the known rela-tionship between H3K9me3 and constitutive heterochroma-tin (46, 47). In contrast, after trabectedin treatment, there was amarked accumulation of H3K9me3 at transcriptional start sites,an effect most obvious when looking at the binding profile(Fig. 4D).

Figure 3.

Trabectedin evicts SWI/SNF fromchromatin in a schedule-dependentmanner. A, Trabectedin evictsSMARCC1 and EWS-FLI1 fromchromatin with high dose(Cmax, 24 nmol/L for 1 hour)followed by incubation in drug-freemedium but not continuous low-dose (AUC, 1 nmol/L continuous; B)exposure in TC32 Ewing sarcomacells. Western blot analysis showingtotal lysate (Total), chromatinfraction (chromatin) with H3histone control (H3) and solublefraction (soluble) with GAPDHcontrol. Lysates collected at 1, 9,and 16 hours. C, ChIP of IgG orSMARCC1 at known EWS-FLI1 andSWI/SNF target genes (MYT1, SOX2,CCND1, NR0B1) in comparison withD, GAPDH locus control following24 nmol/L trabectedin treatmentfor 1 hour (1 h Trab.) followed bycollection immediately or after8 more hours in drug-free medium(9 h Trab.) in TC32 cells. Data arerepresented as percent inputquantitated against a standardcurve. �� , P < 0.0001.






The silencing histone posttranslational modifications werealso associated with the enhancer GGAA repeats, SWI/SNF andEWS-FLI1. There are approximately 26,000 GGAAmicrosatellitesin the genome, almost 70% of these or 18,272 are marked withH3K27me3, H3K9me3 or both within 50 KB of a TSS after high-dose exposure to trabectedin (Fig. 4E). In addition, there was anenrichment of these marks at EWS-FLI1 target genes. We recentlypublished a list of 116 induced EWS-FLI1 targets found in mul-tiple datasets in the literature (26). Eighty-three of the 116genes inthis list were associated with GGAA microsatellites within 50 KBof the start site (Fig. 4F). Of this list of 83 targets, 76 of the 83 or92%were marked with H3K27me3, H3K9me3 or both followingtrabectedin treatment (Fig. 4G). Finally, themostwell-established

microsatellite-associated EWS-FLI1 target gene, NR0B1, wasfound to have a large H3K9me3 peak at the TSS, immediatelyadjacent to the known SWI/SNF-binding site in the region(Fig. 4H). Importantly, we confirmed the presence of bothH3K27me3 and H3K9me3 using ChIP-PCR in TC32 cells. Inaddition, we showed a similar enrichment in an additional cellline, TC252 Ewing sarcoma cells (Supplementary Fig. S3). Similarenrichment of both H3K27me3 and H3K9me3 epigenetic silenc-ing marks was observed at a number of additional well-established EWS-FLI1 target genes including RCOR1, PPP1R1A,MEIS1, WRN, EZH2, BCL11B, LOX, and PRKCB (SupplementaryFigs. S4 and S5). In addition, high-dose trabectedin treatment alsocaused the enrichment of H3K9me3 and H3K27me3 at genomic

Figure 4.

Trabectedin treatment reverses thepioneering activity of EWS-FLI1.A, Venn diagram of the totalnumber of H3K27me3 (left) andH3K9me3 (right) peaks asmeasured by chIP and sequencing(ChIP-seq) following treatment withDMSO solvent, 24 nmol/Ltrabectedin for 1 hour (1 hour Trab.),or 24 nmol/L trabectedin for 1 hourfollowed by an 8-hour recovery indrug-free media (9-hour Trab.) inTC32 Ewing sarcoma cells. Heatmapdisplaying the genome-widedistribution of H3K27me3 (B) orH3K9me3 (C) peaks relative totranscriptional start sites (TSS)following 24 nmol/L trabectedin for1 hour (Hour 1), or 24 nmol/Ltrabectedin for 1 hour followed by 8hours in drug-free media (Hour 9).D, Genome-wide distribution ofreads of H3K9me3 peaks relative toTSS following 24 nmol/Ltrabectedin for 1 hour (Hour 1), or 24nmol/L trabectedin for 1 hourfollowed by 8 hours in drug-freemedia (Hour 9). E, Total number ofGGAAmicrosatellites marked(� 50 KB) with H3K9me3 (15,400,dark green), H3K27me3 (2767, lightgreen), both (105, darkest green) orneither (8443, gray) after treatmentwith 24 nmol/L trabectedin for 1hour. F,Number of EWS-FLI1 targetgenes containing GGAAmicrosatellite sequences within50kb of TSS. G, Total number ofGGAAmicrosatellites associatedwith EWS-FLI1 target genes marked(� 50 KB) with H3K9me3 (30,blue), H3K27me3 (6, light blue),both (40, dark blue), or neither(7, gray) after treatment with24 nmol/L trabectedin for 1 hour.H,Genome browser tracks ofH3K9me3 at TSS followingindicated solvent or trabectedintreatments at the NR0B1 gene.

