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

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Author Manuscript Published OnlineFirst on February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Dependent Manner

Authors: Matt L. Harlow1†,†††, Maggie H. Chasse2,†††, Elissa A. Boguslawski2, Katie M. Sorensen2, Jenna M. Gedminas2,5,6, Susan M. Kitchen-Goosen2, Scott B. Rothbart2, Cenny Taslim3, Stephen L. Lessnick3,4, Anderson S. Peck2††, Zachary B. Madaj2, Megan J. Bowman2‡, Patrick J. Grohar2,5,6*

Affiliations: 1Department of Cancer Biology, Vanderbilt University, Nashville, TN, 37235, USA. 2Van Andel Research Institute, Grand Rapids, MI, 49503, USA. 3Center for Childhood Cancer and Blood Diseases, Nationwide Children’s Hospital Research Institute, Columbus, OH, USA. 4Division of Pediatric Hematology/Oncology/BMT, The Ohio State University College of Medicine, Columbus, OH, USA. 5Michigan State University, Department of Pediatrics, East Lansing, MI, USA. 6Helen DeVos Children’s Hospital, Division of Pediatric Hematology/Oncology, Grand Rapids, MI, USA.

* [email protected], Phone: 616-234-5000. † Current Address: Dana-Farber Cancer Institute, Boston, MA, 02215, USA. ‡ Current Address: Ball Horticultural Company, West Chicago, IL, 60185, USA. †† Current Address: Bamf Health, Grand Rapids, MI ††† These authors contributed equally

Key Words: Ewing sarcoma, EWS-FLI1, SWI/SNF, Pediatric Cancer, Sarcoma

Running Title: Trabectedin inhibits EWS-FLI1

Financial Support: PJG 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 Reach Award from Alex’s Lemonade Stand Foundation (PJG). The work is also supported by internal funds from the Van Andel Institute (PJG, SBR, ZVM, MJB). Additional support is from Hyundai Hope on Wheels (JMG), the NIH/NIGMS (R35GM124736)(SBR) and the NIH/NCI U54CA231641, R01CA183776 (SLL).

Conflict of Interest: The authors declare no potential conflicts of interest.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Purpose: The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the re-evaluation of this drug in the clinic in

Ewing sarcoma patients.

Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using

biochemical fractionation and chromatin immunoprecipitation sequencing (CHIP-Seq). We link

the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, western blot analysis and cell viability assays. Finally, we quantitate target suppression within the 3-dimensional architecture of the tumor in vivo using 18F-FLT imaging.

Results: Trabectedin evicts the SWI/SNF chromatin remodeling complex from chromatin and

redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and

H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of

trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo,

low dose irinotecan is required to improve the magnitude, penetrance and duration of target

suppression in the 3-dimensional architecture of the tumor leading to differentiation of the Ewing

sarcoma xenograft into benign mesenchymal tissue.

Conclusions: These data provide the justification to evaluate trabectedin in the clinic on a short

infusion schedule in combination with low dose irinotecan with 18F-FLT PET imaging in Ewing

sarcoma patients.

Statement of Translational Relevance:

This paper provides the basis for a clinical trial to evaluate trabectedin in combination with low dose irinotecan as an EWS-FLI1 targeted therapy. The clinical suppression of EWS-FLI1 has not been achieved despite a known dependence on this target for more than 20 years. In addition, trabectedin has failed in the disease in a phase II study. These data provide an explanation for the failed phase II, a schedule change that will improve the therapeutic suppression of EWS-FLI1 and evidence 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 EWS-FLI1 suppression in patients. In addition, we establish a novel mechanism of trabectedin as an inhibitor of the SWI/SNF chromatin remodeling complex which is mutated in approximately 25% of all human cancers.

Introduction

Oncogenic transcription factors are dominant oncogenes for a large number of leukemias and solid tumors in both the pediatric and adult populations (1-3). These proteins are challenging drug targets because the active site lacks a traditional druggable domain and most transcription factors interact with complex networks of proteins. Nevertheless, compounds that have successfully targeted specific transcription such as ATRA and arsenic trioxide in acute promyelocytic (APL) are effective in the clinic (4-6).

Ewing sarcoma is a bone and soft tissue sarcoma that is absolutely dependent on the EWS-FLI1 transcription factor for cell survival (7). This fusion transcription factor, formed by the t(11;22)(q24;12) chromosomal translocation, both drives proliferation and blocks differentiation

(8,9). EWS-FLI1 acts as a pioneer transcription factor and binds to repetitive regions of the genome called GGAA microsatellites (10-13). Once bound, the protein exhibits phase transition properties to establish these microsatellites as enhancers to drive gene expression (14). This requires a complex network of protein interactions and relies heavily on the ATP-dependent chromatin remodeling complex, SWI/SNF to maintain chromatin in an open state (14,15).

Therefore, it is likely that reversal of EWS-FLI1 activity would lead to widespread changes in chromatin structure and restore the differentiation program. However, it is not clear if the effective targeting of EWS-FLI1 requires a blockade of SWI/SNF activity or if the pioneer transcription factor activity of EWS-FLI1 is reversible genome-wide.

We have previously shown that the natural product trabectedin interferes with the activity of the

EWS-FLI1 transcription factor (16). We showed that trabectedin reverses expression of the

EWS-FLI1 gene signature. In addition, we cloned EWS-FLI1 into another cellular context, induced an EWS-FLI1 driven promoter luciferase construct, and then rescued this induction with trabectedin (16). These findings were consistent with early preclinical and clinical experience with the drug which suggested a heightened sensitivity of Ewing sarcoma to trabectedin (17,18).

