trabectedin inhibits ews-fli1 and evicts swi/snf from ... · addition, trabectedin has failed in...
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Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule
Dependent Manner
Authors: Matt L. Harlow1†,†††
, Maggie H. Chasse2,†††
, Elissa A. Boguslawski2, Katie M.
Sorensen2, Jenna M. Gedminas
2,5,6, Susan M. Kitchen-Goosen
2, Scott B. Rothbart
2, Cenny
Taslim3, Stephen L. Lessnick
3,4, Anderson S. Peck
2††, Zachary B. Madaj
2, Megan J. Bowman
2‡,
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.
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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 18
F-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 18
F-FLT PET imaging in Ewing
sarcoma patients.
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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 18
F-
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.
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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
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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
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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.
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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 –
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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
65C followed by 10-minute incubation at 95C. 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 37C for 30 minutes followed by Proteinase K at 65C
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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
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(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
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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 18
F-FLT (18
F-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
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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
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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
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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).
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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
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(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
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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
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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,
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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 18
F-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 18
F-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 18
F-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.
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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
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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.
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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
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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 18
F-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
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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 18
F-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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References:
1. Dang CV. MYC, metabolism, cell growth, and tumorigenesis. Cold Spring Harb Perspect
Med 2013;3(8) doi 10.1101/cshperspect.a014217.
2. Sizemore GM, Pitarresi JR, Balakrishnan S, Ostrowski MC. The ETS family of
oncogenic transcription factors in solid tumours. Nat Rev Cancer 2017;17(6):337-51 doi
10.1038/nrc.2017.20.
3. Bradner JE, Hnisz D, Young RA. Transcriptional Addiction in Cancer. Cell
2017;168(4):629-43 doi 10.1016/j.cell.2016.12.013.
4. Zhu J, Koken MH, Quignon F, Chelbi-Alix MK, Degos L, Wang ZY, et al. Arsenic-
induced PML targeting onto nuclear bodies: implications for the treatment of acute
promyelocytic leukemia. Proc Natl Acad Sci U S A 1997;94(8):3978-83.
5. Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, et al. In vitro studies on cellular and
molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute
promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-
2 expression and modulation of PML-RAR alpha/PML proteins. Blood 1996;88(3):1052-
61.
6. Yoshida H, Kitamura K, Tanaka K, Omura S, Miyazaki T, Hachiya T, et al. Accelerated
degradation of PML-retinoic acid receptor alpha (PML-RARA) oncoprotein by all-trans-
retinoic acid in acute promyelocytic leukemia: possible role of the proteasome pathway.
Cancer Res 1996;56(13):2945-8.
7. Maksimenko A, Malvy C. Oncogene-targeted antisense oligonucleotides for the
treatment of Ewing sarcoma. Expert Opin Ther Targets 2005;9(4):825-30 doi
10.1517/14728222.9.4.825.
8. Kauer M, Ban J, Kofler R, Walker B, Davis S, Meltzer P, et al. A molecular function
map of Ewing's sarcoma. PLoS One 2009;4(4):e5415 doi 10.1371/journal.pone.0005415.
9. Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suva ML, et al. EWS-FLI1
utilizes divergent chromatin remodeling mechanisms to directly activate or repress
enhancer elements in Ewing sarcoma. Cancer Cell 2014;26(5):668-81 doi
10.1016/j.ccell.2014.10.004.
10. Kinsey M, Smith R, Lessnick SL. NR0B1 is required for the oncogenic phenotype
mediated by EWS/FLI in Ewing's sarcoma. Mol Cancer Res 2006;4(11):851-9 doi
10.1158/1541-7786.MCR-06-0090.
11. Gangwal K, Lessnick SL. Microsatellites are EWS/FLI response elements: genomic
"junk" is EWS/FLI's treasure. Cell Cycle 2008;7(20):3127-32 doi 10.4161/cc.7.20.6892.
12. Johnson KM, Mahler NR, Saund RS, Theisen ER, Taslim C, Callender NW, et al. Role
for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing
sarcoma anchorage independent growth. Proc Natl Acad Sci U S A 2017;114(37):9870-5
doi 10.1073/pnas.1701872114.
13. Johnson KM, Taslim C, Saund RS, Lessnick SL. Identification of two types of GGAA-
microsatellites and their roles in EWS/FLI binding and gene regulation in Ewing
sarcoma. PLoS One 2017;12(11):e0186275 doi 10.1371/journal.pone.0186275.
