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The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcemic… Wang, Yemin; Chen, Shary Yuting; Karnezis, Anthony N.; Colborne, Shane; Santos, Nancy; Lang, Jessica; Hendricks, William P. D.; Orlando, Krystal; Yap, Damian; Kommoss, Friedrich; Bally, Marcel; Morin, Gregg B.; Hunstman, David G.; Trent, Jeffrey M.; Weissman, Bernard E.; Hunstman, David G. Apr 30, 2017

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		 1	The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcemic type  Running title: Targeting EZH2 in SCCOHT  Yemin Wang1, Shary Yuting Chen1, Anthony N. Karnezis1, Shane Colborne2, Nancy Dos Santos3, Jessica D. Lang4, William P.D. Hendricks4, Krystal A. Orlando5, Damian Yap1, Friedrich Kommoss1, Marcel B. Bally3, Gregg B. Morin3,6, Jeffrey M. Trent4, Bernard E. Weissman5, David G. Huntsman1,7,#   1Department of Pathology and Laboratory Medicine, University of British Columbia and Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada.  2Michael Smith Genome Science Centre, British Columbia Cancer Agency, Vancouver, BC, Canada.  3Department of Experimental Therapeutics, British Columbia Cancer Research Centre, Vancouver, BC, Canada. 4Division of Integrated Cancer Genomics, Translational Genomics Research Institute (TGen), Phoenix, AZ, USA.  5Department of Pathology and Laboratory Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA. . 6Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 7Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, 		 2	Canada  Corresponding author  David Huntsman, MD, FRCPC, FCCMG Dr. Chew Wei Memorial Professor of Gynaecologic Oncology, UBC Professor, Departments of Pathology and Lab Medicine and Obstetrics and Gynaecology, UBC Distinguished Scientist, Department of Molecular Oncology, BC Cancer Research Centre  #4111-675 West 10th Avenue, Vancouver, BC V5Z 1L3 Canada  Phone: 604.675.8205; Email: dhuntsma@bccancer.bc.ca  Conflict of interest: None of the authors report any conflicts of interest. Word Count for main text (limit 4000): 3984 		 3	Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodeling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodeling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumor samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells.  In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis and neuron-like differentiation. EPZ-6438 suppressed tumor growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease.   Keywords: SCCOHT, SWI/SNF, chromatin remodeling complex, differentiation, EZH2, SMARCA4, ovarian cancer  		 4	Introduction Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), a rare but highly aggressive ovarian malignancy with unknown cellular origin, occurs both sporadically and in families [1,2]. Unlike most common ovarian cancers, SCCOHT primarily affects women in their teens and twenties [3-5]. Surgical debulking followed by adjuvant chemotherapy is the mainstay of therapy for SCCOHT. However, recurrence is generally rapid, and the prognosis is dismal – about two-thirds of patients with advanced stage disease die within two years of diagnosis [5,6]. Therefore, more effective treatment options are urgently needed. Despite that Otte et al. have recently discovered c-Met inhibitors as potential targeted therapy agents for a subset of SCCOHT [7], the biology-driven therapeutic options for SCCOHT remain to be explored.  Several research teams, including ourselves, have independently identified inactivating, often homozygous or bi-allelic, mutations of the SMARCA4 gene in over 90% of SCCOHT cases [8-11], which leads to loss of SMARCA4 protein in the majority of SCCOHT tumors and cell lines [8-11]. Unlike common malignancies, no recurrent somatic, non-silent mutations besides those in SMARCA4 have been detected by paired exome or whole-genome sequencing analysis in SCCOHT [8-10,12]. Therefore, the inactivating mutations in SMARCA4 appear to be the primary driver in SCCOHT tumorigenesis and may help inform novel treatment strategies for SCCOHT. SMARCA4 is one of the two mutually exclusive ATPases of the SWI/SNF multi-subunit chromatin-remodeling complex, which uses ATP hydrolysis to destabilize histone-DNA interactions and mobilize nucleosomes. The SWI/SNF complex localizes near transcriptional regulatory elements and regions critical for chromosome organization to regulate the expression of many genes involved in cell cycle control, differentiation and chromosome organization [13,14]. Several subunits of the SWI/SNF complex, such as SMARCA4, SMARCB1, ARID1A, 		 5	PBRM1, are frequently mutated and inactivated in a variety of cancers [14-16]. This highlights the broader potential utility of effective targeted therapies for patients with a defective SWI/SNF complex. Recently, several studies reported that SMARCA4-deficient lung cancer cell lines relied on the activities of SMARCA2, the mutually exclusive ATPase, for proliferation [17,18], raising the possibility of selectively targeting SMARCA2 as therapeutic approaches for these patients. However, all SMARCA4-negative SCCOHT tumors and tumor-derived cell lines also lack the expression of SMARCA2 without apparent mutations in the SMARCA2 gene [19], indicating the need for developing different biologically informed treatment approaches for SCCOHT. The interplay between the SWI/SNF complex and the Polycomb repressive complex 2 (PRC2) was originally demonstrated through genetic studies in Drosophila [20]. Mouse studies revealed that tumorigenesis driven by SMARCB1 loss was ablated by the simultaneous loss of EZH2, the catalytic subunit of PRC2 that trimethylates lysine 27 of histone H3 (H3K27) to promote transcriptional silencing [21]. Therefore, EZH2 has emerged as a putative therapeutic target for SMARCB1-deficient malignant rhabdoid tumors (MRTs), ARID1A-deficient ovarian clear cell carcinomas, SMARCA4-deficient lung cancers and PBRM1-deficient renal cancers, although the non-catalytic activity of EZH2 was likely responsible for the therapeutic potential in some cases [21-23]. Therefore, we set out to address whether targeting EZH2 is a feasible strategy for treating SMARCA4-deficient SCCOHT. We discovered that EZH2 is abundantly expressed in SCCOHT and its inhibition robustly suppressed SCCOHT cell growth, induced apoptosis and neuron-like differentiation, and delayed tumor growth in mouse xenograft models of SCCOHT.  		 