Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule- Dependent Manner Matt L

Published OnlineFirst February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule- dependent Manner Matt 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,and Patrick J. Grohar2,3,4

Abstract

Purpose: The successful clinical translation of compounds Results: Trabectedin evicts the SWI/SNF chromatin- that target speciﬁc oncogenic transcription factors will require remodeling complex from chromatin and redistributes an understanding of the mechanism of target suppression to EWS-FLI1 in the nucleus leading to a marked increase optimize the dose and schedule of administration. We have in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. previously shown trabectedin reverses the gene signature of These effects only occur at high concentrations of trabec- the EWS-FLI1 transcription factor. In this report, we establish tedin leading to suppression of EWS-FLI1 target genes the mechanism of suppression and use it to justify the reeval- and a loss of cell viability. In vivo, low-dose irinotecan uation of this drug in the clinic in patients with Ewing sarcoma. is required to improve the magnitude, penetrance, and Experimental Design: We demonstrate a novel epigenetic duration of target suppression in the three-dimensional mechanism of trabectedin using biochemical fractionation architecture of the tumor leading to differentiation of and chromatin immunoprecipitation sequencing. We link the Ewing sarcoma xenograft into benign mesenchymal the effect to drug schedule and EWS-FLI1 downstream target tissue. expression using confocal microscopy, qPCR, Western Conclusions: These data provide the justiﬁcation to eval- blot analysis, and cell viability assays. Finally, we quantitate uate trabectedin in the clinic on a short infusion schedule in target suppression within the three-dimensional architec- combination with low-dose irinotecan with 18F-FLT PET ture of the tumor in vivo using 18F-FLT imaging. imaging in patients with Ewing sarcoma.

Introduction tional druggable domain and most transcription factors interact with complex networks of proteins. Nevertheless, compounds Oncogenic transcription factors are dominant oncogenes for that have successfully targeted speciﬁc transcription, such as a large number of leukemias and solid tumors in both the ATRA and arsenic trioxide in acute promyelocytic (APL), are pediatric and adult populations (1–3). These proteins are effective in the clinic (4–6). challenging drug targets because the active site lacks a tradi- Ewing sarcoma is a bone and soft-tissue sarcoma that is absolutely dependent on the EWS-FLI1 transcription factor for 1Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee. cell survival (7). This fusion transcription factor, formed by the 2Van Andel Research Institute, Grand Rapids, Michigan. 3Department of Pedi- t(11;22)(q24;12) chromosomal translocation, both drives 4 atrics, Michigan State University, East Lansing, Michigan. Division of Pediatric proliferation and blocks differentiation (8, 9). EWS-FLI1 acts Hematology/Oncology, Helen DeVos Children's Hospital, Grand Rapids, Michi- 5 as a pioneer transcription factor and binds to repetitive regions gan. Center for Childhood Cancer and Blood Diseases, Nationwide Children's – Hospital Research Institute, Columbus, Ohio. 6Division of Pediatric Hematology/ of the genome called GGAA microsatellites (10 13). Once Oncology/BMT, The Ohio State University College of Medicine, Columbus, Ohio. bound, the protein exhibits phase-transition properties to establish these microsatellites as enhancers to drive gene Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). expression (14). This requires a complex network of protein interactions and relies heavily on the ATP-dependent chroma- M.L. Harlow and M.H. Chasse contributed equally to this article. tin-remodeling complex, SWI/SNF to maintain chromatin in Current address for M.L. Harlow: Dana-Farber Cancer Institute, Boston, Massa- an open state (14, 15). Therefore, it is likely that reversal of chusetts; current address for A.S. Peck, Bamf Health, Grand Rapids, Michigan; EWS-FLI1activitywouldleadtowidespreadchangesinchro- and current address for M.J. Bowman, Ball Horticultural Company, West Chi- cago, Illinois. matin structure and restore the differentiation program. How- ever, it is not clear whether the effective targeting of EWS-FLI1 Corresponding Author: Patrick J. Grohar, Van Andel Research Institute, 333 requires a blockade of SWI/SNF activity or whether the pioneer Bostwick Ave NE, Grand Rapids, MI 49503. Phone: 616-234-5000; Fax: 616-234- 5309; E-mail: [email protected] transcription factor activity of EWS-FLI1 is reversible genome- wide. Clin Cancer Res 2019;25:3417–29 We have previously shown that the natural product trabectedin doi: 10.1158/1078-0432.CCR-18-3511 interferes with the activity of the EWS-FLI1 transcription fac- 2019 American Association for Cancer Research. tor (16). We showed that trabectedin reverses expression of the