Most notably, a patient with treatment-refractory Ewing sarcoma achieved a durable complete response with single agent trabectedin treatment in the phase I study. In contrast, the phase II study in Ewing sarcoma was negative and only 1 out of 10 patients responded to the drug (19).

However, the drug was administered on a different schedule in the negative phase II study.

Therefore, it is possible that a detailed understanding of the mechanism of EWS-FLI1 suppression by trabectedin would allow us to optimize the schedule of administration and achieve the therapeutic suppression of EWS-FLI1 in the clinic.

Like many natural products, trabectedin has a complicated mechanism of action (20,21). The compound is known to generate DNA damage and poison various repair pathways, block specific transcription factors such as the FUS-CHOP transcription factor, and exert cytotoxicity with preference for specific cell types such as Tumor Associated Macrophages (TAM), myxoid liposarcoma cells, and Ewing sarcoma cells (22-24)

In this study, we define the mechanism of EWS-FLI1 suppression to establish trabectedin as a bona fide EWS-FLI1 inhibitor. We show that the drug redistributes EWS-FLI1 within the nucleus and at the same time evicts the SWI/SNF chromatin remodeling complex to trigger an epigenetic switch, leading to global increases in H3K27me3 and H3K9me3 with preference for

GGAA microsatellites and EWS-FLI1 target genes. Importantly, these effects are concentration

dependent, and lead to sustained target suppression only if a threshold concentration of drug is

exceeded. This mechanistic insight is consistent with the clinical experience with the drug where

this threshold was exceeded in the phase I and patients responded, but not the negative phase II

study. Finally, target suppression is amplified and sustained in vivo in combination with the

topoisomerase inhibitor irinotecan to cause a complete histological change in the tumor and

differentiation into benign mesenchymal tissues.

Materials and Methods

Cell Culture: TC32, A673 cells were obtained from Dr. Lee Helman and TC252, SK-N-MC,

EW8 from Dr. Tim Triche (Both at Children’s Hospital of Los Angeles). Cell identity was confirmed by STR profiling (DDC Medical; last test 10/24/18). They were cultured at 37 °C

pathogen free with 5% CO2 inRPMI-1640 (Gibco) with 10% fetal bovine serum (Gemini Bio-

Products), 2 mM L-glutamine, and 100 U/mL and 100 μg/mL penicillin and streptomycin

(Gibco).

Western Blotting: 1.5 million cells (TC32, A673) or 3 million cells (TC252, EW8, SK-N-MC)

were incubated with drug, washed in PBS, and lysed in 4% lithium dodecyl sulfate (LDS) buffer.

30 micrograms were resolved on a NuPage 4-12% Bis-Tris gradient gel (Invitrogen) in 1X

NuPage MOPS SDS Running Buffer (Invitrogen) after diluting detergent and quantitating by

bicinchoninic acid (BCA) assay (Pierce, Thermo-Scientific). The protein was transferred overnight to nitrocellulose at 20 V in 1X Tris-Glycine-SDS Buffer (Bio-Rad) with 20%

methanol. The membranes were blocked in 5% milk in TBS-T, and probed with WRN, NR0B1,

GAPDH (Abcam) or EZH2 (Cell Signaling) antibodies.

Quantitative RT-PCR: RNA was collected using the RNeasy kit (Qiagen), immediately

reverse-transcribed using a high-capacity reverse transcriptase kit (Life Technologies) at 25 °C

for 10 min, 37 °C for 120 min, and 85 °C for 10 min. The products were quantitated using qPCR,

SYBR green (Bio-Rad), and the following program: 95 °C for 10 min, 95 °C for 15 s, 55 °C for

15 s, and 72 °C for 1 min, for 40 cycles. Expression was determined from three independent

experiments relative to GAPDH and solvent control using standard ddCt methods.

Luciferase Assays: Stable cell lines containing an EWS-FLI1-driven NR0B1 luciferase or

constitutively active CMV control (25) were incubated with drug in white, flat-bottom 96-well plates (Costar) for 8 h. Cells were lysed in 100 μL of Steady-Glo (Promega) and

bioluminescence was measured on a BioTek plate reader (Winooski, VT).

Cell Proliferation Assays: IC50s were determined by non-linear regression (Prism GraphPad)

as the average of three independent experiments using standard MTS assay CellTiter 96

(Promega). The results were confirmed with real-time proliferation assays on the Incucyte

ZoomTM as previously described (26).

Confocal microscopy: TC32 cells were incubated with DMSO or trabectedin in a Nunc Lab-

Tek II Chamber Slide (Thermo Scientific), fixed in 4% paraformaldehyde in PBS, washed, lysed

in 1% Triton-X100, and blocked in 5% goat serum. Cells were incubated with primary antibody

(18 h), secondary antibody (1 h) and DAPI (10 minutes), mounted in Vecta Shield mounting

media (Vecta Laboratories);(Primary antibodies: nucleolin, Abcam – 1:1000; HA-tag, Abcam –

1:500; FLI1, Abcam – 1:100; N-terminal EWSR1, Cell Signaling – 1:1000) (Secondary antibodies: Cy5-conjugated anti-mouse IgG: Vector – 1:400, FITC-conjugated anti-Rabbit IgG:

Millipore – 1:200) (DAPI Sigma Aldrich – 1:10,000). All images were obtained with standardized settings on a Zeiss 510 confocal microscope.

