Published OnlineFirst September 6, 2017; DOI: 10.1158/1535-7163.MCT-17-0397 Small Molecule Therapeutics Molecular Cancer Therapeutics Death by HDAC Inhibition in Synovial Sarcoma Cells Aimee N. Laporte1, Neal M. Poulin1, Jared J. Barrott2, Xiu Qing Wang1, Alireza Lorzadeh3, Ryan Vander Werff4, Kevin B. Jones2, T. Michael Underhill4, and Torsten O. Nielsen1 Abstract Conventional cytotoxic therapies for synovial sarcoma pro- group targets were reactivated, including tumor suppressor vide limited benefit,andnodrugsspecifically targeting the CDKN2A, and proapoptotic transcriptional patterns were causative SS18-SSX fusion oncoprotein are currently available. induced. Functional analyses revealed that ROS-mediated Histone deacetylase (HDAC) inhibition has been shown in FOXO activation and proapoptotic factors BIK, BIM, and BMF previous studies to disrupt the synovial sarcoma oncoprotein were important to apoptosis induction following HDAC inhi- complex, resulting in apoptosis. To understand the molecular bition in synovial sarcoma. HDAC inhibitor pathway activa- effects of HDAC inhibition, RNA-seq transcriptome analysis tion results in apoptosis and decreased tumor burden follow- fl/fl was undertaken in six human synovial sarcoma cell lines. ing a 7-day quisinostat treatment in the Pten ;hSS2 mouse HDAC inhibition induced pathways of cell-cycle arrest, neu- model of synovial sarcoma. This study provides mechanistic ronal differentiation, and response to oxygen-containing spe- support for a particular susceptibility of synovial sarcoma to cies, effects also observed in other cancers treated with this HDAC inhibition as a means of clinical treatment. Mol Cancer class of drugs. More specific to synovial sarcoma, polycomb Ther; 16(12); 2656–67. Ó2017 AACR. Introduction Current cytotoxic therapies, including doxorubicin and ifo- sphamide, provide limited benefit for synovial sarcoma patients. Synovial sarcoma is an aggressive soft tissue malignancy that Following surgery and radiation, patients remain at high risk for primarily affects adolescents and young adults (1). It is charac- both early and late metastases, and despite the use of multimodal terized by the driving chimeric oncoprotein SS18-SSX, derived therapies the mortality rate remains approximately 50% within 10 from the t(X;18)(p11.2;q11.2) translocation (2). The SS18-SSX years of diagnosis (6). Thus, there is a significant need for targeted fusion oncoprotein has been proposed to interact in place of therapies against synovial sarcoma. native SS18, leading to aberrant SWI/SNF–mediated transcrip- Histone deacetylase (HDAC) inhibition has been shown to tion (3), and to act as a nidus for abnormal assembly of elicit apoptosis in synovial sarcoma models, as well as to disrupt polycomb group members (PcG) and transcription factors that SS18-SSX–mediated repressive complexes (4, 7). This inhibition lead to transcriptional repression by EZH2-mediated H3K27 was observed to elicit reactivation of repressed gene targets, trimethylation (4). Expression of tumor suppressor genes including CDKN2A (4, 7, 8). As a result, a phase II study of a EGR1 and CDKN2A has been shown to be directly repressed pan-HDAC inhibitor, pracinostat, was initiated in translocation- by SS18-SSX, whereas the antiapoptotic protein BCL2 is char- associated sarcomas, from which stable disease was achieved in acteristically upregulated (5) leading to a proliferative and three of three assessable SS18-SSX–positive patients (9). While antiapoptotic phenotype. this study had to be terminated prematurely due to unexpected interruptions in the supply of the studied compound, initial results did warrant further investigation. 1Faculty of Medicine, Vancouver Coastal Health Research Institute, University of Histone acetylation status is an important element in the British Columbia, Vancouver, British Columbia, Canada. 2Huntsman Cancer regulation of gene expression. HDAC inhibition has been Institute, University of Utah, Salt Lake City, Utah. 3Department of Microbiology shown to induce cell-cycle arrest (10), senescence (11), differ- and Immunology, Michael Smith Laboratories Centre for High-Throughput entiation (12), and apoptosis (13) in cancer cells, generally Biology, University of British Columbia, Vancouver, British Columbia, Canada. 