6	Materials and methods SCCOHT tissue microarray and immunohistochemistry SCCOHT tissue microarrays (TMA), as described previously [19], were cut at 4 µm thickness onto Superfrost+ glass slides, and stained for EZH2 (#612667, BD Transduction Laboratories, Mississauga, ON, Canada) on a Ventana Discovery XT autostainer (Ventana Medical Systems, Tucson, AZ, USA). The EZH2 staining was scored by a pathologist (A.N.K.) as negative (<1% of tumor cells showing definite, i.e. moderate to strong, nuclear staining), positive variable (1-50% of tumor cells) or positive diffuse (>50% of tumor cells). Cell culture BIN67, SCCOHT-1, COV434, and G401 cells were grown in DMEM/F-12 supplemented with 10% FBS. ES-2, RMG1, OVCAR-8, NOY1, OVISE and OVTOKO cells were cultured in RPMI supplemented with 10% FBS. All the cells were maintained at 37°C in a humidified 5% CO2-containing incubator. All cell lines have been certified by STR analysis, tested regularly for Mycoplasma and used for the study within six months of thawing. Proteomics Cells were lysed in 100mM HEPES buffer (pH 8.5) containing 1% SDS and 1x protease inhibitor cocktail (Roche). After chromatin degradation by benzonase, reduction and alkylation of disulfide bonds by dithiothreitol and iodoacetamide, samples were cleaned up and prepared for trypsin digestion using SP3-CTP method [24]. Briefly, proteins were digested for 14 hours at 37°C followed by SP3 beads removal. Tryptic peptides from each sample were individually labeled with TMT 10-plex labels, pooled and fractionated into 12 fractions by high pH RP-HPLC, desalted, orthogonally separated and analyzed using and Easy-nLC 1000 coupled to a Thermo Scientific Orbitrap Fusion mass spectrometer operating in MS3 mode. Raw MS data 		 7	were processed and peptide sequences were elucidated using Sequest HT in Proteome Discoverer software (v2.1.0.62), searching against the UniProt Human Proteome database.  Mouse xenografts Animal handling, care, and treatment procedures were performed according to guidelines approved by the Animal Care Committee of the University of British Columbia (A14-0290). Briefly, BIN67 (1x107 cells/mouse) or SCCOHT-1 cells (4x106 cells/mouse) were injected with a 1:1 mix of matrigel (Corning) in a final volume of 200 µl subcutaneously into the back of NRG (NOD.Rag1KO.IL2RγcKO) mice. Mice were randomized to treatment arms once the average tumor volume reached 100mm3. EPZ-6438 (ActiveBiochem) was formulated in 0.5% NaCMC with 0.1% Tween-80 in water. For the BIN67 xenograft model, mice were treated by oral administration of vehicle or EPZ-6438 (100 or 200 mg/kg) twice daily (BID, 0800/1600h) for eight days, halted for six days due to body weight loss in all groups, and then resumed with once daily (QD) dosing for additional three weeks or until the humane endpoint (i.e. tumor volume reached 800 mm3). For the SCCOHT-1 xenograft model, mice were treated by oral administration of vehicle or EPZ-6438 (200 mg/kg, QD) for three weeks or until reaching a humane endpoint. Tumor volume and mouse weight were measured thrice weekly. Tumor volume was calculated as length x (width)2 x 0.52. Statistical analysis The student's t test was used to evaluate the significant difference between two groups in all experiments except proteomics. The Peptide Expression Change Averaging (PECA) analysis [25] was performed for comparing the proteomic profiles of BIN67 cells treated with or without EPZ-6438 to generate signal log ratio values (log fold-change), p-values and false discovery rate (fdr) adjusted p-values (p.fdr) using peptide level signal values. The PECA analysis [25] was 		 8	performed for comparing the proteomic profiles of BIN67 cells treated with or without EPZ-6438. Survival curves and IC50 of drug treatment were determined by PRISM software. A p-value or p.fdr-value (for proteomics data) < 0.05 was considered significant.  Additional Material and Methods are available in the Supplemental Information online.  Results EZH2 is abundantly expressed in SCCOHT The poorly differentiated state of SCCOHT and the critical role of EZH2 in the maintenance of embryonic and tissue stem cells [26], together with the recently discovered antagonism between the SWI/SNF complex and EZH2 [23], imply that EZH2 may play a role in SCCOHT tumorigenesis. We first determined the expression levels of EZH2 in SCCOHT primary tumor samples and cell lines. Using gene expression profiles extracted from microarray data of four primary SCCOHT samples and two normal ovaries [19], we observed that only the expression of EZH2, but not other polycomb group (PcG) proteins, was significantly elevated in SCCOHT tumors compared to normal ovaries (Fig. 1A). Western blotting analysis demonstrated that two SCCOHT cell lines, BIN67 and SCCOHT-1, expressed EZH2 protein at a level comparable to or higher than that in several other ovarian cancer cell lines and the MRT cell line G401 (Fig. 1B). COV434, a cell line originally designated as a juvenile granulosa cell tumor cell line [27] but recently redefined as a SCCOHT cell line with dual deficiency of SMARCA4 and SMARCA2 (manuscript in preparation), also abundantly expressed EZH2 protein (Fig. 1B). Next, we performed EZH2 immunohistochemistry (IHC) on TMA of 24 primary tumors. Seventy-nine % (19/24) displayed strong diffuse EZH2 staining with variable staining in the remainder (Fig. 1C). 		 9	To test whether loss of SMARCA4 upregulates EZH2, we re-introduced SMARCA4 into SCCOHT cell lines. Western blotting revealed that EZH2 was substantially downregulated by SMARCA4 re-expression alongside a reduction in histone H3 lysine residue K27 trimethylation (H3K27me3) levels in all three SCCOHT cell lines (Fig. 1D). These data suggest that loss of SMARCA4 leads to EZH2 upregulation in SCCOHT.  SCCOHT cells are sensitive to EZH2 inhibition To determine whether SCCOHT relies on EZH2 for proliferation, we ablated the expression of EZH2 with two specific EZH2 shRNAs, either of which led to a significant reduction of H3K27me3 levels (Fig. 2A). Depletion of EZH2 significantly inhibited the growth of BIN67, COV434 and SCCOHT-1 cells, but not of ES-2 cells, an ovarian cancer cell line with intact SMARCA4 and SMARCA2 (Fig. 2B), suggesting that EZH2 is a therapeutic target specifically in SMARCA4/A2-deficient SCCOHT cells. Next, we determined whether SCCOHT cell lines are responsive to the inhibition of EZH2 catalytic activity with two pharmaceutical inhibitors, GSK126 and EPZ-6438, both of which are being tested in clinical trials. Western blotting analysis confirmed that each inhibitor potently suppressed histone H3K27Me3 levels in SCCOHT cells at either 0.1 or 0.5 µM (Fig. 2C and 2D). In a six-day drug treatment assay, three SCCOHT cell lines were significantly more sensitive to either GSK126 or EPZ-6438 than other ovarian cancer cell lines tested (Fig. 2E and 2F). Among three SCCOHT cell lines, SCCOHT-1 cells were about 5 or 10 fold more sensitive than the other two lines and the MRT cell line G401 to the treatment of GSK126 or EPZ-6438, respectively (Fig. 2E). Concordant with the drastic anti-proliferative effects observed in MRT lines after a two-week exposure to EPZ-6438 [28], the growth of BIN67 and COV434, which 		 10	displayed lower sensitivity than SCCOHT-1, was strongly suppressed by prolonged exposure to each inhibitor at either 1µM or below (Supplemental Fig. S1). However, neither SMARCA4- nor SMARCA4/SMARCA2-dual-deficient lung tumor cell lines were as sensitive to EZH2 inhibitors as the SCCOHT cell lines (Supplemental Fig. S2).    