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Translational Relevance Materials and Methods Cell culture This article provides the basis for a clinical trial to evaluate TC32, A673 cells were obtained from Dr. Lee Helman and trabectedin in combination with low-dose irinotecan as an TC252, SK-N-MC, EW8 from Dr. Tim Triche (both at Children's EWS-FLI1–targeted therapy. The clinical suppression of EWS- Hospital of Los Angeles, Los Angeles, CA). Cell identity was FLI1 has not been achieved despite a known dependence on confirmed by short tandem repeat profiling (DDC Medical; last this target for more than 20 years. In addition, trabectedin has test October 24, 2018). They were cultured at 37C pathogen free failed in the disease in a phase II study. These data provide an with 5% CO in RPMI-1640 (Gibco) with 10% FBS (Gemini Bio- explanation for the failed phase II, a schedule change that will 2 Products), 2 mmol/L L-glutamine, and 100 U/mL and 100 mg/mL improve the therapeutic suppression of EWS-FLI1 and evi- penicillin and streptomycin (Gibco). dence that low-dose irinotecan improves the magnitude, penetrance, and duration of EWS-FLI1 suppression in vivo.We demonstrate the utility of 18F-FLT to serve as a biomarker of Western blotting EWS-FLI1 suppression in patients. In addition, we establish 1.5 million cells (TC32, A673) or 3 million cells (TC252, a novel mechanism of trabectedin as an inhibitor of the EW8, SK-N-MC) were incubated with drug, washed in PBS, and SWI/SNF chromatin-remodeling complex that is mutated in lysed in 4% lithium dodecyl sulfate (LDS) buffer. Thirty micro- – approximately 25% of all human cancers. 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 at EWS-FLI1 gene signature. In addition, we cloned EWS-FLI1 into 20 V in 1 Tris-Glycine-SDS Buffer (Bio-Rad) with 20% meth- another cellular context, induced an EWS-FLI1–driven promot- anol. The membranes were blocked in 5% milk in TBS-T, and er luciferase construct, and then rescued this induction with probed with WRN, NR0B1, GAPDH (Abcam), or EZH2 (Cell trabectedin (16). These findings were consistent with early Signaling Technology) antibodies. preclinical and clinical experience with the drug that suggested a heightened sensitivity of Ewing sarcoma to trabectedin (17, 18). Most notably, a patient with treatment-refractory Quantitative RT-PCR Ewing sarcoma achieved a durable complete response with RNA was collected using the RNeasy Kit (Qiagen), immediately single-agent trabectedin treatment in the phase I study. In reverse-transcribed using a High-Capacity Reverse Transcriptase Kit (Life Technologies) at 25C for 10 minutes, 37C for 120 contrast, the phase II study in Ewing sarcoma was negative and only 1 of 10 patients responded to the drug (19). However, minutes, and 85 C for 10 minutes. The products were quantitated the drug was administered on a different schedule in the using qPCR, SYBR green (Bio-Rad), and the following program: 95C for 10 minutes, 95C for 15 seconds, 55C for 15 seconds, negative phase II study. Therefore, it is possible that a detailed understanding of the mechanism of EWS-FLI1 suppression by and 72 C for 1 minute, for 40 cycles. Expression was determined trabectedin would allow us to optimize the schedule of admin- from three independent experiments relative to GAPDH and DD istration and achieve the therapeutic suppression of EWS-FLI1 solvent control using standard Ct methods. in the clinic. Like many natural products, trabectedin has a complicated Luciferase assays mechanism of action (20, 21). The compound is known to Stable cell lines containing an EWS-FLI1–driven NR0B1 lucif- generate DNA damage and poison various repair pathways, block erase or constitutively active CMV control (25) were incubated specific transcription factors such as the FUS-CHOP transcription with drug in white, flat-bottom 96-well plates (Costar) for 8 factor, and exert cytotoxicity with preference for specific cell types hours. Cells were lysed in 100 mL of Steady-Glo (Promega) and such as tumor-associated macrophages (TAM), myxoid liposar- bioluminescence was measured on a BioTek plate reader. coma cells, and Ewing sarcoma cells (22–24) In this study, we define the mechanism of EWS-FLI1 sup- Cell proliferation assays pression to establish trabectedin as a bona fide EWS-FLI1 IC50s were determined by nonlinear regression (GraphPad inhibitor. We show that the drug redistributes EWS-FLI1 within Prism) as the average of three independent experiments using the nucleus and at the same time evicts the SWI/SNF chroma- standard MTS assay CellTiter 96 (Promega). The results were tin-remodeling complex to trigger an epigenetic switch, leading confirmed with real-time proliferation assays on the Incucyte to global increases in H3K27me3 and H3K9me3 with prefer- Zoom as described previously (26). ence for GGAA microsatellites and EWS-FLI1 target genes. Importantly, these effects are concentration dependent, and Confocal microscopy lead to sustained target suppression only if a threshold con- TC32 cells were incubated with DMSO or trabectedin in a Nunc centration of drug is exceeded. This mechanistic insight is Lab-Tek II Chamber Slide (Thermo Fisher Scientific), fixed in 4% consistent with the clinical experience with the drug where paraformaldehyde in PBS, washed, lysed in 1% Triton-X100, and this threshold was exceeded in the phase I and patients blocked in 5% goat serum. Cells were incubated with primary responded, but not the negative phase II study. Finally, target antibody (18 hours), secondary antibody (1 hour), and DAPI suppression is amplified and sustained in vivo in combination (10 minutes), mounted in VectaShield mounting media (Vector with the topoisomerase inhibitor irinotecan to cause a com- Laboratories; primary antibodies: nucleolin, Abcam, 1:1,000; plete histologic change in the tumor and differentiation into HA-tag, Abcam, 1:500; FLI1, Abcam, 1:100; N-terminal EWSR1, benign mesenchymal tissues. Cell Signaling Technology, 1:1,000; secondary antibodies:

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Cy5-conjugated anti-mouse IgG, Vector Laboratories, 1:400, were removed from called peaks using BEDtools intersect FITC-conjugated anti-rabbit IgG, Millipore, 1:200; DAPI Sigma (v 2.27.1; refs. 28, 29). Peak intersections were also determined Aldrich, 1:10,000). All images were obtained with standardized using BEDtools. SMARCC1 (BAF155) ChIP-seq data were down- settings on a Zeiss 510 confocal microscope. loaded from NCBI-GEO (GSE94278; ref. 6) and processed using the same software and parameters. Peak annotation was com- Chromatin immunoprecipitation pleted using the ChIPseeker package in R (v 1.14.2; ref. 30). 10 million TC32 cells were incubated with trabectedin or Additional figures were generated using deepTools (v 2.3.6) and DMSO for the indicated time, washed, cross-linked in 1% form- Intervene (v 0.6.2; refs. 31, 32). aldehyde for 10 minutes, and quenched with 0.2 mol/L glycine. The cells were collected in cold PBS with 1 protease inhibitor Nuclear fractionation (Sigma Aldrich), lysed in 20 mmol/L Tri-HCl (pH 7.5), 85 mmol/L 2.5 million TC32 cells were incubated with DMSO control for KCl, and 0.5% NP-40 for 15 minutes on ice with dounce homog- the indicated times and collected or replaced with drug-free media enizing. Chromatin was sheared with the E220 evolution focused for 8 hours (9 hours total) or 15 hours (16 hours total). Cells were sonicator (Covaris) for 10 minutes. Ten micrograms solubilized washed in PBS and incubated in CSK buffer (100 mmol/L NaCl, chromatin was immunoprecipitated with 1 mg mouse IgG (Abcam 300 mmol/L sucrose, 3 mmol/L MgCl2, 0.1% Triton X-100, Roche #18394), or H3K27me3 (Abcam #6002), 1 mg rabbit IgG (Cell COmplete EDTA-free tablet, 10 nmol/L Pipes, pH 7.0 with Signaling Technology #2729S) or 1 mg H3K9me3 (Abcam #8898), NaOH) for 20 minutes on ice (33). The total fraction was collected and 2 mg rabbit IgG or 1 mg SMARCC1/BAF155 (Cell Signaling and the soluble fraction was collected by centrifugation at Technology #11956S). Antibody–chromatin complexes were 1,300 g for 5 minutes at 4C. The nuclear insoluble pellets immunoprecipitated with Magna ChIP Protein AþG magnetic were resuspended with CSK buffer, incubated on ice for 10 beads (EMD Millipore) and washed. DNA was eluted with 100 minutes, and then the chromatin fraction was collected by cen- mmol/L NaHCO3, 1% SDS, and 1 proteinase K for 2 hours at trifugation at 1,300 g for 5 minutes at 4 C (33). Total protein 65C followed by 10-minute incubation at 95C. Chromatin was quantitated using Bradford assay (Bio-Rad Protein Assay Dye immunoprecipitation (ChIP) DNA was purified with QiaQuick Reagent Concentrate). Chromatin protein and soluble protein Purification Kit (Qiagen). Purified SMARCC1 ChIP DNA was quantitation were calculated from total protein quantitation. analyzed with ChIP-qPCR, described below. Purified H3K27me3 Total protein and chromatin protein were incubated with CSK and H3K9me3 ChIP DNA was submitted for 2 75 bp sequencing buffer plus Pierce Universal Nuclease (Thermo Fisher Scientific) and analyzed as described below. for 20 minutes on ice. Ten micrograms of each protein sample were resolved as described above (see Western blotting). ChIP with quantitative PCR Solubilized chromatin was treated with RNAse A at 37C for Xenograft experiments 30 minutes followed by Proteinase K at 65C for 2 hours, puri- Two million TC32 cells were injected intramuscularly in the fied with the QiaQuick purification kit (Qiagen), and quantified gastrocnemius of female 8- to 10-week-old female homozygous using SYBR Green relative to a standard curve of DNA generated nude mice (Crl; Nu-Foxn1Nu; Van Andel Research Institute, Grand with input DNA from the respective sample independently for Rapids, MI) and established to a minimum diameter of 0.5 cm. each primer set. qPCR was performed with the following primer Five cohorts of mice were treated with vehicle (n ¼ 6), trabectedin sets (MYT1, NR0B1, SOX2, CCND1) using published primer (n ¼ 9; 0.18 mg/kg i.v. on days 1 and 8), irinotecan (n ¼ 7; 5 mg/kg sequences (14). intraperitoneal on days 2 and 4), the combination trabectedin plus irinotecan (n ¼ 7; same dose route and schedule as the single- ChIP Sequencing agent treatments). Tumor volume was measured daily and deter- Libraries for input and immunoprecipitated samples were mined using the equation (D d2)/6 3.12 (where D is the prepared by the Van Andel Genomics Core from 10 ng of input maximum diameter and d is the minimum diameter). All experi- material and either 10 ng or all available IP material using the ments were performed in accordance with the guidelines and KAPA Hyper Prep Kit (v5.16; Kapa Biosystems). Prior to PCR regulation of, and approved by the Van Andel Institute (VAI) amplification, end-repaired and A-tailed DNA fragments were Institutional Animal Care and Use Committee (IACUC). Inves- ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific). tigators were not blinded to the treatment groups. Quality and quantity of the libraries were by Agilent DNA High Sensitivity ChIP (Agilent Technologies, Inc.), QuantiFluor dsDNA 18F-FLT PET imaging System (Promega Corp.), and Kapa Illumina Library Quantifica- Mice were anesthetized with 2% isoflurane in oxygen, injection tion qPCR assays (Kapa Biosystems). 50 bp, paired-end sequenc- with approximately 25 mCi 18F-FLT (18F-30-Deoxy-30-Fluorothy- ing was performed on an Illumina NovaSeq sequencer using a midine; Spectron MRC) and given 1-hour uptake time while 100-bp S1 sequencing kit (Illumina Inc.). Base calling was done by conscious before 10-minute imaging on a GENISYS4 pet scanner Illumina RTA v3.0 software and output of RTA was demultiplexed (Sofie Biosciences) and a 6-minute NanoSPECT/CT (Bioscan and converted to FastQ format with Illumina Bcl2fastq2 v2.20.0. Inc.). PET reconstruction was performed using 3D maximum- likelihood expectation-maximization algorithm for 60 iterations ChIP-Seq bioinformatic analysis and CT reconstruction utilized filtered back-projection with a H3K27me3 and H3K9me3 ChIP sequencing (ChIP-seq) and Shepp–Logan filter. Data visualization and analysis utilized Osirix input reads were aligned to human genome (hg19) using MD (Pixmeo SARL) and the R statistical programming language. BWA-MEM v 0.7.15 and peaks were called using MACS2 Reconstructed images were normalized for exact uptake time, (v 2.1.1.20160309) compared with input using the broad param- actual injected dose, and residual dose remaining in the tail when eter and a q-value of 0.01 (27). Known ENCODE blacklist regions applicable. Tumor uptake changes over time were assessed using