4 leading to a decrease in tumor burden (14). While maintaining Department of Cellular and Physiological Sciences, Biomedical Research Cen- some pathway commonalities, the distinct mechanism of tre, University of British Columbia, Vancouver, British Columbia, Canada. HDAC inhibitor–induced cell death may be specifictothe Note: Supplementary data for this article are available at Molecular Cancer underlying oncogenic context. With efficacy observed in revers- Therapeutics Online (http://mct.aacrjournals.org/). ing the abnormal epigenetic status of several cancer subtypes, a Corresponding Author: Torsten O. Nielsen, University of British Columbia, 855 variety of HDAC inhibitors are currently under clinical inves- W. 12th Avenue, Vancouver, British Columbia V5Z 1M9, Canada. Phone: 604-875- tigation. Since 2006, five clinical drugs have been approved as 4111, ext. 62649; Fax: 604-875-5707; E-mail: [email protected] treatments for advanced T-cell lymphoma or multiple myeloma, doi: 10.1158/1535-7163.MCT-17-0397 while several additional HDAC-inhibiting compounds are cur- Ó2017 American Association for Cancer Research. rently under investigation in solid tumor clinical trials (14). 2656 Mol Cancer Ther; 16(12) December 2017 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 6, 2017; DOI: 10.1158/1535-7163.MCT-17-0397 HDAC Inhibition in Synovial Sarcoma Cells Quisinostat (15), used in this study, is a second-generation Reads of quality 10 or greater were enumerated on the sense HDAC inhibitor showing promising efficacy against synovial strand for the exonic regions of each gene. Fragments Per Kilobase sarcoma in preclinical experiments (16). of exon per Million mapped reads (FPKM) values were calculated Although strong and specific responses to HDAC inhibition using the robust normalization method of DESeq2, and variance have been consistently observed in synovial sarcoma, the mech- stabilized log fold changes were calculated using the regularized anism of apoptosis induction remains unclear. Advancing under- log transformation of DESeq2 (24). DESeq2 was used to deter- standing of this relationship may lead to improved targeted mine significance values for differential expression between trea- therapy development and improved trial design, as well as aid ted and untreated cells, specifying pairwise comparison of drug in further elucidating the important pathways implicated in effects across cell lines. A significance threshold of 0.01 was synovial sarcoma oncogenesis. In this study, we used next-gen- selected for adjusted P values. Gene enrichment analyses were eration sequencing analyses of six human synovial sarcoma cell performed for gene ontologies using PANTHER with Bonferroni lines to investigate the mechanisms of induced cell-cycle arrest correction (25), and for the MsigDB (molecular signatures data- and cell death in synovial sarcoma following HDAC inhibition by base) v5.2 (26) using the hypergeometric test. quisinostat, a relatively new, orally available HDAC inhibitor that Raw count data for the TCGA sarcoma dataset were obtained was the top hit in a synovial sarcoma drug screen (16). from Genomic Data Commons (https://gdc.cancer.gov/node/ 31). FPKM, log2 fold changes, and significance values were deter- Materials and Methods mined using DESeq2, comparing synovial sarcoma to all other sarcoma types. Cell culture and chemicals The following human synovial sarcoma cell lines were kindly Public microarray studies provided: SYO-1 (ref. 17; Dr. Akira Kawai, National Cancer Centre The gene expression ominibus was surveyed for studies of Hospital, Tokyo, Japan, 2004), FUJI (ref. 18; Dr. Kazuo Naga- HDAC inhibition at lethal doses in cancer cell lines, and five shima, Hokkaido University School of Medicine, Sapporo, Japan, studies on more recent microarray platforms were selected for 2003), YaFuss (19), and HS-SY-II (ref.20; Dr. Marc Ladanyi, comparison [prostatic adenocarcinoma: GSE74418, neuroblas- Memorial Sloan Kettering Cancer Centre, New York, NY, toma: GSE49158, NUT midline carcinoma: GSE18668, ovarian 2014), MoJo (ref. 21; Dr. K. Jones, University of Utah, Salt Lake carcinoma GSE53603, atypical teratoid rhabdoid tumor (AT/RT): City, UT, 2014), and Yamato-SS (ref. 22; Dr. K. Itoh, Osaka GSE37373]. Expression data were downloaded from the pro- Medical Center for Cancer and Cardiovascular Diseases, Japan, cessed data files supplied to Gene Expression Omnibus (GEO; 2013). Sarcoma cell lines were maintained in RPMI1640 medium series matrix files), and differential expression was determined supplemented with 10% FBS (Life Technologies) and the presence using uniform criteria for each study using Limma (27). Gene IDs of a disease-defining SS18-SSX fusion oncogene was confirmed by were mapped to the RNA-seq dataset using the supplied platform RT-PCR analysis. Mycoplasma testing was undertaken every six specifications. Expression data for GEO studies was ranked weeks by RT-PCR analysis and any cell lines found positive were according to log fold change of treatment versus control, and 2 discarded. All cells were grown at 37 C, 95% humidity, and 5% Gene Set Enrichment Analysis (GSEA version 2.2.3) was used to CO2. Pharmacologic