EPZ-6438 suppresses tumor growth in a mouse SCCOHT xenograft model Next, we employed mouse subcutaneous xenografts of SCCOHT cells to evaluate in vivo efficacy of EPZ-6438, which, of the two inhibitors, displayed more selectivity in SCCOHT cells in vitro (Fig. 2D). First, we dosed BIN67 xenograft-bearing mice with EPZ-6438 (100 mg/kg or 200 mg/kg, n=10) twice a day [28-30]. BIN67 xenografts harvested three hours following the EPZ-6438 treatment displayed a significantly lower amount of histone H3K27me3 than vehicle-treated tumors (Supplemental Fig. S3), demonstrating suppression of EZH2 enzymatic activity by EPZ-6438. Accordingly, EPZ-6438 at either 100 or 200 mg/kg suppressed tumor growth compared to those treated with vehicle (Fig. 3A, day 28, P <0.01, P <0.01, respectively). Unexpectedly, mice from all the treatment groups displayed significant weight loss due to dehydration (Supplemental Fig. S4A). After stopping dosing for 6 days to allow recovery from dehydration, the tumors in mice exposed to EPZ-6438 grew back from about 100 mm3 to 300 mm3 (Fig. 3A, day 34). To further test whether the once daily treatment with EPZ-6438 was effective in suppressing tumor growth, we resumed treatment with a daily single dose schedule until mice reached humane endpoints. This schedule at 100 mg/kg failed to delay tumor growth (Fig. 3A, days 34-48), while single daily dosing with 200 mg/kg EPZ-6438 retarded growth after 10 days (Fig. 3A). Though overall survival did not differ between two dosages, both dosages significantly improved the median survival of mice to humane endpoint from 37 days (vehicle) to 46 days 		 11	(100mg/kg) and 48 days (200mg/kg) mainly due to the benefit from the first week of twice daily dosing (Fig. 3B, P = 0.0036 and 0.0017, respectively).   Next, we tested the efficacy of EPZ-6438 in the SCCOHT-1 xenograft model. Since SCCOHT-1 cells displayed much higher sensitivity to EZH2 inhibitors than any other cell lines (Fig. 2E), we dosed the mice with 200 mg/kg EPZ-6438 once daily (n=6), which produced only mild effect on the mouse body weight (Supplemental Fig. S4B). Tumor growth was effectively suppressed by the once daily treatment of EPZ-6438 at 200 mg/kg wherein the tumor growth doubling time (tumor volume from 150 mm3 to 300 mm3) increased from 5 days (vehicle) to 17 days (EPZ-6438) (Fig. 3C). The average tumor weight was also reduced by 50% in the EPZ-6438-treated group compared to the vehicle-treated group at the end of three weeks of treatment (Fig. 3D, p < 0.05). These data suggest that once daily treatment of EPZ-6438 at 200 mg/kg significantly slowed down the growth of SCCOHT-1 xenografts.  Effects of EPZ-6438 on the proteome of BIN67 cells To identify the molecular mechanisms driving growth inhibition caused by EZH2 inhibitors in SCCOHT cells, we determined the proteomic profiles of BIN67 cells treated with EPZ-6438 or vehicle using mass spectrometry. Unsupervised hierarchical clustering resulted in clustering based on the treatment conditions (Supplemental Fig. S5) with approximately 8.4% (571/6768) and 7.4% (498/6768) being significantly upregulated or downregulated, respectively, in BIN67 cells treated with 1 µM EPZ-6438 for 7 days versus those treated with DMSO (Fig. 4A and Supplemental Table S1 and S2, pfdr<0.05 and log2FC>mean+SD or <mean-SD). Ingenuity pathway analysis (IPA) of the significantly altered proteins by EPZ-6438 revealed a significant enrichment of proteins involved in biological functions, such as “Cell cycle”, “Cellular Assembly 		 12	and Organization” and “Cellular development” (Fig. 4B, Supplemental Table S3). Particularly, both “development of neurons” and several processes related to neuronal development, such as “formation of cellular protrusions”, “microtubule dynamics” and “neuritogeneis”, were predicted to be enhanced significantly in EPZ-6438-treated BIN67 cells (Fig. 4C, Supplemental Table S3, z>2). Clustering of the enriched proteins confirmed that many of the identified proteins involved in microtubule dynamics, formation of celluar protrusions and organization of cytoskeleton or cytoplasm were also involved in development of neurons (Supplemental Fig. S6), supporting their engagement in neuron development. These data suggest that EPZ-6438 not only altered the cell cycle of SCCOHT cells, but also triggered significant changes in cell organization and assembly, leading to neuron-like differentiation.    EPZ-6438 induces SCCOHT cell cycle arrest and apoptosis  Next, we used flow cytometry to validate the effect of EPZ-6438 on cell cycle distribution of SCCOHT cells. FACS analysis demonstrated that 1 µM EPZ-6438 treatment induced cell cycle arrest at G1 in a time-dependent manner in BIN67 and COV434 cells (Fig. 5A), and also triggered cell death (sub-G1 population) beginning at 7 days after drug exposure in both BIN67 and COV434 cell lines, consistent with a mild induction of apoptosis predicted by proteomic data (Supplemental Table S4). In contrast, 1 µM EPZ-6438 induced cell death more rapidly (3 days) and potently in SCCOHT-1 cells (Fig. 5A), consistent with the lower IC50 in this cell line than others (Fig. 2E). To precisely monitor the induction of apoptosis, we measured the activation of caspase-3/7 through fluorescent microscopy coupled with live cell imaging (Fig. 5B). This revealed that EPZ-6438 rapidly induced cell apoptosis in SCCOHT-1 cells after 48 hours of EPZ-6438 treatment at either 0.3 or 1 µM, whereas apoptosis in BIN67 and COV434 cells increased 		 13	gradually beginning at about 120 hours of exposure to 1 µM EPZ-6438 (Fig. 5C). Next, we employed western blotting to determine the expression of some cell cycle and apoptosis regulating genes that were significantly altered by EPZ-6438 in BIN67 cells by proteomic profiling. We confirmed that the expression of CDKN1A (p21) and BAD (Log2FC =0 .7 and 1.4, respectively, Supplemental Table S1) was gradually induced by EPZ-6438, while that of Myc (Log2 FC = -0.9, Supplemental Table S2) was potently down-regulated by EPZ-6438 in all three SCCOHT cell lines (Fig. 5D). In contrast, the expression of CDKN2A (p16) (Log2 FC = 0.2, Supplemental Table S1), which is known to play an important role in EZH2 inhibitor-triggered growth arrest [31], was only significantly induced upon EPZ-6438 treatment in SCCOHT-1 cells (Fig. 5D).   