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Chemotherapy-Induced Distal Enhancers Drive Transcriptional Programs to Maintain the Chemoresistant State in Ovarian Cancer

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Chemotherapy-Induced Distal Enhancers Drive Transcriptional Programs to Maintain the Chemoresistant State in Ovarian Cancer Author Manuscript Published OnlineFirst on July 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0215 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title: Chemotherapy-induced distal enhancers drive transcriptional programs to maintain the chemoresistant state in ovarian cancer. Authors: Stephen Shang1, Jiekun Yang1, Amir A. Jazaeri2, Alexander James Duval1, Turan Tufan1, Natasha Lopes Fischer1, Mouadh Benamar1,3, Fadila Guessous3, Inyoung Lee1, Robert M. Campbell4, Philip J. Ebert4, Tarek Abbas1,3, Charles N. Landen5, Analisa Difeo6, Peter C. Scacheri6, Mazhar Adli1# 1 Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 1340 JPA, Pinn Hall, Charlottesville, VA 22908, USA 2 Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 3 Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA 4 Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA 5 Department of Obstetrics and Gynecology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA 6 Department of Genetics and Genome Sciences, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA. # Correspondence: Mazhar Adli, Ph.D. Email: [email protected] Address: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, 1340 JPA, Pinn Hall, Rm: 640, Charlottesville, Virginia, 22902 1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0215 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SEs) and their gene targets that coordinate and maintain the transcriptional program of the platinum- resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor (TF) that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. INTRODUCTION The American Cancer Society estimates 22,240 new cases of ovarian cancer (OC) in 2018 (1). Unfortunately, the five-year survival rate of OC remains less than fifty percent. Thus, nearly 14,000 women in the USA and 160,000 worldwide die of OC each year (2). Epithelial ovarian cancers, which account for nearly 90% of all OC diagnoses are associated with worse prognosis (3). They originate mainly from the epithelial cells of fallopian tubes (4, 5) and areas of endometriosis (6), among others. Critically, 75% of the patients with epithelial OC are high grade serous ovarian cancer (HGSOC) (7) that are more challenging to effectively treat. The frontline therapy for OC involves the combination of cytoreductive surgery followed by platinum and taxane-based chemotherapy. Platinum-based compounds such as cisplatin induce increased DNA damage through interstrand cross-links and cell death in proliferative cancerous cells (7, 8). Despite the high rate of initial response to therapy, the duration of response declines over time and a vast majority of patients succumb to chemotherapy- resistant ovarian cancer (9-12). Recent genomic approaches have shed significant light on the genetic risk factors of OC. Low- grade ovarian tumors often harbor BRAF, KRAS, BRCA1/2, and PTEN mutations whereas high-grade tumors are uniformly characterized by TP53 mutations (13, 14). Apart from the antiangiogenic agent bevacizumab, and partially effective PARP inhibitors for patients with BRCA1/2 mutations (15), targeted therapies are lacking for ovarian cancer. Although specific genetic alterations such as reversion of germline BRCA1/2 mutations and inactivating mutations in tumor suppressor RB1, NF1, RAD51B, and PTEN genes were noted in some chemoresistant patients (16), the molecular network that drives and 2 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0215 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. maintains the chemoresistant state in ovarian cancer is largely unknown. In addition to genetic alterations, epigenetic regulation of proximal promoters and distal enhancers are critical determinants of cellular identities. Alterations in the chromatin landscape are increasingly recognized as hallmarks of malignant cellular states (17-19). Due to technical limitations, previous ovarian cancer epigenetic studies primarily focused on targeted DNA methylation at individual gene promoters. Although these studies implicate differential methylation at multiple genes, such as MLH1 (20), SFRP1 (21), BRCA1 (16), MAL (22), FANCF (23) with chemoresistance, there have been limited attempts to comprehensively map differentially regulated gene promoters and distal enhancers in ovarian cancer. In this study, we aimed to identify molecular drivers of chemoresistance in ovarian cancer through unbiased epigenomic and transcriptional profiling across multiple cellular models of ovarian cancer. We aimed to map differentially regulated proximal promoters and distal enhancers in multiple cellular models of ovarian cancer. By integrating genome-wide maps of a well-characterized epigenetic mark of active regulatory genomic elements with gene expression profiles, we aimed to identify differentially regulated proximal promoters and distal regulatory elements that are specifically associated with chemoresistance across multiple OC cell lines. To this end, we generated multiple isogenic cellular models of cisplatin resistance and performed ChIP-Seq analysis of the Histone H3, Lysine 27 acetylation (H3K27ac) epigenetic mark, which is deposited to active enhancers and promoters. By integrating ChIP-Seq maps with RNA-Seq gene expression profiles across naïve and chemoresistant cellular counterparts, we found that the chemoresistant state is associated with largely cell type-specific sets of distal enhancer elements. Critically, we found significant upregulation of distal enhancer clusters known as super- enhancers (SE) in resistant cells. Small molecule epigenetic drugs that target enhancers and super- enhancers result in significant decrease in the expression of their target genes and an increase in cisplatin sensitivity in chemoresistant HGSOC cells. Our findings identified, in addition to known drivers of chemoresistance, SOX9 as a critical SE-regulated transcription factor (TF) that plays a critical role in chemoresistance across multiple ovarian cancer cell lines. MATERIALS and METHODS: Cell culture Human ovarian cancer OVCAR4, CAOV3, OV81 and COV362 cell lines were cultured in complete medium consisting of RPMI 1640, 20 % heat-inactivated FBS, 1% Pen/Strep. SKOV3 Cells were cultured in complete medium consisting of McCoy’s 5A, 10% heat-inactivated FBS (FBS, Sigma Aldrich), 1% Pen/Strep (100U/ml penicillin, 100μg/ml streptomycin (PAA Laboratories GmbH). Cells were cultured incubator at 37 °C in a humidified atmosphere consisting of 5 % CO2 and 95 % air. The cells were originally obtained from ATCC and monitored periodically for mycoplasma contamination. The cells were validated using FTA Sample Collection Kits for Human Cell Authentication Service (ATCC). 3 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 29, 2019; DOI: 10.1158/0008-5472.CAN-19-0215 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Creating Cisplatin Resistant and Resensitized Cell Lines Cells were grown in their respective culture media and passaged for at least two generations after thawing to ensure proper viability. When the cells reached 80% confluency, they were split into two 6cm plates with 40% confluency. Cells were treated with an initial dose of 1 μM cisplatin in 3 mL complete media. After 4 hours, the media for both control and treated cells were aspirated and replaced washed with an equal volume of PBS twice before replacing with drug-free complete media. Cells were allowed to recover for two passages, and treated with the same or increasing dose of cisplatin depending on the viability levels. Once the cells gain resistance, either the dose was increased or cells were periodically treated with cisplatin to maintain the chemoresistant state. Resistant SKOV3, cells reached a maximum concentration of 20 μM cisplatin tolerance, OV81 cells reached a maximum
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