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sites previously associated with SWI/SNF at MYT1, CCND1, andSOX2 (Supplementary Fig. S6). Importantly, silencing ofSMARCC1 reduces cell viability inEwing sarcoma cells and furtherpotentiates the activity of the drug in an analogous fashion tosilencing of EWS-FLI1 (Supplementary Fig. S7).

Trabectedin requires irinotecan to improve suppression ofEWS-FLI1 in the three-dimensional architecture of a tumor

We have previously shown that trabectedin is particularlyeffective in Ewing sarcoma in combination with extremely lowdoses of irinotecan (35). Because irinotecan is known to impacttranscription, we sought to determine whether the function ofirinotecan in this combination is to improve the magnitude,penetrance, or duration of EWS-FLI1 suppression. We have pre-viously shown that 18F-FLT PET reflects EWS-FLI1 activity because

EWS-FLI1 drives the expression of the proteins responsible foractivity in Ewing cells, ENT1/ENT2 and TK1 (48).

Treatment of mice bearing Ewing sarcoma xenografts withtrabectedin suppressed EWS-FLI1 activity and caused a loss of18F-FLT PET activity. Peak suppression occurred 6–24 hours aftertreatment and the xenograft recovered PET avidity by 54–72hours(Fig. 5A). To investigate EWS-FLI1 suppression in the three-dimensional architecture of the tumor, we used the signal fromevery voxel in the tumor to mathematically reconstruct the tumorto determine the distribution of EWS-FLI1 suppression. Again, wefound striking 18F-FLT PET signal in control tumors (Fig. 5B) andmarked suppression of EWS-FLI1most evident in the X, Y, Z planecross-sections of the trabectedin-treated tumors (Fig. 5C). After 24hours, control animals had a mean signal 20% higher thantrabectedin-treated animals (P < 0.0001; 95%CI, 12.9–27.6).

Figure 5.

Trabectedin suppresses EWS-FLI1activity as measured by 18F-FLTimaging. A,Mice bearing TC32Ewing sarcoma xenografts in rightgastrocnemius show suppression of18F-FLT signal 6 to 54 hours aftertreatment with trabectedin, but notvehicle control. The bladder showshigh 18F-FLT signal across allsamples due to excretion of tracer.B, High PET avidity of twomice 24hours after treatment with vehicle(day 2). Data are a three-dimensional reconstruction of thetumor (tumor) followed by cross-sections in the X, Y, and Z axes. C,Suppression of 18F-FLT PET avidityin two mice 24 hours aftertreatment with 0.18 mg/kg oftrabectedin (day 2). Data are athree-dimensional reconstructionof the tumor (tumor) followed bycross-sections in the X, Y, and Zaxes. Scale indicates signalintensity.D, 3D reconstruction andsingle cross-section of tumors atmultiple time points followingtreatment with vehicle (1 hour),trabectedin (1 hour), irinotecan (24and 48 hours), or the combinationof trabectedin and irinotecan. Rowsindicate time and treatments,columns represent 3Dreconstruction and single cross-section for each of the treatmentgroups. The intensity scale is thesame as B and C.






Interestingly, we found marked variability in the distribution ofEWS-FLI1 suppression among the animals in the cohort in threedimensions, the magnitude, and even the duration of EWS-FLI1suppression (Figs. 5B and C; Supplementary Fig. S8). It is notablethat these tumors all came from the same cell line,were implantedat the same time, at the identical cell number, and treated with theidentical dose of trabectedin (and all trabectedin was delivered orlocal toxicity would be obvious). Nevertheless, the variability wasremarkable and consistent with the heterogeneity in response totreatment that we have consistently observed across cohorts ofmice regardless of therapy. We were able to rescue this variablesuppression in vivo, by adding irinotecan, which improved theamplitude, penetrance and duration of EWS-FLI1 suppressionlikely accounting for the favorable clinical experience with thiscombination (Fig. 5D). In addition, this suppression correlatedwith effects on tumor growth and striking regressions of tumorwere observed with the combination therapy as previouslyreported (ref. 35; Supplementary Fig. S9). The most strikingexample was the day 8 animals (combo treatment in Fig. 4) thathad complete suppression of target and complete regression oftumor while trabectedin and irinotecan recovered signal at 102

hours (Fig. 5D, �). The average number of voxels with signal abovebackground for trabectedin and irinotecan animals was 3,346.17and 977.72, respectively [95% CIs (722.8–15,490.5); (192.4–4,968.1)]; compared with 24.3 for animals treated with both(95% CI, 4.2–141.1; P¼ 0.0003, 0.0063, respectively). However,as early as day 5, the animals showed little to no evidence of18F-FLT activity suggesting a change in the tissue from highlyproliferative malignant tissue to benign consistent with a sus-tained release in the EWS-FLI1–mediated differentiation block.