EPZ-6438 induces neuron-like differentiation in SCCOHT cells In agreement with the prediction of induced neuron development in BIN67 cells from proteomic profiling, the surviving SCCOHT cells displayed a neuron-like morphology after prolonged exposure to 1 µM EPZ-6438 (Fig. 6A). In contrast, SCCOHT cells exposed to cytotoxic agents, such as cisplatin, etoposide, and paclitaxel, displayed no morphology change other than fragmentation during cell death (Supplemental Fig. S7). Immunofluorescence confirmed that the expression of MAP2, a specific marker of neuronal differentiation [32], was increased upon EPZ-6438 treatment in a time-dependent manner in BIN67 cells (Fig. 6B), consistent with increased expression with treatment observed in the proteomic data (Log2 FC = 2.2, Supplementary Table S2).  Western blot analysis further confirmed that the expression levels of two neuronal markers (MAP2 and TUBB3 (βIII tubulin), Log2FC=1.2, Supplementary Table S2) were induced by EPZ-6438 treatment (Fig. 6C). In agreement with the induction of differentiation, the expression 		 14	of EZH2 (Log2FC=-0.4, Supplementary Table S1) also dropped significantly upon EPZ-6438 treatment in a time-dependent manner (Fig. 6C). Therefore, our data show that unlike cytotoxic agents, EZH2 inhibitors can induce expression of markers of neuronal differentiation of SCCOHT cells.    Discussion Transformation of normal cells requires acquisition of hallmark characteristics of cancer including survival and proliferation even in the presence of counteracting signals. These features usually include extensive rewiring of cellular signaling networks driven by mutations and deregulation of oncogenes and tumor suppressors [33], leading to a strict reliance on either an oncogenic driver event (oncogene addiction) or the activity of certain gene products that are not essential in normal cells (non-oncogene addiction or synthetic lethality) [34]. Our findings suggest that inactivation of SMARCA4 in SCCOHT’s unknown precursor cells may rewire their cellular signaling network to be dependent on the catalytic activity of the histone methyltransferase EZH2 in transcriptional repression. In support of this notion, previous studies have shown that SMARCA4 and the associated SWI/SNF chromatin-remodeling complex can suppress EZH2 either directly [21] or through repression of E2F transcription factors [35-37].  Concordantly, re-expression of SMARCA4 in SCCOHT cell lines lowered the expression of EZH2 and reduced the global level of histone H3K27me3, suggesting that SMARCA4 loss may promote SCCOHT tumorigenesis, at least partially, through the direct up-regulation of EZH2 expression.  In addition to this work, several other studies have demonstrated the requirement for the methyltransferase activity of EZH2 in cancers with a defective subunit of the SWI/SNF complex.  		 15	However, two ARID1A-deficient ovarian clear cell carcinoma cell lines (OVISE and OVTOKO) and several lung cancer cell lines with SMARCA4 deficiency or SMARCA4/SMARCA2 dual deficiency did not respond or responded poorly to EPZ-6438 treatment in our study (Fig. 2C and Supplemental Fig. S3). Therefore, the absence of subunits of the SWI/SNF complex alone does not predict sensitivity to inhibition of EZH2 catalytic activity. Whether the efficacy of EZH2 inhibitors depends upon additional genetic or epigenetic features of the tumor remains unclear.  Of note, both SCCOHT and MRT cell lines [28] that respond to EZH2 inhibitors lack the core subunits of SWI/SNF complex (either SMARCA4/A2 or SMARCA2/SMARCB1 deficiency), while ARID1A-deficient ovarian clear cell carcinoma cells usually retain an active complex that includes ARID1B [38].  Similarly, most SMARCA4-deficient ovarian and lung carcinomas retain SMARCA2 expression and its associated chromatin remodeling activity. Accordingly, it has been reported that ARID1A-deficient ovarian cancer and SMARCA4-deficient lung cancer depend on ARID1B and SMARCA2, respectively, for maintaining their oncogenic property [17,18,38]. Furthermore, most tumors with SMARCA4 or ARID1A loss usually display a higher mutation burden in comparison to the minimally disturbed genomes of either SCCOHT or MRT [8,39-41].  The lack of additional mutations in the latter tumors implicates a dependence upon other epigenetic changes such as EZH2 overexpression.  Thus, SMARCA4 inactivation in the absence of SMARCA2 may rewire the cellular signaling network to be dependent on EZH2-mediated oncogenesis while additional mutations possibly negate this requirement in other cancers.  Although the potency of EZH2 inhibitors has been demonstrated in several cancer types, the molecular mechanisms are still not well understood. EZH2 is the catalytic component of the PRC2 complex that mediates the transcriptional repression of targets by trimethylation of histone H3 at lysine residue 27 in their promoters. Therefore, the PRC2 complex could promote 		 16	tumorigenesis by specifically repressing tumor-suppressor genes, including the major tumor suppressor locus CDKN2A, through recruitment of DNA methyltransferases. Accordingly, treatment with EZH2 inhibitors can rescue the expression of p16 in cancer cells, such as MRT cells and leukemia stem cells [28,42]. Unlike MRT cells, in only one of the three SCCOHT cell lines (SCCOHT-1) was p16 expression altered upon EPZ-6438 treatment. The other two cell lines expressed substantial amounts of p16, which were not further induced upon the EPZ-6438 treatment. Therefore, a different mechanism may underlie the efficacy of EPZ-6438 in the BIN67 and COV434 cell lines.  Proteome analysis revealed that EPZ-6438 altered about 15% of the proteome in BIN67 cells with a significant enrichment of proteins involved in cell cycle control and development of neurons. These results implicate these pathways as the major biological events behind the cellular response to EZH2 inhibitors in SCCOHT cells. Particularly, we discovered that the expression of several cell cycle control genes, such as Myc, were repressed by EPZ-6438. Although the PRC2 complex may upregulate the expression of these genes indirectly through suppressing the negative regulators of these genes, it has been suggested that the transcription of Myc can be activated directly by the PRC2 complex in glioblastoma cancer stem cells [43]. Given that Myc activation is a hallmark of tumor initiation and maintenance, suppression of Myc may have a significant contribution to the efficacy of EZH2 inhibitors in SCCOHT and other cancers, such as MRT and glioblastoma [43].  