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percentage injected dose per mL (%ID/mL) and mean and max- To test this hypothesis, we pulsed cells with compound imum standardized uptake value. then changed medium to evaluate the impact of brief exposures to trabectedin on cell viability, EWS-FLI1 activity, and down- – Tissue staining and IHC stream target expression. This is possible because trabectedin Five-micron sections of formalin-fixed, paraffin-embedded tis- DNA adducts are known to be repaired and cleared from treated sue were mounted on charged slides and stained with hematox- cells (34). We treated cells with the identical exposure of ¼ ylin and eosin (H&E; Ventana Symphony). For IHC, antigen drug (AUC Concentration time; 600 nmol/L/hr), but at retrieval was performed on the PT Link platform on the Dako varying maximal concentrations, removed the drug from medi- Autostainer Plus instrument or manually using Dako Target um, and measured the effect on viability using real-time Retrieval System citrate buffer. Following blocking, tissue sections microscopy. We observed sustained suppression of cell viability were incubated with either SP7 Osterix antibody (Abcam, over time with as little as 1 hour of exposure if a 10 nmol/L 1:2,000) or MTCO2 antibody (Abcam, 1:800), washed, then concentration threshold was exceeded (Fig. 1A). To see whether incubated in secondary antibody (polyclonal goat anti-rabbit this threshold translates to suppression of EWS-FLI1 activity, HRP or EnvisionþSystem HRP-labeled polymer anti-rabbit, we repeated the experiment and evaluated the effect on EWS- Dako, 1:100) and developed with Dako Liquid DABþ Substrate FLI1 activity 18 hours later using a NR0B1 promoter-driven Chromogen System. Collagen staining was performed via Picro luciferase construct (16, 35). The activity of this NR0B1-pro- fi Sirius Red Stain Kit (Connective Tissue Stain, Abcam). moter driven luciferase is highly speci c for EWS-FLI1 because it contains an EWS-FLI1–responsive GGAA microsatellite in the promoter that is the proper length to induce transcrip- Project statistics tion (13, 36). CRISPR/Cas9 elimination of this microsatellite All qPCR data are normalized to solvent (expression data) or eliminates NR0B1 expression and while both FLI1 and EWS- input (CHIP data) as fold change from three independent FLI1 can bind this region, only EWS-FLI1 can activate NR0B1 experiments. The P value was determined by two-sided Student expression (12, 37, 38). We found marked suppression of t test or one-way ANOVA using the Dunnett test for multiple NR0B1 luciferase 18 hours after drug removal with no impact comparisons. For PET imaging, the signal above background on a constitutively active CMV-driven control again with a 10 was determined by a mixed-effects Poisson regression with nmol/L threshold concentration that reflects the phase I, high random intercepts for each animal and false-discovery rate C exposure observed in patients (Fig. 1B). fi max adjusted. Background signal was de ned as the average signal To directly compare the impact of exposure on mRNA expres- from a similar sized region in the contralateral limb. Treatment sion of target genes, we performed the same experiments and group differences were determined by a log-transformed linear evaluated mRNA expression of three target genes, NR0B1, EZH2, mixed-effects regression with random intercepts for each ani- and WRN at 24 hours (35, 39, 40). We treated the cells for 1 nmol/L mal and false-discovery rate adjusted. All hypotheses were two- for 24 hours to maximize the likelihood that lower dose over time sided, significance level set at 0.05, and performed using R (AUC) would block target expression as this is 2 the GI50 of the v3.4.4. Data are plotted with signal broken out into "high," drug that we have previously established (16). Target suppression "medium," and "low," which are the tertiles of the vehicle's was found only with a high Cmax (Cmax; 24 nmol/L for 1 hour), but signal above background at hour 1. not with a sustained lower dose exposure (AUC; 1 nmol/L for 24 hours) despite the fact that the identical total exposure was used in both treatments (Fig. 1C). These effects extended to the protein Results level where again only the Cmax exposure, but not the AUC Suppression of EWS-FLI1 by trabectedin requires high serum exposure, led to a loss of expression of NR0B1, EZH2, and WRN concentrations in two different Ewing sarcoma cell lines (Fig. 1D). This was To determine whether the schedule of administration may a generalized effect on EWS-FLI1 activity and suppression of correlate with EWS-FLI1 suppression and clinical response in NR0B1 expression was observed in three additional Ewing sarcoma patients with Ewing sarcoma, we modeled the effects of drug cell lines, SK-N-MC, EW8, and TC252 cells with a high Cmax exposure on cell viability and EWS-FLI1 activity in vitro. In the exposure (Cmax), but not with prolonged but identical exposure pediatric phase I study, trabectedin was administered over 3 hours (AUC; Fig. 1E). and accumulated to a high maximal serum concentration (Cmax) Finally, to firmly establish the schedule dependence of these of either 6 ng/mL (7.8–12.2 nmol/L at 1.1 mg/m2 dose) or effects, we evaluated the effect of drug treatment on cell viability as 10.5 ng/mL [13.8–20.4 nmol/L) at 1.3 mg/m2 dose] but lower a function of AUC. Full dose–response curves were washed out at AUC of 39 ng/mL/hr. In contrast, when administered as a 24-hour variable time points and the effect on viability was determined 48 infusion in the phase II study, a greater exposure of 112 ng/mL/hr hours later (Fig. 1F). As long as a threshold concentration was was achieved at the expense of a lower serum Cmax of only 2.5 achieved, as little as 6 minutes of drug exposure suppressed cell ng/mL (3.2 nmol/L). Interestingly, 2 of 3 patients with Ewing viability leading therefore minimizing the AUC needed to sup- sarcoma responded to the drug in the phase I (high Cmax) and only press proliferation, leading to a shift in the curve to the left (Fig. 1F; 1 of 10 patients with Ewing sarcoma responded with stable ref. 41). These effects are specific for trabectedin as a similar disease in the phase II study despite a substantially higher expo- relationship with Cmax was not found with an alternative EWS- sure (AUC) of the tumor to the drug (17, 19). These data suggest FLI1 inhibitor, mithramycin (25). Even at high concentrations that tumor response correlates with concentration (Cmax), not that exceed what is required to suppress EWS-FLI1, the suppres- total exposure (AUC). Because Ewing sarcoma is dependent on sion of viability by mithramycin exactly correlated with AUC EWS-FLI1, it suggests that a threshold concentration is required to regardless of concentration/time of exposure (Supplementary Fig. block target and impact viability. S1A and S1B).

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Figure 1. The suppression of EWS-FLI1 by trabectedin is concentration dependent. A, Direct comparison of identical exposures of trabectedin for the indicated time followed by replacement with drug-free medium in TC32 cells (exposure ¼ concentration time). Greater suppression of cell viability (percent conﬂuence) occurs above a 10 nmol/L threshold (10 nmol/L, 60 minutes) relative to solvent control (solvent). B, Sustained suppression of EWS-FLI1 activity as measured by NR0B1-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-free medium. C, Sustained suppression of EWS-FLI1 target genes (EZH2, WRN, NR0B1) favors high concentration (Cmax) exposure to drug. Data are direct comparison of 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) exposure DDC asmeasured by qPCR fold change relative to GAPDH (2 t). , P < 0.0001. D and E, Western blot analysis in 5 Ewing sarcoma cell lines comparing the effect of

Solvent (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 10 concentrations 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, 6 minutes of drug exposure leads to sustained effects on viability 48 hours after drug is removed as indicated by the red curve.