Chromatin Immunoprecipitation (ChIP): 10 million TC32 cells were incubated with trabectedin or DMSO for the indicated time, washed, cross-linked in 1% formaldehyde for 10 minutes and quenched with 0.2 M glycine. The cells were collected in cold PBS with 1X protease inhibitor (Sigma Aldrich), lysed in 20 mM Tri-HCl (pH 7.5), 85 mM KCl, and 0.5%

NP-40 for 15 minutes on ice with dounce homogenizing. Chromatin was sheared with the E220 evolution focused sonicator (Covaris) for 10 minutes. 10 µg solubilized chromatin was immunoprecipitated with 1 µg mouse IgG (Abcam #18394), or H3K27me3 (Abcam #6002), 1

µg rabbit IgG (Cell Signaling #2729S) or 1 µg H3K9me3 (Abcam #8898), 2 µg rabbit IgG or 1

µg SMARCC1/BAF155 (Cell Signaling #11956S). Antibody-chromatin complexes were immunoprecipitated with Magna ChIP Protein A+G magnetic beads (EMD Millipore) and washed. DNA was eluted with 100mM NaHCO3, 1% SDS, and 1x proteinase K for 2-hours at

65C followed by 10-minute incubation at 95C. ChIP DNA was purified with QiaQuick purification kit (Qiagen). Purified SMARCC1 ChIP DNA was analyzed with ChIP-qPCR, described below. Purified H3K27me3 and H3K9me3 ChIP DNA was submitted for 2x75bp sequencing and analyzed as described below.

Chromatin Immunoprecipitation with quantitative PCR (ChIP-qPCR): Solubilized chromatin was treated with RNAse A at 37C for 30 minutes followed by Proteinase K at 65C

for 2 h, purified with the QiaQuick purification kit (Qiagen) and quantified using SYBR green relative to a standard curve of DNA generated with input DNA from the respective sample independently for each primer set. qPCR was performed with the following primer sets (MYT1,

NR0B1, SOX2, CCND1) using published primer sequences (27).

Chromatin Immunoprecipitation Sequencing (ChIP-Seq): Libraries for input and immunoprecipitated samples were prepared by the Van Andel Genomics Core from 10 ng of input material and either 10 ng or all available IP material using the KAPA Hyper Prep Kit

(v5.16) (Kapa Biosystems, Wilmington, MA USA). Prior to PCR amplification, end repaired and

A-tailed DNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific,

Austin, TX, USA). Quality and quantity of the libraries were by Agilent DNA High Sensitivity

ChIP (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI,

USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). 50 bp, paired end sequencing was performed on an Illumina NovaSeq sequencer using a 100 bp S1 sequencing kit (Illumina Inc., San Diego, CA, USA). Base calling was done by Illumina RTA v3.0 software and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq2 v2.20.0.

ChIP-Seq Bioinformatic Analysis: H3K27me3 and H3K9me3 ChIP-seq and input reads were aligned to human genome (hg19) using BWA-MEM v 0.7.15 and peaks were called using

MACS2 (v 2.1.1.20160309) compared to input using the broad parameter and a q-value of 0.01

(28). Known ENCODE blacklist regions were removed from called peaks using BEDtools intersect (v 2.27.1)(29,30). Peak intersections were also determined using BEDtools. SMARCC1

(BAF155)ChIP-seq data were downloaded from NCBI-GEO (GSE94278)(6) and processed

using the same software and parameters. Peak annotation was completed using the ChIPseeker

package in R (v 1.14.2)(31). Additional figures were generated using deepTools (v 2.3.6) and

Intervene (v 0.6.2)(32,33).

Nuclear Fractionation: 2.5 million TC32 cells were incubated with DMSO control for the

indicated times and collected or replaced with drug-free media for 8 h (9-hous total) or 15 h (16-

hours total). Cells were washed in PBS, incubated in CSK buffer (100mM NaCl, 300mM

sucrose, 3mM MgCl2, 0.1% Triton X-100, Roche COmplete EDTA-free tablet, 10nM Pipes, pH

7.0 with NaOH) for 20 minutes on ice (34). The total fraction was collected and the soluble fraction was collected by centrifugation at 1,300 g for 5 minutes at 4oC. The nuclear insoluble pellets were re-suspended with CSK buffer, incubated on ice for 10 minutes, then the chromatin fraction was collected by centrifugation at 1,300 g for 5 minutes at 4oC (34). Total protein was

quantitated using Bradford assay (Bio-Rad Protein Assay Dye Reagent Concentrate). Chromatin

protein and soluble protein quantitation were calculated from total protein quantitation. Total

protein and chromatin protein were incubated with CSK buffer plus Pierce Universal Nuclease

(Thermo Fisher Scientific) for 20 minutes on ice. 10 g of each protein sample were resolved as

described above (see Western Blotting).

Xenograft Experiments: Two million TC32 cells were injected intramuscularly in the

gastrocnemius of female 8-10-week old female homozygous nude mice (Crl; Nu-Foxn1Nu)(Van

Andel Research Institute, Grand Rapids, MI) and established to a minimum diameter of 0.5 cm.