Furthermore, we also observed a time-dependent induction of BAD pro-apoptotic protein in three SCCOHT cells following EPZ-6438 treatment. By inactivating the function of the anti-apoptotic proteins Bcl-2 and Bcl-xl, BAD can promote apoptosis [44]. Our data together with the previous study showing that BAD was up-regulated upon EZH2 depletion by shRNA in lung cancer cell lines [45] suggest that it may play a key role in EZH2 inhibitor-		 17	triggered apoptosis. In addition to causing cell cycle arrest and apoptosis, prolonged exposure to EZH2 inhibitors also drove the differentiation of SCCOHT cells towards the neuronal lineage. Consistent with the neuron-like differentiation, EPZ-6438 caused a late induction of p21 (Fig. 4F), a cyclin kinase inhibitor that may drive neural precursor cells into cycle exit and differentiation [46]. It will be interesting to determine whether depletion of p21 can prevent the neuron-like differentiation caused by EZH2 inhibitors.  A neuron-like morphology change has also been reported in G401 cells upon EPZ-6438 treatment [28]. Both genomic analysis and mouse transgenic models have provided evidence that some MRT may arise from neural precursor cells [41,47]. Interestingly, some studies suggest that SCCOHT is the MRT of ovary. Therefore, our present finding, that EZH2 inhibition induced markers of neuronal differentiation of SCCOHT, suggest that SCCOHT may develop from multi-potent stem cells inside the ovary with the capability of undergoing neuronal differentiation.  In support of this model, extensive IHC analysis of multiple sections of two SCCOHT cases previously revealed rare foci of immature teratoma [11], which mainly contain primitive neuroepithelium in their immature region [48]. Future studies are therefore needed to explore the possible cellular origin of SCCOHT from neural precursor cells.  SCCOHT is a rare but extremely aggressive disease for young women.  No effective treatment strategies have been developed for fighting this lethal disease. In the present study, we provide support that the methyltransferase EZH2 is universally expressed in SCCOHT and may serve as a potential therapeutic option for young women with this deadly disease.  Importantly, Epizyme pharmaceutics has recently launched a phase 1 clinical trail for evaluating the toxicity of EPZ-6438 in solid tumors with SMARCB1 or SMARCA4 deficiency (clinicaltrials.gov).  Their interim results showed that EPZ-6438 (Tazemetostat) was well tolerated and elicited a 		 18	partial response by RECIST criteria in two SCCOHT patients previously treated with chemotherapy (www.epizyme.com).  Therefore, identifying other targeted therapy strategies and determining the efficacy of combining EZH2 inhibitors with other putative targeted therapies for SCCOHT treatment remains a high priority.  Acknowledgements We acknowledge for the technical support from Sarah Maines-Bandiera, Winnie Yang, Christine Chow, Nicole Wretham, Dana Masin, Hong Yan, Jenna Rawji and Chris Ke-dong Wang. We thank Drs. Barbara Vanderhyden and Ralf Hass for providing BIN67 and SCCOHT-1 cells, respectively. This work was supported by research funds from the Canadian Cancer Society Research Institute (#703458, to D.G.H.) and the National Institute of Health (1R01CA195670-01, to D.G.H., J.T. and B.W.), the Terry Fox Research Institute Initiative New Frontiers Program in Cancer (D.G.H.), the British Columbia Cancer Foundation, the Marsha Rivkin Center for Ovarian Cancer Research, the Ovarian Cancer Alliance of Arizona, the Small Cell Ovarian Cancer Foundation, and philanthropic support to the TGen Foundation.  Statement of author contributions Y.W. designed and performed experiments, analyzed data and wrote the manuscript. S.Y.C. performed experiments and analyzed data. A.N.K. performed IHC, analyzed data and edited the manuscript. S.C. performed mass spectrometry experiments, analyzed data and wrote the manuscript. N.D.S helped the design of xenograft studies and analyzed data. J.L., W.P.D.H., K.A.O., B.E.W., G.B.M. and J.F.T. provided thoughtful discussion and edited the manuscript. 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(A) The expression of PRC2 complex proteins in three SCCOHT versus two normal ovaries were extracted out from our previous Agilent microarray analyses (GE access No.: GSE49887 and GSE66434). (B) The expression of EZH2 protein in multiple cell lines was analyzed by Western blotting analysis. (C) The expression of EZH2 protein in primary SCCOHT samples was determined using immunohistochemistry on TMA. Representative images of strong or variable staining are shown. (D) SCCOHT cell lines were infected with lentivirus expressing either GFP or SMARCA4. Cells were harvested 72 hours post infection for determining the effect of SMARCA4 re-expression on EZH2 and histone H3 K27me3 by Western blotting. * P<0.001.  Figure 2. SCCOHT cells are sensitive to EZH2 depletion and suppression. (A, B) Cells were infected with lentivirus expressing control scramble shRNA or EZH2 shRNA followed by puromycin selection for 48 hours. Cells were then reseeded in 24-well plates for 6 days before being fixed and quantitated by crystal violet staining assay. (C, D) Cells were treated as indicated for 3 days for Western blot analysis of histone H3K27me3 level. (E) Cells were seeded in 96-well plates, treated with EZH2 inhibitors GSK126 or EPZ-6438 at indicated doses and incubated for 6 days and measured for cell survival by crystal violet assay. (F) The IC50s of cell lines to EZH2 inhibitors in (E) were compared between SCCOHT lines and other ovarian lines. Note: 20µM was assigned to the cell lines that were not responsive to EZH2 inhibitors at 10µM. * P<0.01, ** P<0.001  		 24	Figure 3. In vivo efficacy of EPZ-6438 in BIN67 mouse xenograft model. (A-B) The efficacy of EPZ-6438 was evaluated in BIN67-derived mouse subcutaneous xenograft model. Tumor volume and the overall survival of each treated group until the study endpoint (see Material and Methods for details) were plotted against the days post cell inoculation, respectively. (C, D) The efficacy of EPZ-6438 was evaluated in SCCOHT-1-derived mouse subcutaneous xenograft model. Tumor volume of either vehicle or 200 mg/kg EPZ-6438-treated group was plotted against the days post cell inoculation (C). Final tumor weight was determined and compared between two treated groups (D). * P<0.05, ** P<0.01, *** P<0.001  Figure 4. The effect of EPZ-6438 on the proteome of BIN67 cells. (A) Volcano plot of the proteome of BIN67 cells exposed to EPZ-6438 or vehicle. Cells were treated with either DMSO or 1µM EPZ-6438 for 7 days and then processed for proteomic profiling. Peptide data were subjected to PECA analysis for identification of significantly altered proteins (p.fdr<0.05 and Log2FC>mean+SD or <mean-SD). (B) IPA analysis of significantly altered proteins caused by EPZ-6438 identified top affected biological functions by EPZ-6438 treatment. Any biological function with an activation z-score greater than 2 or less than -2 was predicted to be significantly increased or decreased by IPA analysis. (C) IPA analysis identified significantly increased cellular activities related to neuronal development. (D) Clustering analysis of proteins in significantly altered biological functions.  