14. Boulay G, Sandoval GJ, Riggi N, Iyer S, Buisson R, Naigles B, et al. Cancer-Specific
Retargeting of BAF Complexes by a Prion-like Domain. Cell 2017;171(1):163-78 e19
doi 10.1016/j.cell.2017.07.036.
Research. on February 1, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511
15. Selvanathan SP, Graham GT, Erkizan HV, Dirksen U, Natarajan TG, Dakic A, et al.
Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing.
Proc Natl Acad Sci U S A 2015;112(11):E1307-16 doi 10.1073/pnas.1500536112.
16. Grohar PJ, Griffin LB, Yeung C, Chen QR, Pommier Y, Khanna C, et al. Ecteinascidin
743 interferes with the activity of EWS-FLI1 in Ewing sarcoma cells. Neoplasia
2011;13(2):145-53.
17. Lau L, Supko JG, Blaney S, Hershon L, Seibel N, Krailo M, et al. A phase I and
pharmacokinetic study of ecteinascidin-743 (Yondelis) in children with refractory solid
tumors. A Children's Oncology Group study. Clin Cancer Res 2005;11(2 Pt 1):672-7.
18. Scotlandi K, Perdichizzi S, Manara MC, Serra M, Benini S, Cerisano V, et al.
Effectiveness of Ecteinascidin-743 against drug-sensitive and -resistant bone tumor cells.
Clin Cancer Res 2002;8(12):3893-903.
19. Baruchel S, Pappo A, Krailo M, Baker KS, Wu B, Villaluna D, et al. A phase 2 trial of
trabectedin in children with recurrent rhabdomyosarcoma, Ewing sarcoma and non-
rhabdomyosarcoma soft tissue sarcomas: a report from the Children's Oncology Group.
Eur J Cancer 2012;48(4):579-85 doi 10.1016/j.ejca.2011.09.027.
20. D'Incalci M, Badri N, Galmarini CM, Allavena P. Trabectedin, a drug acting on both
cancer cells and the tumour microenvironment. Br J Cancer 2014;111(4):646-50 doi
10.1038/bjc.2014.149.
21. Germano G, Frapolli R, Belgiovine C, Anselmo A, Pesce S, Liguori M, et al. Role of
macrophage targeting in the antitumor activity of trabectedin. Cancer Cell
2013;23(2):249-62 doi 10.1016/j.ccr.2013.01.008.
22. Aune GJ, Takagi K, Sordet O, Guirouilh-Barbat J, Antony S, Bohr VA, et al. Von
Hippel-Lindau-coupled and transcription-coupled nucleotide excision repair-dependent
degradation of RNA polymerase II in response to trabectedin. Clin Cancer Res
2008;14(20):6449-55 doi 10.1158/1078-0432.CCR-08-0730.
23. Forni C, Minuzzo M, Virdis E, Tamborini E, Simone M, Tavecchio M, et al. Trabectedin
(ET-743) promotes differentiation in myxoid liposarcoma tumors. Mol Cancer Ther
2009;8(2):449-57 doi 10.1158/1535-7163.MCT-08-0848.
24. Soares DG, Escargueil AE, Poindessous V, Sarasin A, de Gramont A, Bonatto D, et al.
Replication and homologous recombination repair regulate DNA double-strand break
formation by the antitumor alkylator ecteinascidin 743. Proc Natl Acad Sci U S A
2007;104(32):13062-7 doi 10.1073/pnas.0609877104.
25. Grohar PJ, Woldemichael GM, Griffin LB, Mendoza A, Chen QR, Yeung C, et al.
Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-
Throughput Screening. J Natl Cancer Inst 2011;103(12):962-78.
26. Harlow ML, Maloney N, Roland J, Guillen Navarro MJ, Easton MK, Kitchen-Goosen
SM, et al. Lurbinectedin Inactivates the Ewing Sarcoma Oncoprotein EWS-FLI1 by
Redistributing It within the Nucleus. Cancer Res 2016 doi 10.1158/0008-5472.CAN-16-
0568.
27. Boulay G, Sandoval GJ, Riggi N, Iyer S, Buisson R, Naigles B, et al. Cancer-Specific
Retargeting of BAF Complexes by a Prion-like Domain. Cell 2017 doi
10.1016/j.cell.2017.07.036.
28. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based
analysis of ChIP-Seq (MACS). Genome Biol 2008;9(9):R137 doi 10.1186/gb-2008-9-9-
r137.