Five cohorts of mice were treated with vehicle (n=6), trabectedin (n=9)(0.18 mg/kg

intravenously on days 1 and 8), irinotecan (n=7) (5 mg/kg intraperitoneal on days 2 & 4), the combination trabectedin plus irinotecan (n=7) (same dose route and schedule as the single agent treatments). Tumor volume was measured daily and determined using the equation (D x d2)/6 ×

3.12 (where D is the maximum diameter and d is the minimum diameter). All experiments were performed in accordance with the guidelines and regulation of, and approved by the Van Andel

Institute (VAI) Institutional Animal Care and Use Committee (IACUC). Investigators were not blinded to the treatment groups.

18F-FLT PET Imaging: Mice were anesthetized with 2% isoflurane in Oxygen, injection with

~25 uCi 18F-FLT (18F-3′-Deoxy-3′-Fluorothymidine)(Spectron MRC, South Bend, IN, USA) and given 1-hour uptake time while conscious before 10-minute imaging on a GENISYS4 pet scanner (Sofie Biosciences, Culver City, CA, USA) and a 6-minute NanoSPECT/CT (Bioscan

Inc., Washington DC, USA). PET reconstruction was performed using 3D maximum-likelihood expectation-maximization algorithm for 60 iterations and CT reconstruction utilized filtered back-projection with a Shepp-Logan filter. Data visualization and analysis utilized Osirix MD

(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 when applicable. Tumor uptake changes over time were assessed using percentage injected dose per mL (%ID/mL) and mean and maximum standardized uptake value.

Tissue Staining and Immunohistochemistry: 5-micrometer sections of FFPE tissue were mounted on charged slides, stained with Hematoxylin and Eosin (Ventana Symphony). For immunohistochemistry, antigen retrieval was performed on the PT Link platform on the Dako

Autostainer Plus instrument or manually using Dako Target Retrieval System citrate buffer.

Following blocking, tissue was incubated with SP7 Osterix antibody (Abcam, 1:2000) or

MTCO2 antibody (Abcam 1:800), washed and then secondary antibody (Polyclonal Goat Anti-

Rabbit HRP or Envision+System HRP labelled polymer Anti-Rabbit, Dako 1:100) and developed with Dako Liquid DAB+ Substrate Chromogen System. Collagen staining was performed via Picro Sirius Red Stain Kit (Connective Tissue Stain, Abcam).

Project Statistics: All qPCR data is normalized to solvent (expression data) or input (CHIP data) as fold change from 3-independent experiments. The P-value was determined by two-sided student’s t-test or one-way ANOVA using the Dunnett test for multiple comparisons. For PET imaging, the signal above background was determined by a mixed-effects Poisson regression with random intercepts for each animal and false-discovery rate adjusted. Background signal was defined as the average signal from a similar sized region in the contralateral limb. Treatment group differences were determined by a log-transformed linear mixed-effects regression with random intercepts for each animal and false-discovery rate adjusted. All hypotheses were two- sided, significance level set at 0.05, and performed using R v3.4.4. Data are plotted with signal broken out into ‘high’, ‘medium’, and ‘low’; which are the tertiles of the vehicle’s signal above background at hour 1.

Results

Suppression of EWS-FLI1 by trabectedin requires high serum concentrations.

To determine if the schedule of administration may correlate with EWS-FLI1 suppression and clinical response in Ewing sarcoma patients, we modeled the effects of drug exposure on cell

viability and EWS-FLI1 activity in vitro. In the pediatric phase I study, trabectedin was administered over 3 hours and accumulated to a high maximal serum concentration (Cmax) of either 6 ng/mL (7.8 to 12.2 nM at 1.1 mg/m2 dose) or 10.5 ng/mL (13.8 nM to 20.4 nM) at 1.3 mg/m2 dose) but lower AUC of 39 ng/mL*hr. In contrast, when administered as a 24-hour infusion in the phase II study, a greater exposure of 112 ng/mL*hr was achieved at the expense of a lower serum Cmax of only 2.5 ng/mL (3.2 nM). Interestingly, 2 out of 3 Ewing sarcoma patients responded to the drug in the phase I (high Cmax) and only 1 out of 10 Ewing sarcoma patients responded with stable disease in the phase II study despite a substantially higher exposure (AUC) of the tumor to the drug (17,19). These data suggest that tumor response correlates with concentration (Cmax), not total exposure (AUC). Since Ewing sarcoma is dependent on EWS-FLI1, it suggests that a threshold concentration is required to block target and impact viability.

To test this hypothesis, we pulsed cells with compound then changed medium to evaluate the impact of brief exposures to trabectedin on cell viability, EWS-FLI1 activity and downstream target expression. This is possible because trabectedin DNA adducts are known to be repaired and cleared from treated cells (35). We treated cells with the identical exposure of drug (AUC =

Concentration * time; 600 nmol/L*hr) but at varying maximal concentrations, removed the drug from medium, and measured the effect on viability using real time microscopy. We observed sustained suppression of cell viability over time with as little as 1 hour of exposure if a 10 nM concentration threshold was exceeded (Fig. 1A). In order to see if this 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 driven luciferase construct (16,36). The

activity of this NR0B1-promoter driven luciferase is highly specific for EWS-FLI1 because it

contains an EWS-FLI1 responsive GGAA microsatellite in the promoter that is the proper length

to induce transcription (13,37). CRISPR/Cas9 elimination of this microsatellite eliminates

NR0B1 expression and while both FLI1 and EWS-FLI1 can bind this region, only EWS-FLI1

can activate NR0B1 expression (12,38,39). We found marked suppression of NR0B1 luciferase

18 hours after drug removal with no impact on a constitutively active CMV driven control again

with a 10 nM threshold concentration that reflects the phase I, high Cmax exposure observed in

patients (Fig. 1B).