Figure 5. EPZ-6438 induces SCCOHT cell cycle arrest and apoptosis. (A) FACS analysis of cell cycle profiles in SCCOHT cells after DMSO or 1µM EPZ-6438 treatment for the shown times. (B, C) SCCOHT cell lines were treated with 1µM EPZ-6438 and cultured in the presence 		 25	of a cell-permeable fluorescent dye for monitoring activated caspase-3/7 activity with IncuCyte live cell imaging system (see Materials and Methods). Apoptotic index was calculated by dividing the overall fluorescent object counts to cell numbers under each condition and plotted over incubation time. (D) The effects of EPZ-6438 (BIN67/COV434: 1µM; SCCOHT-1: 0.25µM) on the expression of Myc, BAD, p16 and p21 proteins was determined by western blotting.   Figure 6. EPZ-6438 induces neuronal differentiation in SCCOHT cells. (A) Morphology of SCCOHT cells after prolonged exposure to EPZ-6438. Cells were treated with EPZ-6438 at either 1µM (BIN67/COV434) or 0.25µM (SCCOHT-1) for 12 days and characterized by phase contrast microscopy. (B) BIN67 cells were fixed and immunostained for MAP2, a selective neuronal marker. (C) Western blotting for neuronal proteins from SCCOHT cells treated with EPZ-6438 at either 1µM (BIN67/COV434) or 0.25µM (SCCOHT-1) for the days shown in the panel. Vinculin serves as a loading control.      		 1	The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcemic type  Running title: Targeting EZH2 in SCCOHT  Yemin Wang1, Shary Yuting Chen1, Anthony N. Karnezis1, Shane Colborne2, Nancy Dos Santos3, Jessica D. Lang4, William P.D. Hendricks4, Krystal A. Orlando5, Damian Yap1, Friedrich Kommoss1, Marcel B. Bally3, Gregg B. Morin3,6, Jeffrey M. Trent4, Bernard E. Weissman5, David G. Huntsman1,7,#   1Department of Pathology and Laboratory Medicine, University of British Columbia and Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada.  2Michael Smith Genome Science Centre, British Columbia Cancer Agency, Vancouver, BC, Canada.  3Department of Experimental Therapeutics, British Columbia Cancer Research Centre, Vancouver, BC, Canada. 4Division of Integrated Cancer Genomics, Translational Genomics Research Institute (TGen), Phoenix, AZ, USA.  5Department of Pathology and Laboratory Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA. . 6Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 7Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, 		 2	Canada  Corresponding author  David Huntsman, MD, FRCPC, FCCMG Dr. Chew Wei Memorial Professor of Gynaecologic Oncology, UBC Professor, Departments of Pathology and Lab Medicine and Obstetrics and Gynaecology, UBC Distinguished Scientist, Department of Molecular Oncology, BC Cancer Research Centre  #4111-675 West 10th Avenue, Vancouver, BC V5Z 1L3 Canada  Phone: 604.675.8205; Email: dhuntsma@bccancer.bc.ca  Conflict of interest: None of the authors report any conflicts of interest. Word Count for main text (limit 4000): 3984 		 3	Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodeling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodeling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumor samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells.  In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis and neuron-like differentiation. EPZ-6438 suppressed tumor growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease.   Keywords: SCCOHT, SWI/SNF, chromatin remodeling complex, differentiation, EZH2, SMARCA4, ovarian cancer  		 4	Introduction Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), a rare but highly aggressive ovarian malignancy with unknown cellular origin, occurs both sporadically and in families [1,2]. Unlike most common ovarian cancers, SCCOHT primarily affects women in their teens and twenties [3-5]. Surgical debulking followed by adjuvant chemotherapy is the mainstay of therapy for SCCOHT. However, recurrence is generally rapid, and the prognosis is dismal – about two-thirds of patients with advanced stage disease die within two years of diagnosis [5,6]. Therefore, more effective treatment options are urgently needed. Despite that Otte et al. have recently discovered c-Met inhibitors as potential targeted therapy agents for a subset of SCCOHT [7], the biology-driven therapeutic options for SCCOHT remain to be explored.  Several research teams, including ourselves, have independently identified inactivating, often homozygous or bi-allelic, mutations of the SMARCA4 gene in over 90% of SCCOHT cases [8-11], which leads to loss of SMARCA4 protein in the majority of SCCOHT tumors and cell lines [8-11]. Unlike common malignancies, no recurrent somatic, non-silent mutations besides those in SMARCA4 have been detected by paired exome or whole-genome sequencing analysis in SCCOHT [8-10,12]. Therefore, the inactivating mutations in SMARCA4 appear to be the primary driver in SCCOHT tumorigenesis and may help inform novel treatment strategies for SCCOHT. SMARCA4 is one of the two mutually exclusive ATPases of the SWI/SNF multi-subunit chromatin-remodeling complex, which uses ATP hydrolysis to destabilize histone-DNA interactions and mobilize nucleosomes. The SWI/SNF complex localizes near transcriptional regulatory elements and regions critical for chromosome organization to regulate the expression of many genes involved in cell cycle control, differentiation and chromosome organization [13,14]. Several subunits of the SWI/SNF complex, such as SMARCA4, SMARCB1, ARID1A, 		 5	PBRM1, are frequently mutated and inactivated in a variety of cancers [14-16]. This highlights the broader potential utility of effective targeted therapies for patients with a defective SWI/SNF complex. Recently, several studies reported that SMARCA4-deficient lung cancer cell lines relied on the activities of SMARCA2, the mutually exclusive ATPase, for proliferation [17,18], raising the possibility of selectively