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. 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. 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. 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. 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. 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. 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 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. 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