To directly compare the impact of exposure on mRNA expression of target genes, we performed

the same experiments and evaluated mRNA expression of three target genes, NR0B1, EZH2, and

WRN at 24 hours (36,40,41). We treated the cells for 1 nM for 24 hours to maximize the

likelihood that lower dose over time (AUC) would block target expression as this is 2X the GI50 of the drug that we have previously established (16). Target suppression was found only with a high Cmax (Cmax; 24 nM for 1 hour) but not with a sustained lower dose exposure (AUC; 1 nM for 24 hours) despite the fact that the identical total exposure was used in both treatments (Fig.

1C). These effects extended to the protein level where again only the Cmax exposure, but not the

AUC exposure, led to a loss of expression of NR0B1, EZH2, and WRN in two different Ewing

sarcoma cell lines (Fig. 1D). This was a generalized effect on EWS-FLI1 activity and

suppression of NR0B1 expression was observed in 3 additional Ewing sarcoma cell 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, in order to firmly establish the schedule dependence of these effects, we evaluated the

effect of drug treatment on cell viability as a function of AUC. Full dose response curves were

washed out at variable time points and the effect on viability was determined 48 hours later (Fig.

1F). As long as a threshold concentration was achieved, as little as 6 minutes of drug exposure

suppressed cell viability leading therefore minimizing the AUC needed to suppress proliferation,

leading to a shift in the curve to the left (Fig. 1F)(42). These effects are specific for trabectedin as a similar relationship with Cmax was not found with an alternative EWS-FLI1 inhibitor, mithramycin (25). Even at high concentrations that exceed what is required to suppress EWS-

FLI1, the suppression of viability by mithramycin exactly correlated with AUC regardless of concentration/time of exposure (Supplemental Fig. S1A, S1B).

EWS-FLI1 redistributes in the nucleus to the nucleolus only with high serum

concentrations. We have previously shown that treatment of Ewing sarcoma cells with

trabectedin and a second-generation analog redistributes EWS-FLI1 within the nucleus to the nucleolus (26). Therefore, we investigated if the Cmax exposure was required for nucleolar

redistribution and if it would be sustained following drug removal. A short 24 nM 1-hour pulse

of drug caused EWS-FLI1 to redistribute within the nucleus to the nucleolus (Fig. 2A). This

effect persisted following drug removal consistent with the sustained suppression of targets

described above. It is notable that the penetrance of the effect within the population of cells

decreases over time (data not shown). The effect was concentration dependent and a similar redistribution of EWS-FLI1 was not seen with 1 nM treatment even after 24 hours of exposure at this concentration consistent with the requirement for high concentrations to inhibit EWS-FLI1

(Fig. 2B). The effect was not dependent on TP53 status as a similar redistribution of EWS-FLI1 was seen only with high dose exposure (24 nM for 1 hour) in the A673 cell line (Fig. 2C).

Re-distribution of EWS-FLI1 coincides with loss of SWI/SNF binding to chromatin.

A recent report has shown that the activity of EWS-FLI1 requires the recruitment of the ATP- dependent SWI/SNF chromatin remodeling complex to open chromatin and allow EWS-FLI1 to act as a pioneer transcription factor (27). In addition, it is known that both trabectedin and

SWI/SNF bind the minor groove of DNA (43,44). Therefore, in order to determine the impact of drug treatment on the chromatin binding of EWS-FLI1 and SWI/SNF, we again pulsed the cells

with drug and biochemically fractionated the cells into chromatin bound or soluble fractions. We

found that indeed, the redistribution of EWS-FLI1 led to less binding of EWS-FLI1 to

chromatin. However, even more impressive was the immediate eviction of SMARCC1

(BAF155) from chromatin that occurred within an hour of treatment with trabectedin (Fig. 3A).

In both cases, this eviction was accompanied by accumulation of SMARCC1 and EWS-FLI1 in the soluble fraction; an effect that persisted after drug removal (Fig. 3A). Importantly, this effect only occurred at relatively high concentrations of trabectedin; the identical concentration

associated with target suppression and nucleolar redistribution of EWS-FLI1. Neither SWI/SNF

or EWS-FLI1 were evicted from chromatin at 1 nM even with prolonged exposure (Fig. 3B). To confirm that these effects occurred at EWS-FLI1 target genes and SWI/SNF binding sites in the

genome, we used chromatin immunoprecipitation and qPCR to quantitate the impact of drug

treatment on binding at previously identified EWS-FLI1 and SMARCC1 binding sites (from an

independent study (14)). We confirmed loss of binding of SMARCC1 to chromatin at several

key loci (Fig. 3C). Importantly, SMARCC1 binds throughout the genome, so as an additional

control, we mapped and immunoprecipitated SMARCC1 at GAPDH. While GAPDH could be immunoprecipitated, binding of SMARRC1 at this site was not impacted by drug treatment suggesting the importance of EWS-FLI1 to this effect of trabectedin (Fig. 3D). It is notable that identical inputs were loaded into all immunoprecipitations (Supplemental Fig. S2A).