Research. on February 1, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511
29. Consortium EP. An integrated encyclopedia of DNA elements in the human genome.
Nature 2012;489(7414):57-74 doi 10.1038/nature11247.
30. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic
features. Bioinformatics 2010;26(6):841-2 doi 10.1093/bioinformatics/btq033.
31. Yu G, Wang LG, He QY. ChIPseeker: an R/Bioconductor package for ChIP peak
annotation, comparison and visualization. Bioinformatics 2015;31(14):2382-3 doi
10.1093/bioinformatics/btv145.
32. Ramirez F, Dundar F, Diehl S, Gruning BA, Manke T. deepTools: a flexible platform for
exploring deep-sequencing data. Nucleic Acids Res 2014;42(Web Server issue):W187-91
doi 10.1093/nar/gku365.
33. Ramirez F, Ryan DP, Gruning B, Bhardwaj V, Kilpert F, Richter AS, et al. deepTools2: a
next generation web server for deep-sequencing data analysis. Nucleic Acids Res
2016;44(W1):W160-5 doi 10.1093/nar/gkw257.
34. Rothbart SB, Krajewski K, Nady N, Tempel W, Xue S, Badeaux AI, et al. Association of
UHRF1 with methylated H3K9 directs the maintenance of DNA methylation. Nat Struct
Mol Biol 2012;19(11):1155-60 doi 10.1038/nsmb.2391.
35. Soares DG, Machado MS, Rocca CJ, Poindessous V, Ouaret D, Sarasin A, et al.
Trabectedin and its C subunit modified analogue PM01183 attenuate nucleotide excision
repair and show activity toward platinum-resistant cells. Mol Cancer Ther
2011;10(8):1481-9 doi 10.1158/1535-7163.MCT-11-0252.
36. Grohar PJ, Segars LE, Yeung C, Pommier Y, D'Incalci M, Mendoza A, et al. Dual
targeting of EWS-FLI1 activity and the associated DNA damage response with
trabectedin and SN38 synergistically inhibits Ewing sarcoma cell growth. Clin Cancer
Res 2014;20(5):1190-203 doi 10.1158/1078-0432.CCR-13-0901.
37. Kinsey M, Smith R, Iyer AK, McCabe ER, Lessnick SL. EWS/FLI and its downstream
target NR0B1 interact directly to modulate transcription and oncogenesis in Ewing's
sarcoma. Cancer Res 2009;69(23):9047-55 doi 10.1158/0008-5472.CAN-09-1540.
38. Gangwal K, Sankar S, Hollenhorst PC, Kinsey M, Haroldsen SC, Shah AA, et al.
Microsatellites as EWS/FLI response elements in Ewing's sarcoma. Proc Natl Acad Sci U
S A 2008;105(29):10149-54 doi 10.1073/pnas.0801073105.
39. Gangwal K, Close D, Enriquez CA, Hill CP, Lessnick SL. Emergent Properties of
EWS/FLI Regulation via GGAA Microsatellites in Ewing's Sarcoma. Genes Cancer
2010;1(2):177-87 doi 10.1177/1947601910361495.
40. Riggi N, Suva ML, Suva D, Cironi L, Provero P, Tercier S, et al. EWS-FLI-1 expression
triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells.
Cancer Res 2008;68(7):2176-85 doi 10.1158/0008-5472.CAN-07-1761.
41. Richter GH, Plehm S, Fasan A, Rossler S, Unland R, Bennani-Baiti IM, et al. EZH2 is a
mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and
neuro-ectodermal differentiation. Proc Natl Acad Sci U S A 2009;106(13):5324-9 doi
10.1073/pnas.0810759106.
42. Twelves C, Hoekman K, Bowman A, Vermorken JB, Anthoney A, Smyth J, et al. Phase I
and pharmacokinetic study of Yondelis (Ecteinascidin-743; ET-743) administered as an
infusion over 1 h or 3 h every 21 days in patients with solid tumours. Eur J Cancer
2003;39(13):1842-51.
43. Pommier Y, Kohlhagen G, Bailly C, Waring M, Mazumder A, Kohn KW. DNA
sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by
Research. on February 1, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511
ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate
Ecteinascidia turbinata. Biochemistry 1996;35(41):13303-9 doi 10.1021/bi960306b.
44. Quinn J, Fyrberg AM, Ganster RW, Schmidt MC, Peterson CL. DNA-binding properties
of the yeast SWI/SNF complex. Nature 1996;379(6568):844-7 doi 10.1038/379844a0.