Importantly, this type of analysis would simply not be possiblewith traditional IHC or PCR approaches to evaluate target sup-pression as it allowed us to evaluate the distribution of suppres-sion in the same animal over time. Finally, it is notable thatsustained suppression of EWS-FLI1with this combination led to arelease in the differentiation block and the tumor showed evi-dence of differentiation down a number ofmesenchymal lineagesand human collagen, osteoblasts, and fat were identified in thexenograft (Fig. 6). It is notable that the mouse is known toremodel and replace benign human tissue with mouse tissue andso the penetrance of the differentiation phenotype is difficult toestablish (49). While the cell of origin of Ewing sarcoma is not

Figure 6.

Combination treatment oftrabectedin and irinotecan inducesdifferentiation of TC32 Ewingsarcoma cells in vivo. A, (left toright) 4� and 20�magnification ofH&E staining of TC32 IM xenografttumor 3 days after treatment withvehicle (control) or trabectedin andirinotecan (treated). 60�magnification of MTCO2 humanmitochondrial stain and 60� SP7Osterix osteoblast stain showinghuman cells expressing SP7 intreated but not control. B, (left toright) 4� and 20�magnification ofH&E staining of TC32 IM xenograft.60�magnification of MTCO2humanmitochondrial stain and 60�PicroSirius Red stain indicatingspecific human collagen cells 5 daysafter treatment with vehicle(control) or trabectedin andirinotecan (treated). C, (left toright) 4� and 20�magnification ofH&E staining of TC32 IM xenograft.20� and 60�magnification ofMTCO2 humanmitochondrial stainshowing human adipocyte. Fivedays after treatment with vehicle(control) trabectedin and irinotecan(treated).

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known, current thinking favors a mesenchymal or neural crestorigin (39, 50–52). Therefore, sustained suppression of EWS-FLI1allows restoration of the differentiation programbut this programis relatively unorganized leading to mesenchymal confusion.

DiscussionThis study highlights the importance of drugmechanism to the

drug development process. Compounds with broad cytotoxicityprofiles canbedeveloped for specific indications if they inhibit thedominant oncogene of a specific tumor. However, the successfulimplementation of therapies of this type absolutely requires thatthe mechanism of suppression be optimized for a specific onco-gene and a defined cell context.

In this study, we show that the therapeutic suppression of thedominant oncogene of Ewing sarcoma, EWS-FLI1, requires a highconcentration of trabectedin in serum. We model this exposurepreclinically and show in vitro and in vivo that the drug is able toinhibit EWS-FLI1.

The drug redistributes EWS-FLI1 in the nucleus, displacesSWI/SNF from chromatin, and triggers an epigenetic switchdriving an increase in H3K27me3 and H3K9me3 with preferencefor EWS-FLI1 target genes. However, these effects absolutelyrequire high concentrations of drug in serum and do not occurat lower concentrations even with prolonged exposure.

These observations are important because they justify theinvestigation of trabectedin in Ewing sarcoma on a short-infusion schedule in combination with low-dose irinotecan.It has been known for more than 20 years that Ewing sarcomacells are dependent on EWS-FLI1 (53). However, the therapeu-tic suppression of EWS-FLI1 has not been achieved in clinic. Inaddition, trabectedin was previously evaluated in Ewing sarco-ma as a 24-hour infusion in a phase II study because thisschedule was shown to be more active in other sarcomatypes (54). However, the data in this article suggest that ashorter 1-hour infusion schedule may increase activity in Ewingsarcoma because the drug would accumulate to serum concen-trations above a threshold that we define in this article as beinghigh enough to inhibit the dominant oncogene, EWS-FLI1 (41).This blockade of EWS-FLI1 is amplified and sustained incombination with low-dose irinotecan. Because this tumorabsolutely depends on EWS-FLI1, it is likely that this studywould show clinical activity. Therefore, this study justifies thefurther exploration of this compound on an alternative 1-hourinfusion schedule in this tumor in combination with low-doseirinotecan. Perhaps the most important observation in thisstudy is that even within sarcoma, different schedules of activecompounds may be more effective in particular subtypes.

This study also provides important insight into the mecha-nism of action of trabectedin, a compound that has foundunique activity in a number of sarcomas. Trabectedin has acomplicated mechanism of action including both generatingDNA damage and poisoning-specific DNA damage repaircomplexes, poisoning-specific transcription factors such asEWS-FLI1 and FUS-CHOP, and specifically targeting tumor-associated macrophages (55, 56). In this study, we add dis-placement of SWI/SNF from chromatin to this mechanism. It islikely that this mechanism contributes to the broad cytotoxicityprofile of this compound as SWI/SNF is mutated in up to 25%of human cancer and commonly altered either functionally orthrough mutation in sarcoma.