SWI/SNF eviction reverses the pioneering transcription factor activity of EWS-FLI1.

A link between SWI/SNF and EWS-FLI1 and the establishment of GGAA microsatellites as enhancers has already been established (9,14). Therefore, we were interested in determining if the histone modifications at EWS-FLI1 targeting changed from enhancer marks (K3K27ac,

H3K4me1) to marks associated with epigenetically silenced chromatin (H3K27me3, H3K9me3).

We treated cells with trabectedin (Cmax exposure), washed out the drug, and then performed chromatin immunoprecipitation of H3K9me3 and H3K27me3 at 1- and 9-hours following drug removal. We chose these time points because they both featured the redistribution of EWS-FLI1 and loss of SMARCC1 binding to chromatin (Fig. 2A, 3A, 3B). We found that high dose trabectedin led to the marked accumulation of both H3K27me3 and H3K9me3 epigenetic marks throughout the genome (Fig. 4A). This effect was most prominent with H3K9me3 as the number of peaks increased from 1104 peaks in solvent to 28,901 by hour 1, with an additional 7957 peaks by hour 9. In addition, we found an enrichment of both marks at transcriptional start sites

(Fig. 4B, C). There was an enrichment of H3K27me3 marks at transcriptional start sites with drug treatment consistent with a known antagonism between SWI/SNF and the PRC2 complex

(Fig. 4B, Supplemental Fig. S2B)(45,46). There was also a major increase in H3K9me3 enrichment at transcriptional start sites (Fig. 4C). Indeed, pre-treatment there was little association between H3K9me3 and transcriptional start sites consistent with the known

relationship between H3K9me3 and constitutive heterochromatin (47,48). In contrast, after

trabectedin treatment, there was a marked accumulation of H3K9me3 at transcriptional start

sites, an effect most obvious when looking at the binding profile (Fig. 4D).

The silencing histone post-translational modifications were also associated with the enhancer

GGAA repeats, SWI/SNF and EWS-FLI1. There are approximately 26,000 GGAA

microsatellites in the genome, almost 70% of these or 18,272 are marked with H3K27me3,

H3K9me3 or both within 50 KB of a TSS after high dose exposure to trabectedin (Fig. 4E). In

addition, there was an enrichment of these marks at EWS-FLI1 target genes. We recently

published a list of 116 induced EWS-FLI1 targets found in multiple data sets in the literature

(49). 83 of the 116 genes in this list were associated with GGAA microsatellites within 50 KB of

the start site (Fig. 4F). Of this list of 83 targets, 76 of the 83 or 92% were marked with

H3K27me3, H3K9me3 or both following trabectedin treatment (Fig. 4G). Finally, the most well-

established microsatellite associated EWS-FLI1 target gene, NR0B1, was found to have a large

H3K9me3 peak at the TSS, immediately adjacent to the known SWI/SNF binding site in the

region (Fig. 4H). Importantly, we confirmed the presence of both H3K27me3 and H3K9me3

using ChIP-PCR in TC32 cells. In addition, we showed a similar enrichment in an additional

cell line, TC252 Ewing sarcoma cells (Supplemental Fig. S3). Similar enrichment of both

H3K27me3 and H3K9me3 epigenetic silencing marks was observed at a number of additional

well-established EWS-FLI1 target genes including RCOR1, PPP1R1A, MEIS1, WRN, EZH2,

BCL11B, LOX, and PRKCB (Supplemental Fig. S4, S5). In addition, high dose trabectedin treatment also caused the enrichment of H3K9me3 and H3K27me3 at genomic sites previously associated with SWI/SNF at MYT1, CCND1, and SOX2 (Supplemental Fig. S6). Importantly,

silencing of SMARCC1 reduces cell viability in Ewing sarcoma cells and further potentiates the activity of the drug in an analogous fashion to silencing of EWS-FLI1 (Supplemental Fig. S7)

Trabectedin requires irinotecan to improve suppression of EWS-FLI1 in the three- dimensional architecture of a tumor. We have previously shown that trabectedin is particularly effective in Ewing sarcoma in combination with extremely low doses of irinotecan

(36). Since, irinotecan is known to impact transcription, we sought to determine if the function of irinotecan in this combination is to improve the magnitude, penetrance or duration of EWS-

FLI1 suppression. We have previously shown that 18F-FLT PET reflects EWS-FLI1 activity because EWS-FLI1 drives the expression of the proteins responsible for activity in Ewing cells,

ENT1/ENT2 and TK1 (50).

Treatment of mice bearing Ewing sarcoma xenografts with trabectedin suppressed EWS-FLI1 activity and caused a loss of 18F-FLT PET activity. Peak suppression occurred 6-24 hours after treatment and the xenograft recovered PET avidity by 54-72 hours (Fig. 5A). In order to investigate EWS-FLI1 suppression in the 3-dimensional architecture of the tumor, we used the signal from every voxel in the tumor to mathematically reconstruct the tumor to determine the distribution of EWS-FLI1 suppression. Again, we found striking 18F-FLT PET signal in control tumors (Fig. 5B) and marked suppression of EWS-FLI1 most evident in the X, Y, Z plane cross- sections of the trabectedin treated tumors (Fig. 5C). After 24 hours control animals had a mean signal 20% higher than trabectedin treated animals (p<0.0001, 95%CI[12.9,27.6]). Interestingly, we found marked variability in the distribution of EWS-FLI1 suppression among the animals in the cohort in 3-dimensions, the magnitude and even the duration of EWS-FLI1 suppression (Fig.