45. Kadoch C, Williams RT, Calarco JP, Miller EL, Weber CM, Braun SM, et al. Dynamics
of BAF-Polycomb complex opposition on heterochromatin in normal and oncogenic
states. Nat Genet 2017;49(2):213-22 doi 10.1038/ng.3734.
46. Wilson BG, Wang X, Shen X, McKenna ES, Lemieux ME, Cho YJ, et al. Epigenetic
antagonism between polycomb and SWI/SNF complexes during oncogenic
transformation. Cancer Cell 2010;18(4):316-28 doi 10.1016/j.ccr.2010.09.006.
47. Rea S, Eisenhaber F, O'Carroll D, Strahl BD, Sun ZW, Schmid M, et al. Regulation of
chromatin structure by site-specific histone H3 methyltransferases. Nature
2000;406(6796):593-9 doi 10.1038/35020506.
48. Martens JH, O'Sullivan RJ, Braunschweig U, Opravil S, Radolf M, Steinlein P, et al. The
profile of repeat-associated histone lysine methylation states in the mouse epigenome.
EMBO J 2005;24(4):800-12 doi 10.1038/sj.emboj.7600545.
49. Harlow ML, Maloney N, Roland J, Guillen-Navarro MJ, Easton MK, Kitchen-Goosen
SM, et al. Lurbinectedin inactivates the Ewing sarcoma oncoprotein EWS-FLI1 by
redistributing it within the nucleus. Cancer Res 2016 doi 10.1158/0008-5472.CAN-16-
0568.
50. Osgood CL, Tantawy MN, Maloney N, Madaj ZB, Peck A, Boguslawski E, et al. 18F-
FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-
FLI1 Activity and Ewing Sarcoma. Sci Rep 2016;6:33926 doi 10.1038/srep33926.
51. Proia DA, Kuperwasser C. Reconstruction of human mammary tissues in a mouse model.
Nat Protoc 2006;1(1):206-14 doi 10.1038/nprot.2006.31.
52. von Levetzow C, Jiang X, Gwye Y, von Levetzow G, Hung L, Cooper A, et al. Modeling
initiation of Ewing sarcoma in human neural crest cells. PLoS One 2011;6(4):e19305 doi
10.1371/journal.pone.0019305.
53. Hu-Lieskovan S, Zhang J, Wu L, Shimada H, Schofield DE, Triche TJ. EWS-FLI1 fusion
protein up-regulates critical genes in neural crest development and is responsible for the
observed phenotype of Ewing's family of tumors. Cancer Res 2005;65(11):4633-44 doi
10.1158/0008-5472.CAN-04-2857.
54. Torchia EC, Jaishankar S, Baker SJ. Ewing tumor fusion proteins block the
differentiation of pluripotent marrow stromal cells. Cancer Res 2003;63(13):3464-8.
55. Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y. EWS-Fli1 antisense
oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive
neuroectodermal tumor cells. J Clin Invest 1997;99(2):239-47 doi 10.1172/JCI119152.
56. Demetri GD, Chawla SP, von Mehren M, Ritch P, Baker LH, Blay JY, et al. Efficacy and
safety of trabectedin in patients with advanced or metastatic liposarcoma or
leiomyosarcoma after failure of prior anthracyclines and ifosfamide: results of a
randomized phase II study of two different schedules. J Clin Oncol 2009;27(25):4188-96
doi 10.1200/JCO.2008.21.0088.
57. Larsen AK, Galmarini CM, D'Incalci M. Unique features of trabectedin mechanism of
action. Cancer Chemother Pharmacol 2016;77(4):663-71 doi 10.1007/s00280-015-2918-
1.
Research. on February 1, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3511
58. Galmarini CM, D'Incalci M, Allavena P. Trabectedin and plitidepsin: drugs from the sea
that strike the tumor microenvironment. Mar Drugs 2014;12(2):719-33 doi
10.3390/md12020719.
Research. on February 1, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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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
(AUC) exposure as measured by qPCR fold change relative to GAPDH (2ddCt
) ****, 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
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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 18
F-FLT imaging. (A)
Mice bearing TC32 Ewing sarcoma xenografts in right gastrocnemius show suppression of 18
F-
FLT signal 6 to 54 hours after treatment with trabectedin but not vehicle control. The bladder
shows high 18
F-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 18
F-
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).
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Published OnlineFirst February 5, 2019.Clin Cancer Res Matt L Harlow, Maggie H. Chasse, Elissa A Boguslawski, et al. Chromatin in a Schedule Dependent MannerTrabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from
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