targeting SMARCA2 as therapeutic approaches for these patients. However, all SMARCA4-negative SCCOHT tumors and tumor-derived cell lines also lack the expression of SMARCA2 without apparent mutations in the SMARCA2 gene [19], indicating the need for developing different biologically informed treatment approaches for SCCOHT. The interplay between the SWI/SNF complex and the Polycomb repressive complex 2 (PRC2) was originally demonstrated through genetic studies in Drosophila [20]. Mouse studies revealed that tumorigenesis driven by SMARCB1 loss was ablated by the simultaneous loss of EZH2, the catalytic subunit of PRC2 that trimethylates lysine 27 of histone H3 (H3K27) to promote transcriptional silencing [21]. Therefore, EZH2 has emerged as a putative therapeutic target for SMARCB1-deficient malignant rhabdoid tumors (MRTs), ARID1A-deficient ovarian clear cell carcinomas, SMARCA4-deficient lung cancers and PBRM1-deficient renal cancers, although the non-catalytic activity of EZH2 was likely responsible for the therapeutic potential in some cases [21-23]. Therefore, we set out to address whether targeting EZH2 is a feasible strategy for treating SMARCA4-deficient SCCOHT. We discovered that EZH2 is abundantly expressed in SCCOHT and its inhibition robustly suppressed SCCOHT cell growth, induced apoptosis and neuron-like differentiation, and delayed tumor growth in mouse xenograft models of SCCOHT.  		 6	Materials and methods SCCOHT tissue microarray and immunohistochemistry SCCOHT tissue microarrays (TMA), as described previously [19], were cut at 4 µm thickness onto Superfrost+ glass slides, and stained for EZH2 (#612667, BD Transduction Laboratories, Mississauga, ON, Canada) on a Ventana Discovery XT autostainer (Ventana Medical Systems, Tucson, AZ, USA). The EZH2 staining was scored by a pathologist (A.N.K.) as negative (<1% of tumor cells showing definite, i.e. moderate to strong, nuclear staining), positive variable (1-50% of tumor cells) or positive diffuse (>50% of tumor cells). Cell culture BIN67, SCCOHT-1, COV434, and G401 cells were grown in DMEM/F-12 supplemented with 10% FBS. ES-2, RMG1, OVCAR-8, NOY1, OVISE and OVTOKO cells were cultured in RPMI supplemented with 10% FBS. All the cells were maintained at 37°C in a humidified 5% CO2-containing incubator. All cell lines have been certified by STR analysis, tested regularly for Mycoplasma and used for the study within six months of thawing. Proteomics Cells were lysed in 100mM HEPES buffer (pH 8.5) containing 1% SDS and 1x protease inhibitor cocktail (Roche). After chromatin degradation by benzonase, reduction and alkylation of disulfide bonds by dithiothreitol and iodoacetamide, samples were cleaned up and prepared for trypsin digestion using SP3-CTP method [24]. Briefly, proteins were digested for 14 hours at 37°C followed by SP3 beads removal. Tryptic peptides from each sample were individually labeled with TMT 10-plex labels, pooled and fractionated into 12 fractions by high pH RP-HPLC, desalted, orthogonally separated and analyzed using and Easy-nLC 1000 coupled to a Thermo Scientific Orbitrap Fusion mass spectrometer operating in MS3 mode. Raw MS data 		 7	were processed and peptide sequences were elucidated using Sequest HT in Proteome Discoverer software (v2.1.0.62), searching against the UniProt Human Proteome database.  Mouse xenografts Animal handling, care, and treatment procedures were performed according to guidelines approved by the Animal Care Committee of the University of British Columbia (A14-0290). Briefly, BIN67 (1x107 cells/mouse) or SCCOHT-1 cells (4x106 cells/mouse) were injected with a 1:1 mix of matrigel (Corning) in a final volume of 200 µl subcutaneously into the back of NRG (NOD.Rag1KO.IL2RγcKO) mice. Mice were randomized to treatment arms once the average tumor volume reached 100mm3. EPZ-6438 (ActiveBiochem) was formulated in 0.5% NaCMC with 0.1% Tween-80 in water. For the BIN67 xenograft model, mice were treated by oral administration of vehicle or EPZ-6438 (100 or 200 mg/kg) twice daily (BID, 0800/1600h) for eight days, halted for six days due to body weight loss in all groups, and then resumed with once daily (QD) dosing for additional three weeks or until the humane endpoint (i.e. tumor volume reached 800 mm3). For the SCCOHT-1 xenograft model, mice were treated by oral administration of vehicle or EPZ-6438 (200 mg/kg, QD) for three weeks or until reaching a humane endpoint. Tumor volume and mouse weight were measured thrice weekly. Tumor volume was calculated as length x (width)2 x 0.52. Statistical analysis The student's t test was used to evaluate the significant difference between two groups in all experiments except proteomics. The Peptide Expression Change Averaging (PECA) analysis [25] was performed for comparing the proteomic profiles of BIN67 cells treated with or without EPZ-6438 to generate signal log ratio values (log fold-change), p-values and false discovery rate (fdr) adjusted p-values (p.fdr) using peptide level signal values. The PECA analysis [25] was 		 8	performed for comparing the proteomic profiles of BIN67 cells treated with or without EPZ-6438. Survival curves and IC50 of drug treatment were determined by PRISM software. A p-value or p.fdr-value (for proteomics data) < 0.05 was considered significant.  Additional Material and Methods are available in the Supplemental Information online.  Results EZH2 is abundantly expressed in SCCOHT The poorly differentiated state of SCCOHT and the critical role of EZH2 in the maintenance of embryonic and tissue stem cells [26], together with the recently discovered antagonism between the SWI/SNF complex and EZH2 [23], imply that EZH2 may play a role in SCCOHT tumorigenesis. We first determined the expression levels of EZH2 in SCCOHT primary tumor samples and cell lines. Using gene expression profiles extracted from microarray data of four primary SCCOHT samples and two normal ovaries [19], we observed that only the expression of EZH2, but not other polycomb group (PcG) proteins, was significantly elevated in SCCOHT tumors compared to normal ovaries (Fig. 1A). Western blotting analysis demonstrated that two SCCOHT cell lines, BIN67 and SCCOHT-1, expressed EZH2 protein at a level comparable to or higher than that in several other ovarian cancer cell lines and the MRT cell line G401 (Fig. 1B). COV434, a cell line originally designated as a juvenile granulosa cell tumor cell line [27] but recently redefined as a SCCOHT cell line with dual deficiency of SMARCA4 and SMARCA2 (manuscript in preparation), also abundantly expressed EZH2 protein (Fig. 1B). Next, we performed EZH2 immunohistochemistry (IHC) on TMA of 24 primary tumors. Seventy-nine % (19/24) displayed strong diffuse EZH2 staining with variable staining in the remainder (Fig. 1C). 		 