5B, C; Supplemental Fig. S8). It is notable that these tumors all came from the same cell line, were implanted at the same time, at the identical cell number, and treated with the identical dose of trabectedin (and all trabectedin was delivered or local toxicity would be obvious).

Nevertheless, the variability was remarkable and consistent with the heterogeneity in response to treatment that we have consistently observed across cohorts of mice regardless of therapy. We were able to rescue this variable suppression in vivo, by adding irinotecan which improved the amplitude, penetrance and duration of EWS-FLI1 suppression likely accounting for the favorable clinical experience with this combination (Fig. 5D). Additionally, this suppression correlated with effects on tumor growth and striking regressions of tumor were observed with the combination therapy as previously reported (36)(Supplemental Fig. 9). The most striking example was the day 8 animals (combo treatment in Fig. 4) that had complete suppression of target and complete regression of tumor while trabectedin and irinotecan recovered signal at 102 hours (Fig. 5D*). The average number of voxels with signal above background for trabectedin and irinotecan animals was 3346.17 and 977.72, respectively (95% CIs [722.8, 15490.5]; [192.4,

4968.1]); compared to 24.3 for animals treated with both (95% CI [4.2, 141.1]) (P = 0.0003,

0.0063, respectively). However, as early as day 5, the animals showed little to no evidence of

18F-FLT activity suggesting a change in the tissue from highly proliferative malignant tissue to benign consistent with a sustained release in the EWS-FLI1 mediated differentiation block.

Importantly, this type of analysis would simply not be possible with traditional IHC or PCR approaches to evaluate target suppression as it allowed us to evaluate the distribution of suppression in the same animal over time. Finally, it notable that sustained suppression of EWS-

FLI1 with this combination led to a release in the differentiation block and the tumor showed

evidence of differentiation down a number of mesenchymal lineages and human collagen, osteoblasts and fat were identified in the xenograft (Fig. 6). It is notable that the mouse is known to remodel and replace benign human tissue with mouse tissue and so the penetrance of the differentiation phenotype is difficult to establish (51). While the cell of origin of Ewing sarcoma is not known, current thinking favors a mesenchymal or neural crest origin (40,52-54).

Therefore, sustained suppression of EWS-FLI1 allows restoration of the differentiation program but this program is relatively unorganized leading to mesenchymal confusion.

Discussion

This study highlights the importance of drug mechanism to the drug development process.

Compounds with broad cytotoxicity profiles can be developed for specific indications if they inhibit the dominant oncogene of a specific tumor. However, the successful implementation of therapies of this type absolutely requires that the mechanism of suppression be optimized for a specific oncogene and a defined cell context.

In this study, we show that the therapeutic suppression of the dominant oncogene of Ewing sarcoma, EWS-FLI1, requires a high concentration of trabectedin in serum. We model this exposure preclinically and show in vitro and in vivo that the drug is able to inhibit EWS-FLI1.

The drug redistributes EWS-FLI1 in the nucleus, displaces SWI/SNF from chromatin, and triggers an epigenetic switch driving an increase in H3K27me3 and H3K9me3 with preference for EWS-FLI1 target genes. However, these effects absolutely require high concentrations of drug in serum and do not occur at lower concentrations even with prolonged exposure.

These observations are important because they justify the investigation 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 sarcoma cells are dependent on EWS-FLI1 (55).

However, the therapeutic suppression of EWS-FLI1 has not been achieved in clinic. In addition,

trabectedin was previously evaluated in Ewing sarcoma as a 24-hour infusion in a phase II study

because this schedule was shown to be more active in other sarcoma types (56). However, the

data in this manuscript suggests that a shorter 1-hour infusion schedule may increase activity in

Ewing sarcoma because the drug would accumulate to serum concentrations above a threshold

that we define in this manuscript as being high enough to inhibit the dominant oncogene, EWS-

FLI1 (42). This blockade of EWS-FLI1 is amplified and sustained in combination with low-dose irinotecan. Since this tumor absolutely depends on EWS-FLI1 it is likely that this study would show clinical activity. Therefore, this study justifies the further exploration of this compound on

an alternative 1-hour infusion schedule in this tumor in combination with low-dose irinotecan.

Perhaps the most important observation in this study is that even within sarcoma different

schedules of active compounds may be more effective in particular subtypes.

This study also provides important insight into the mechanism of action of trabectedin, a

compound that has found unique activity in a number of sarcomas. Trabectedin has a

complicated mechanism of action including both generating DNA damage and poisoning

specific DNA damage repair complexes, poisoning specific transcription factors such as EWS-

FLI1 and FUS-CHOP, and specifically targeting tumor associated macrophages (57,58). In this

study, we add displacement of SWI/SNF from chromatin to this mechanism. It is likely that this

mechanism contributes to the broad cytotoxicity profile of this compound as SWI/SNF is

mutated in up to 25% of human cancer and commonly altered either functionally or through

mutation in sarcoma.

This study also highlights important features of Ewing sarcoma biology. We confirm the recent

observation that SWI/SNF is important to the biology of EWS-FLI1 and further establish the link

to EWS-FLI1, particularly at the GGAA microsatellites (27). We show that removal of EWS-

FLI1 leads to a cellular response and widespread chromatin silencing particularly with H3K9me3

which favors repetitive sequences and constitutive heterochromatin. The data suggests both

inhibition of EWS-FLI1 and displacement of SWI/SNF are required to reverse activity, however

further work would need to be done to clearly establish this point. In addition, once this reversal is achieved, relatively non-specific blockade can sustain suppression of the target in vivo. The net result is a differentiation endpoint, although this differentiation is unorganized.