EWS-FLI1 redistributes in the nucleus to the nucleolus only olus (Fig. 2A). This effect persisted following drug removal with high serum concentrations consistent with the sustained suppression of targets described We have previously shown that treatment of Ewing sarcoma above. It is notable that the penetrance of the effect within the cells with trabectedin and a second-generation analogue redis- population of cells decreases over time (data not shown). The tributes EWS-FLI1 within the nucleus to the nucleolus (26). effect was concentration dependent and a similar redistribution of Therefore, we investigated whether the Cmax exposure was EWS-FLI1 was not seen with 1 nmol/L treatment even after 24 required for nucleolar redistribution and if it would be sustained hours of exposure at this concentration consistent with the following drug removal. A short 24 nmol/L 1-hour pulse of drug requirement for high concentrations to inhibit EWS-FLI1 caused EWS-FLI1 to redistribute within the nucleus to the nucle- (Fig. 2B). The effect was not dependent on TP53 status as a similar

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Figure 2. Trabectedin redistributes EWS-FLI1 within the nucleus in a schedule-dependent manner. Redistribution of EWS-FLI1 within the nucleus in TC32 Ewing sarcoma cells with high-dose

exposure (Cmax, 24 nmol/L for 1 hour; A), drug removal, and incubation for the indicated time, but not with low-dose continuous exposure (AUC, 1 nmol/L for 24 hours; B). C, Similar redistribution

of EWS-FLI1 only with high Cmax exposure (24 nmol/L for 1 hour) in TP53-mutant A673 cells. Confocal microscopy stained for nucleolin (NCL), EWS-FLI1.

redistribution of EWS-FLI1 was seen only with high dose exposure prolonged exposure (Fig. 3B). To confirm that these effects (24 nmol/L for 1 hour) in the A673 cell line (Fig. 2C). occurred at EWS-FLI1 target genes and SWI/SNF–binding sites in the genome, we used ChIP and qPCR to quantitate the impact of Redistribution of EWS-FLI1 coincides with loss of SWI/SNF drug treatment on binding at previously identified EWS-FLI1 and binding to chromatin SMARCC1-binding sites (from an independent study; ref. 14). We A recent report has shown that the activity of EWS-FLI1 requires confirmed loss of binding of SMARCC1 to chromatin at several the recruitment of the ATP-dependent SWI/SNF chromatin-remo- key loci (Fig. 3C). Importantly, SMARCC1 binds throughout the deling complex to open chromatin and allow EWS-FLI1 to act as a genome, so as an additional control, we mapped and immuno- pioneer transcription factor (14). In addition, it is known that precipitated SMARCC1 at GAPDH. While GAPDH could be both trabectedin and SWI/SNF bind the minor groove of immunoprecipitated, binding of SMARRC1 at this site was DNA (42, 43). Therefore, to determine the impact of drug treat- not impacted by drug treatment suggesting the importance of ment on the chromatin binding of EWS-FLI1 and SWI/SNF, we EWS-FLI1 to this effect of trabectedin (Fig. 3D). It is notable that again pulsed the cells with drug and biochemically fractionated identical inputs were loaded into all immunoprecipitations (Sup- the cells into chromatin bound or soluble fractions. We found that plementary Fig. S2A). indeed, the redistribution of EWS-FLI1 led to less binding of EWS- FLI1 to chromatin. However, even more impressive was the SWI/SNF eviction reverses the pioneering transcription factor immediate eviction of SMARCC1 (BAF155) from chromatin that activity of EWS-FLI1 occurred within an hour of treatment with trabectedin (Fig. 3A). A link between SWI/SNF and EWS-FLI1 and the establishment In both cases, this eviction was accompanied by accumulation of of GGAA microsatellites as enhancers has already been estab- SMARCC1 and EWS-FLI1 in the soluble fraction; an effect that lished (9, 14). Therefore, we were interested in determining persisted after drug removal (Fig. 3A). Importantly, this effect only whether the histone modifications at EWS-FLI1 targeting changed occurred at relatively high concentrations of trabectedin; the from enhancer marks (K3K27ac, H3K4me1) to marks associated identical concentration associated with target suppression with epigenetically silenced chromatin (H3K27me3, H3K9me3). and nucleolar redistribution of EWS-FLI1. Neither SWI/SNF or We treated cells with trabectedin (Cmax exposure), washed out the EWS-FLI1 were evicted from chromatin at 1 nmol/L even with drug, and then performed ChIP of H3K9me3 and H3K27me3 at 1

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Figure 3. Trabectedin evicts SWI/SNF from chromatin in a schedule-dependent manner. A, Trabectedin evicts SMARCC1 and EWS-FLI1 from chromatin with high dose

(Cmax, 24 nmol/L for 1 hour) followed by incubation in drug-free medium but not continuous low- dose (AUC, 1 nmol/L continuous; B) exposure in TC32 Ewing sarcoma cells. Western blot analysis showing total lysate (Total), chromatin fraction (chromatin) with H3 histone control (H3) and soluble fraction (soluble) with GAPDH control. Lysates collected at 1, 9, and 16 hours. C, ChIP of IgG or SMARCC1 at known EWS-FLI1 and SWI/SNF target genes (MYT1, SOX2, CCND1, NR0B1) in comparison with D, GAPDH locus control following 24 nmol/L trabectedin treatment for 1 hour (1 h Trab.) followed by collection immediately or after 8 more hours in drug-free medium (9 h Trab.) in TC32 cells. Data are represented as percent input quantitated against a standard curve. , P < 0.0001.