This study also highlights important features of Ewing sarcomabiology. We confirm the recent observation that SWI/SNF isimportant to the biology of EWS-FLI1 and further establish thelink to EWS-FLI1, particularly at the GGAA microsatellites (14).We show that removal of EWS-FLI1 leads to a cellular responseandwidespread chromatin silencing, particularly withH3K9me3,which favors repetitive sequences and constitutive heterochro-matin. The data suggest both inhibition of EWS-FLI1 and dis-placement of SWI/SNF are required to reverse activity; however,further workwould need to be done to clearly establish this point.In addition, once this reversal is achieved, relatively nonspecificblockade can sustain suppression of the target in vivo. The netresult is a differentiation endpoint, although this differentiation isunorganized.

Finally, this study reports a novel use of 18F-FLT PET imagingas a tool to quantitate target suppression and at the same timevisualize the penetrance and distribution of target suppressionwithin the three-dimensional architecture of the tumor. Indeed,perhaps the most interesting observation in this study is thewidely variable suppression of EWS-FLI1 that occurred withincohorts of mice. The Ewing sarcoma xenografts were establishedfrom the same cell collection and the same flask and tube, with2 million cells in every animal by the same technician on thesame day in one strain of animal. Treatment was also initiatedby the same technician from the same stock of drug and all drugmade it into the circulation as any extravasation of this drugleads to tail necrosis. Yet, despite these similarities, the mag-nitude, penetrance, and even duration of target suppression waswidely variable from one animal to the next. It is likely that thisvariable target suppression is an important factor driving tumorresponse. However, it is not known what the source of thisvariability is; a question we are now starting to investigate.Nevertheless, this study serves as proof of principle to ask thisquestion in a prospective fashion, using schedule-optimizedtrabectedin in combination with low-dose irinotecan, and18F-FLT imaging in patients with Ewing sarcoma in the clinic.

Disclosure of Potential Conflicts of InterestS.L. Lessnick holds ownership interest (including patents) in and is a

consultant/advisory board member for Salarius Pharmaceuticals. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design:M.L. Harlow, M.H. Chasse, M.J. Bowman, P.J. GroharDevelopment of methodology: M.L. Harlow, S.M. Kitchen-Goosen,S.B. Rothbart, A.S. Peck, P.J. GroharAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.L. Harlow, M.H. Chasse, E.A. Boguslawski,K.M. Sorensen, J.M. Gedminas, S.M. Kitchen-Goosen, S.L. Lessnick, A.S. PeckAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.L. Harlow, M.H. Chasse, S.B. Rothbart, C. Taslim,S.L. Lessnick, A.S. Peck, Z.B. Madaj, M.J. Bowman, P.J. GroharWriting, review, and/or revisionof themanuscript:M.L.Harlow,M.H.Chasse,E.A. Boguslawski, K.M. Sorensen, J.M. Gedminas, S.B. Rothbart, S.L. Lessnick,A.S. Peck, Z.B. Madaj, M.J. Bowman, P.J. GroharAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E.A. Boguslawski, S.M. Kitchen-Goosen,C. Taslim, M.J. Bowman, P.J. GroharStudy supervision: P.J. Grohar

AcknowledgmentsThe authors would like to thank Ron Chandler, PhD (Michigan State

University, East Lansing, MI) for helpful discussion. The authors would alsolike to thank Dr. Peter Adamson (Children's Hospital of Philadelphia,






Philadelphia, PA) for helpful advice. We would like to thank Robert Vaughanfrom the Rothbart lab for technical help. We would like to thank Marie Adamsfor technical support and library preparations and the Bioinformatic andBiostatistics Core of the Van Andel Research Institute (Grand Rapids, MI). Theauthors would like to thank Pharma Mar pharmaceutical company for materialused in this proposal. P.J. Grohar is supported by a grant from the NIH(R01-CA188314). Additional support is from the NIH/NCI MHC(F31CA236300). The imaging portion of the study was supported by a ReachAward from Alex's Lemonade Stand Foundation (to P.J. Grohar). The work isalso supported by internal funds from the Van Andel Institute (to P.J.Grohar, S.B. Rothbart, Z.B. Madaj, M.J. Bowman). Additional support is

from Hyundai Hope on Wheels (to J.M. Gedminas), the NIH/NIGMS(R35GM124736; S.B. Rothbart), and the NIH/NCI U54CA231641,R01CA183776 (to S.L. Lessnick).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 26, 2018; revised December 24, 2018; accepted January 23,2019; published first February 5, 2019.

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2019;25:3417-3429. Published OnlineFirst February 5, 2019.Clin Cancer Res Matt L. Harlow, Maggie H. Chasse, Elissa A. Boguslawski, et al. in a Schedule-dependent MannerTrabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin

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