Finally, this study reports a novel use of 18F-FLT PET imaging as a tool to quantitate target

suppression and at the same time visualize the penetrance and distribution of target suppression

within the 3-dimensional architecture of the tumor. Indeed, perhaps the most interesting

observation in this study is the widely variable suppression of EWS-FLI1 that occurred within

cohorts of mice. The Ewing sarcoma xenografts were established from the same cell collection

and the same flask and tube, with 2 million cells in every animal by the same technician on the

same day in one strain of animal. Treatment was also initiated by the same technician from the

same stock of drug and all drug made it into the circulation as any extravasation of this drug

leads to tail necrosis. Yet, despite these similarities, the magnitude, penetrance and even duration

of target suppression was widely variable from one animal to the next. It is likely that this

variable target suppression is an important factor driving tumor response. However, it is not known what the source of this variability is; a question we are now starting to investigate.

Nevertheless, this study serves as proof of principle to ask this question in a prospective fashion, using schedule-optimized trabectedin in combination with low dose irinotecan, and 18F-FLT imaging in Ewing sarcoma patients in the clinic.

Acknowledgments: The authors would like to thank Ron Chandler, PhD (Michigan State

University) for helpful discussion. The authors would also like to thank Dr. Peter Adamson

(Children’s Hospital of Philadelphia) for helpful advice. We would like to thank Robert Vaughan from the Rothbart lab for technical help. We would like to thank Marie Adams for technical support and library preparations and the Bioinformatic and Biostatistics Core of the Van Andel

Research Institute. The authors would like to thank Pharma Mar pharmaceutical company for material used in this proposal.

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Figure Legends: 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 = Concentration*Time). Greater suppression of cell viability (percent confluence) occurs above a 10 nM threshold (10 nM, 60 min) relative to solvent control (solvent). (B) Sustained suppression of EWS-FLI1 activity as measured by NR0B1-Luc (black bars) in comparison to 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 is direct comparison of identical exposure of 24nM trabectedin for 1 hour followed by 23 hours in drug free medium (Cmax) or 1nM trabectedin for 24 hours ddCt (AUC) exposure as measured by qPCR fold change relative to GAPDH (2 ) ****, p- value<0.0001. (D)(E) Western blot 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 = 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 nM. 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.

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 (A) high dose exposure (Cmax, 24 nM for 1 hour), drug removal and incubation for the indicated time but not with (B) low dose continuous exposure (AUC, 1 nM for 24 hours). (C) Similar redistribution of EWS-FLI1 only with high Cmax exposure (24 nM for 1 hour) in TP53 mutant A673 cells. Confocal microscopy stained for nucleolin (NCL), EWS-FLI1.

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 nM for 1 hour) followed by incubation in drug-free medium but not (B) continuous low dose (AUC, 1nM continuous) 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) Chromatin immunoprecipitation of IgG or SMARCC1 at known EWS-FLI1 and SWI/SNF target genes (MYT1, SOX2, CCND1, NR0B1) in comparison to (D) GAPDH locus control following 24 nM trabectedin treatment for 1 hour (1h Trab.) followed by collection immediately or after 8 more hours in drug free medium (9h Trab.) in TC32 cells. Data is represented as percent input quantitated against a standard curve.

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 chromatin immunoprecipitation and sequencing (ChIP-seq) following treatment with DMSO solvent, 24nM trabectedin for 1 hour (1 Hour Trab.), or 24 nM trabectedin for 1 hour followed by an 8 hour recovery in drug-free media (9 Hour Trab.) in TC32 Ewing sarcoma cells (B) Heatmap

displaying the genome-wide distribution of (B) H3K27me3 or (C) H3K9me3 peaks relative to transcriptional start sites (TSS) following 24 nM trabectedin for 1 hour (Hour 1), or 24nM 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 nM trabectedin for 1 hour (Hour 1), or 24nM trabectedin for 1 hour followed by 8 hours in drug-free media (Hour 9) (E) Total number of GGAA microsatellites marked (+/- 50KB) with H3K9me3 (15,400, dark green), H3K27me3 (2767, light green), both (105, darkest green) or neither (8443, grey) after treatment with 24 nM 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 (+/- 50KB) with H3K9me3 (30, blue), H3K27me3 (6, light blue), both (40, dark blue) or neither (7, grey) after treatment with 24 nM trabectedin for 1 hour (H) Genome browser tracks of H3K9me3 at TSS following indicated solvent or trabectedin treatments at the NR0B1 gene.

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 is a 3-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 is a 3-Dimensional reconstruction of the tumor (tumor) followed by cross-sections in the X, Y, and Z axes. Scale indicates signal intensity. (D) 3-D reconstruction and single cross-section of tumors at multiple time points following treatment with vehicle (1-hour), trabectedin (1-hour), irinotecan (24 and 48 hour), or the combination of trabectedin and irinotecan. Rows indicate time and treatments, columns represent 3-dimensional reconstruction and single cross-section for each of the treatment groups. The intensity scale is the same as (B), and (C).

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

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 Published OnlineFirst February 5, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3511

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