and 9 hours following drug removal. We chose these time points treatment consistent with a known antagonism between because they both featured the redistribution of EWS-FLI1 and SWI/SNF and the PRC2 complex (Fig. 4B; Supplementary Fig. loss of SMARCC1 binding to chromatin (Figs. 2A, 3A, and 3B). We S2B; refs. 44, 45). There was also a major increase in H3K9me3 found that high-dose trabectedin led to the marked accumulation enrichment at transcriptional start sites (Fig. 4C). Indeed, of both H3K27me3 and H3K9me3 epigenetic marks throughout pretreatment, there was little association between H3K9me3 the genome (Fig. 4A). This effect was most prominent with and transcriptional start sites consistent with the known rela- H3K9me3 as the number of peaks increased from 1,104 peaks tionship between H3K9me3 and constitutive heterochroma- in solvent to 28,901 by hour 1, with an additional 7,957 peaks by tin (46, 47). In contrast, after trabectedin treatment, there was a hour 9. In addition, we found an enrichment of both marks at marked accumulation of H3K9me3 attranscriptionalstartsites, transcriptional start sites (Fig. 4B and C). There was an enrichment an effect most obvious when looking at the binding proﬁle of H3K27me3 marks at transcriptional start sites with drug (Fig. 4D).

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Figure 4. Trabectedin treatment reverses the pioneering activity of EWS-FLI1. A, Venn diagram of the total number of H3K27me3 (left) and H3K9me3 (right) peaks as measured by chIP and sequencing (ChIP-seq) following treatment with DMSO solvent, 24 nmol/L trabectedin for 1 hour (1 hour Trab.), or 24 nmol/L trabectedin for 1 hour followed by an 8-hour recovery in drug-free media (9-hour Trab.) in TC32 Ewing sarcoma cells. Heatmap displaying the genome-wide distribution of H3K27me3 (B)or H3K9me3 (C) peaks relative to transcriptional start sites (TSS) following 24 nmol/L trabectedin for 1 hour (Hour 1), or 24 nmol/L trabectedin for 1 hour followed by 8 hours in drug-free media (Hour 9). D, Genome-wide distribution of reads of H3K9me3 peaks relative to TSS following 24 nmol/L trabectedin for 1 hour (Hour 1), or 24 nmol/L trabectedin for 1 hour followed by 8 hours in drug-free media (Hour 9). E, Total number of GGAA microsatellites marked ( 50 KB) with H3K9me3 (15,400, dark green), H3K27me3 (2767, light green), both (105, darkest green) or neither (8443, gray) after treatment with 24 nmol/L trabectedin for 1 hour. F, Number of EWS-FLI1 target genes containing GGAA microsatellite sequences within 50kb of TSS. G, Total number of GGAA microsatellites associated with 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 with 24 nmol/L trabectedin for 1 hour. H, Genome browser tracks of H3K9me3 at TSS following indicated solvent or trabectedin treatments at the NR0B1 gene.

The silencing histone posttranslational modiﬁcations were microsatellite-associated EWS-FLI1 target gene, NR0B1, was also associated with the enhancer GGAA repeats, SWI/SNF and found to have a large H3K9me3 peak at the TSS, immediately EWS-FLI1. There are approximately 26,000 GGAA microsatellites adjacent to the known SWI/SNF-binding site in the region in the genome, almost 70% of these or 18,272 are marked with (Fig. 4H). Importantly, we conﬁrmed the presence of both H3K27me3, H3K9me3 or both within 50 KB of a TSS after high- H3K27me3 and H3K9me3 using ChIP-PCR in TC32 cells. In dose exposure to trabectedin (Fig. 4E). In addition, there was an addition, we showed a similar enrichment in an additional cell enrichment of these marks at EWS-FLI1 target genes. We recently line, TC252 Ewing sarcoma cells (Supplementary Fig. S3). Similar published a list of 116 induced EWS-FLI1 targets found in mul- enrichment of both H3K27me3 and H3K9me3 epigenetic silenc- tiple datasets in the literature (26). Eighty-three of the 116 genes in ing marks was observed at a number of additional well- this list were associated with GGAA microsatellites within 50 KB established EWS-FLI1 target genes including RCOR1, PPP1R1A, of the start site (Fig. 4F). Of this list of 83 targets, 76 of the 83 or MEIS1, WRN, EZH2, BCL11B, LOX, and PRKCB (Supplementary 92% were marked with H3K27me3, H3K9me3 or both following Figs. S4 and S5). In addition, high-dose trabectedin treatment also trabectedin treatment (Fig. 4G). Finally, the most well-established caused the enrichment of H3K9me3 and H3K27me3 at genomic

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Figure 5. Trabectedin suppresses EWS-FLI1 activity as measured by 18F-FLT imaging. A, Mice bearing TC32 Ewing sarcoma xenografts in right gastrocnemius show suppression of 18F-FLT signal 6 to 54 hours after treatment with trabectedin, but not vehicle control. The bladder shows high 18F-FLT signal across all samples due to excretion of tracer. B, High PET avidity of two mice 24 hours after treatment with vehicle (day 2). Data are a three- dimensional reconstruction of the tumor (tumor) followed by cross- sections in the X, Y, and Z axes. C, Suppression of 18F-FLT PET avidity in two mice 24 hours after treatment with 0.18 mg/kg of trabectedin (day 2). Data are a three-dimensional reconstruction of the tumor (tumor) followed by cross-sections in the X, Y, and Z axes. Scale indicates signal intensity. D, 3D reconstruction and single cross-section of tumors at multiple time points following treatment with vehicle (1 hour), trabectedin (1 hour), irinotecan (24 and 48 hours), or the combination of trabectedin and irinotecan. Rows indicate time and treatments, columns represent 3D reconstruction and single cross- section for each of the treatment groups. The intensity scale is the same as B and C.

sites previously associated with SWI/SNF at MYT1, CCND1, and EWS-FLI1 drives the expression of the proteins responsible for SOX2 (Supplementary Fig. S6). Importantly, silencing of activity in Ewing cells, ENT1/ENT2 and TK1 (48). SMARCC1 reduces cell viability in Ewing sarcoma cells and further Treatment of mice bearing Ewing sarcoma xenografts with potentiates the activity of the drug in an analogous fashion to trabectedin suppressed EWS-FLI1 activity and caused a loss of silencing of EWS-FLI1 (Supplementary Fig. S7). 18F-FLT PET activity. Peak suppression occurred 6–24 hours after treatment and the xenograft recovered PET avidity by 54–72 hours Trabectedin requires irinotecan to improve suppression of (Fig. 5A). To investigate EWS-FLI1 suppression in the three- EWS-FLI1 in the three-dimensional architecture of a tumor dimensional architecture of the tumor, we used the signal from We have previously shown that trabectedin is particularly every voxel in the tumor to mathematically reconstruct the tumor effective in Ewing sarcoma in combination with extremely low to determine the distribution of EWS-FLI1 suppression. Again, we doses of irinotecan (35). Because irinotecan is known to impact found striking 18F-FLT PET signal in control tumors (Fig. 5B) and transcription, we sought to determine whether the function of marked suppression of EWS-FLI1 most evident in the X, Y, Z plane irinotecan in this combination is to improve the magnitude, cross-sections of the trabectedin-treated tumors (Fig. 5C). After 24 penetrance, or duration of EWS-FLI1 suppression. We have pre- hours, control animals had a mean signal 20% higher than viously shown that 18F-FLT PET reﬂects EWS-FLI1 activity because trabectedin-treated animals (P < 0.0001; 95%CI, 12.9–27.6).

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Interestingly, we found marked variability in the distribution of hours (Fig. 5D, ). The average number of voxels with signal above EWS-FLI1 suppression among the animals in the cohort in three background for trabectedin and irinotecan animals was 3,346.17 dimensions, the magnitude, and even the duration of EWS-FLI1 and 977.72, respectively [95% CIs (722.8–15,490.5); (192.4– suppression (Figs. 5B and C; Supplementary Fig. S8). It is notable 4,968.1)]; compared with 24.3 for animals treated with both that these tumors all came from the same cell line, were implanted (95% CI, 4.2–141.1; P ¼ 0.0003, 0.0063, respectively). However, at the same time, at the identical cell number, and treated with the as early as day 5, the animals showed little to no evidence of identical dose of trabectedin (and all trabectedin was delivered or 18F-FLT activity suggesting a change in the tissue from highly local toxicity would be obvious). Nevertheless, the variability was proliferative malignant tissue to benign consistent with a sus- remarkable and consistent with the heterogeneity in response to tained release in the EWS-FLI1–mediated differentiation block. treatment that we have consistently observed across cohorts of Importantly, this type of analysis would simply not be possible mice regardless of therapy. We were able to rescue this variable with traditional IHC or PCR approaches to evaluate target sup- suppression in vivo, by adding irinotecan, which improved the pression as it allowed us to evaluate the distribution of suppres- amplitude, penetrance and duration of EWS-FLI1 suppression sion in the same animal over time. Finally, it is notable that likely accounting for the favorable clinical experience with this sustained suppression of EWS-FLI1 with this combination led to a combination (Fig. 5D). In addition, this suppression correlated release in the differentiation block and the tumor showed evi- with effects on tumor growth and striking regressions of tumor dence of differentiation down a number of mesenchymal lineages were observed with the combination therapy as previously and human collagen, osteoblasts, and fat were identiﬁed in the reported (ref. 35; Supplementary Fig. S9). The most striking xenograft (Fig. 6). It is notable that the mouse is known to example was the day 8 animals (combo treatment in Fig. 4) that remodel and replace benign human tissue with mouse tissue and had complete suppression of target and complete regression of so the penetrance of the differentiation phenotype is difﬁcult to tumor while trabectedin and irinotecan recovered signal at 102 establish (49). While the cell of origin of Ewing sarcoma is not

Figure 6. Combination treatment of trabectedin and irinotecan induces differentiation of TC32 Ewing sarcoma cells in vivo. A, (left to right) 4 and 20 magnification of H&E staining of TC32 IM xenograft tumor 3 days after treatment with vehicle (control) or trabectedin and irinotecan (treated). 60 magnification of MTCO2 human mitochondrial stain and 60 SP7 Osterix osteoblast stain showing human cells expressing SP7 in treated but not control. B, (left to right) 4 and 20 magnification of H&E staining of TC32 IM xenograft. 60 magnification of MTCO2 human mitochondrial stain and 60 PicroSirius Red stain indicating specific human collagen cells 5 days after treatment with vehicle (control) or trabectedin and irinotecan (treated). C, (left to right) 4 and 20 magnification of H&E staining of TC32 IM xenograft. 20 and 60 magnification of MTCO2 human mitochondrial stain showing human adipocyte. Five days after treatment with vehicle (control) trabectedin and irinotecan (treated).

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known, current thinking favors a mesenchymal or neural crest This study also highlights important features of Ewing sarcoma origin (39, 50–52). Therefore, sustained suppression of EWS-FLI1 biology. We confirm the recent observation that SWI/SNF is allows restoration of the differentiation program but this program important to the biology of EWS-FLI1 and further establish the is relatively unorganized leading to mesenchymal confusion. link to EWS-FLI1, particularly at the GGAA microsatellites (14). We show that removal of EWS-FLI1 leads to a cellular response and widespread chromatin silencing, particularly with H3K9me3, Discussion which favors repetitive sequences and constitutive heterochro- This study highlights the importance of drug mechanism to the matin. The data suggest both inhibition of EWS-FLI1 and dis- drug development process. Compounds with broad cytotoxicity placement of SWI/SNF are required to reverse activity; however, profiles can be developed for specific indications if they inhibit the further work would need to be done to clearly establish this point. dominant oncogene of a specific tumor. However, the successful In addition, once this reversal is achieved, relatively nonspecific implementation of therapies of this type absolutely requires that blockade can sustain suppression of the target in vivo. The net the mechanism of suppression be optimized for a specific onco- result is a differentiation endpoint, although this differentiation is gene and a defined cell context. unorganized. In this study, we show that the therapeutic suppression of the Finally,thisstudyreportsanoveluseof18F-FLT PET imaging dominant oncogene of Ewing sarcoma, EWS-FLI1, requires a high as a tool to quantitate target suppression and at the same time concentration of trabectedin in serum. We model this exposure visualize the penetrance and distribution of target suppression preclinically and show in vitro and in vivo that the drug is able to within the three-dimensional architecture of the tumor. Indeed, inhibit EWS-FLI1. perhaps the most interesting observation in this study is the The drug redistributes EWS-FLI1 in the nucleus, displaces widely variable suppression of EWS-FLI1 that occurred within SWI/SNF from chromatin, and triggers an epigenetic switch cohorts of mice. The Ewing sarcoma xenografts were established driving an increase in H3K27me3 and H3K9me3 with preference from the same cell collection and the same flask and tube, with for EWS-FLI1 target genes. However, these effects absolutely 2 million cells in every animal by the same technician on the require high concentrations of drug in serum and do not occur same day in one strain of animal. Treatment was also initiated at lower concentrations even with prolonged exposure. bythesametechnicianfromthesamestockofdrugandalldrug These observations are important because they justify the made it into the circulation as any extravasation of this drug investigation of trabectedin in Ewing sarcoma on a short- leads to tail necrosis. Yet, despite these similarities, the mag- infusion schedule in combination with low-dose irinotecan. nitude, penetrance, and even duration of target suppression was It has been known for more than 20 years that Ewing sarcoma widely variable from one animal to the next. It is likely that this cells are dependent on EWS-FLI1 (53). However, the therapeu- variable target suppression is an important factor driving tumor tic suppression of EWS-FLI1 has not been achieved in clinic. In response. However, it is not known what the source of this addition, trabectedin was previously evaluated in Ewing sarco- variability is; a question we are now starting to investigate. ma as a 24-hour infusion in a phase II study because this Nevertheless, this study serves asproofofprincipletoaskthis schedule was shown to be more active in other sarcoma question in a prospective fashion, using schedule-optimized types (54). However, the data in this article suggest that a trabectedin in combination with low-dose irinotecan, and shorter 1-hour infusion schedule may increase activity in Ewing 18F-FLT imaging in patients with Ewing sarcoma in the clinic. sarcoma because the drug would accumulate to serum concentrations above a threshold that we define in this article as being Disclosure of Potential Conflicts of Interest high enough to inhibit the dominant oncogene, EWS-FLI1 (41). S.L. Lessnick holds ownership interest (including patents) in and is a This blockade of EWS-FLI1 is amplified and sustained in consultant/advisory board member for Salarius Pharmaceuticals. No potential fl combination with low-dose irinotecan. Because this tumor con icts of interest were disclosed by the other authors. absolutely depends on EWS-FLI1, it is likely that this study would show clinical activity. Therefore, this study justifies the Authors' Contributions further exploration of this compound on an alternative 1-hour Conception and design: M.L. Harlow, M.H. Chasse, M.J. Bowman, P.J. Grohar Development of methodology: M.L. Harlow, S.M. Kitchen-Goosen, infusion schedule in this tumor in combination with low-dose S.B. Rothbart, A.S. Peck, P.J. Grohar irinotecan. Perhaps the most important observation in this Acquisition of data (provided animals, acquired and managed patients, study is that even within sarcoma, different schedules of active provided facilities, etc.): M.L. Harlow, M.H. Chasse, E.A. Boguslawski, compounds may be more effective in particular subtypes. K.M. Sorensen, J.M. Gedminas, S.M. Kitchen-Goosen, S.L. Lessnick, A.S. Peck This study also provides important insight into the mecha- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, nism of action of trabectedin, a compound that has found 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. Grohar unique activity in a number of sarcomas. Trabectedin has a Writing, review, and/or revision of the manuscript: M.L. Harlow, M.H. Chasse, complicated mechanism of action including both generating E.A. Boguslawski, K.M. Sorensen, J.M. Gedminas, S.B. Rothbart, S.L. Lessnick, DNA damage and poisoning-specific DNA damage repair A.S. Peck, Z.B. Madaj, M.J. Bowman, P.J. Grohar complexes, poisoning-specific transcription factors such as Administrative, technical, or material support (i.e., reporting or organizing EWS-FLI1 and FUS-CHOP, and specifically targeting tumor- data, constructing databases): E.A. Boguslawski, S.M. Kitchen-Goosen, associated macrophages (55, 56). In this study, we add dis- C. Taslim, M.J. Bowman, P.J. Grohar Study supervision: P.J. Grohar placementofSWI/SNFfromchromatintothismechanism.Itis likely that this mechanism contributes to the broad cytotoxicity Acknowledgments fi pro leofthiscompoundasSWI/SNFismutatedinupto25% The authors would like to thank Ron Chandler, PhD (Michigan State of human cancer and commonly altered either functionally or University, East Lansing, MI) for helpful discussion. The authors would also through mutation in sarcoma. like to thank Dr. Peter Adamson (Children's Hospital of Philadelphia,

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Philadelphia, PA) for helpful advice. We would like to thank Robert Vaughan from Hyundai Hope on Wheels (to J.M. Gedminas), the NIH/NIGMS from the Rothbart lab for technical help. We would like to thank Marie Adams (R35GM124736; S.B. Rothbart), and the NIH/NCI U54CA231641, for technical support and library preparations and the Bioinformatic and R01CA183776 (to S.L. Lessnick). Biostatistics Core of the Van Andel Research Institute (Grand Rapids, MI). The authors would like to thank Pharma Mar pharmaceutical company for material used in this proposal. P.J. Grohar is supported by a grant from the NIH The costs of publication of this article were defrayed in part by the payment of (R01-CA188314). Additional support is from the NIH/NCI MHC page charges. This article must therefore be hereby marked advertisement in (F31CA236300). The imaging portion of the study was supported by a Reach accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Award from Alex's Lemonade Stand Foundation (to P.J. Grohar). The work is also supported by internal funds from the Van Andel Institute (to P.J. Received October 26, 2018; revised December 24, 2018; accepted January 23, Grohar, S.B. Rothbart, Z.B. Madaj, M.J. Bowman). Additional support is 2019; published ﬁrst February 5, 2019.

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www.aacrjournals.org Clin Cancer Res; 25(11) June 1, 2019 3429

Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner

Matt L. Harlow, Maggie H. Chasse, Elissa A. Boguslawski, et al.

Clin Cancer Res 2019;25:3417-3429. Published OnlineFirst February 5, 2019.

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