9	To test whether loss of SMARCA4 upregulates EZH2, we re-introduced SMARCA4 into SCCOHT cell lines. Western blotting revealed that EZH2 was substantially downregulated by SMARCA4 re-expression alongside a reduction in histone H3 lysine residue K27 trimethylation (H3K27me3) levels in all three SCCOHT cell lines (Fig. 1D). These data suggest that loss of SMARCA4 leads to EZH2 upregulation in SCCOHT.  SCCOHT cells are sensitive to EZH2 inhibition To determine whether SCCOHT relies on EZH2 for proliferation, we ablated the expression of EZH2 with two specific EZH2 shRNAs, either of which led to a significant reduction of H3K27me3 levels (Fig. 2A). Depletion of EZH2 significantly inhibited the growth of BIN67, COV434 and SCCOHT-1 cells, but not of ES-2 cells, an ovarian cancer cell line with intact SMARCA4 and SMARCA2 (Fig. 2B), suggesting that EZH2 is a therapeutic target specifically in SMARCA4/A2-deficient SCCOHT cells. Next, we determined whether SCCOHT cell lines are responsive to the inhibition of EZH2 catalytic activity with two pharmaceutical inhibitors, GSK126 and EPZ-6438, both of which are being tested in clinical trials. Western blotting analysis confirmed that each inhibitor potently suppressed histone H3K27Me3 levels in SCCOHT cells at either 0.1 or 0.5 µM (Fig. 2C and 2D). In a six-day drug treatment assay, three SCCOHT cell lines were significantly more sensitive to either GSK126 or EPZ-6438 than other ovarian cancer cell lines tested (Fig. 2E and 2F). Among three SCCOHT cell lines, SCCOHT-1 cells were about 5 or 10 fold more sensitive than the other two lines and the MRT cell line G401 to the treatment of GSK126 or EPZ-6438, respectively (Fig. 2E). Concordant with the drastic anti-proliferative effects observed in MRT lines after a two-week exposure to EPZ-6438 [28], the growth of BIN67 and COV434, which 		 10	displayed lower sensitivity than SCCOHT-1, was strongly suppressed by prolonged exposure to each inhibitor at either 1µM or below (Supplemental Fig. S1). However, neither SMARCA4- nor SMARCA4/SMARCA2-dual-deficient lung tumor cell lines were as sensitive to EZH2 inhibitors as the SCCOHT cell lines (Supplemental Fig. S2).    EPZ-6438 suppresses tumor growth in a mouse SCCOHT xenograft model Next, we employed mouse subcutaneous xenografts of SCCOHT cells to evaluate in vivo efficacy of EPZ-6438, which, of the two inhibitors, displayed more selectivity in SCCOHT cells in vitro (Fig. 2D). First, we dosed BIN67 xenograft-bearing mice with EPZ-6438 (100 mg/kg or 200 mg/kg, n=10) twice a day [28-30]. BIN67 xenografts harvested three hours following the EPZ-6438 treatment displayed a significantly lower amount of histone H3K27me3 than vehicle-treated tumors (Supplemental Fig. S3), demonstrating suppression of EZH2 enzymatic activity by EPZ-6438. Accordingly, EPZ-6438 at either 100 or 200 mg/kg suppressed tumor growth compared to those treated with vehicle (Fig. 3A, day 28, P <0.01, P <0.01, respectively). Unexpectedly, mice from all the treatment groups displayed significant weight loss due to dehydration (Supplemental Fig. S4A). After stopping dosing for 6 days to allow recovery from dehydration, the tumors in mice exposed to EPZ-6438 grew back from about 100 mm3 to 300 mm3 (Fig. 3A, day 34). To further test whether the once daily treatment with EPZ-6438 was effective in suppressing tumor growth, we resumed treatment with a daily single dose schedule until mice reached humane endpoints. This schedule at 100 mg/kg failed to delay tumor growth (Fig. 3A, days 34-48), while single daily dosing with 200 mg/kg EPZ-6438 retarded growth after 10 days (Fig. 3A). Though overall survival did not differ between two dosages, both dosages significantly improved the median survival of mice to humane endpoint from 37 days (vehicle) to 46 days 		 11	(100mg/kg) and 48 days (200mg/kg) mainly due to the benefit from the first week of twice daily dosing (Fig. 3B, P = 0.0036 and 0.0017, respectively).   Next, we tested the efficacy of EPZ-6438 in the SCCOHT-1 xenograft model. Since SCCOHT-1 cells displayed much higher sensitivity to EZH2 inhibitors than any other cell lines (Fig. 2E), we dosed the mice with 200 mg/kg EPZ-6438 once daily (n=6), which produced only mild effect on the mouse body weight (Supplemental Fig. S4B). Tumor growth was effectively suppressed by the once daily treatment of EPZ-6438 at 200 mg/kg wherein the tumor growth doubling time (tumor volume from 150 mm3 to 300 mm3) increased from 5 days (vehicle) to 17 days (EPZ-6438) (Fig. 3C). The average tumor weight was also reduced by 50% in the EPZ-6438-treated group compared to the vehicle-treated group at the end of three weeks of treatment (Fig. 3D, p < 0.05). These data suggest that once daily treatment of EPZ-6438 at 200 mg/kg significantly slowed down the growth of SCCOHT-1 xenografts.  Effects of EPZ-6438 on the proteome of BIN67 cells To identify the molecular mechanisms driving growth inhibition caused by EZH2 inhibitors in SCCOHT cells, we determined the proteomic profiles of BIN67 cells treated with EPZ-6438 or vehicle using mass spectrometry. Unsupervised hierarchical clustering resulted in clustering based on the treatment conditions (Supplemental Fig. S5) with approximately 8.4% (571/6768) and 7.4% (498/6768) being significantly upregulated or downregulated, respectively, in BIN67 cells treated with 1 µM EPZ-6438 for 7 days versus those treated with DMSO (Fig. 4A and Supplemental Table S1 and S2, pfdr<0.05 and log2FC>mean+SD or <mean-SD). Ingenuity pathway analysis (IPA) of the significantly altered proteins by EPZ-6438 revealed a significant enrichment of proteins involved in biological functions, such as “Cell cycle”, “Cellular Assembly 		 12	and Organization” and “Cellular development” (Fig. 4B, Supplemental Table S3). Particularly, both “development of neurons” and several processes related to neuronal development, such as “formation of cellular protrusions”, “microtubule dynamics” and “neuritogeneis”, were predicted to be enhanced significantly in EPZ-6438-treated BIN67 cells (Fig. 4C, Supplemental Table S3, z>2). Clustering of the enriched proteins confirmed that many of the identified proteins involved in microtubule dynamics, formation of celluar protrusions and organization of cytoskeleton or cytoplasm were also involved in development of neurons (Supplemental Fig. S6), supporting their engagement in neuron development. These data suggest that EPZ-6438 not only altered the cell cycle of SCCOHT cells, but also triggered significant changes in cell organization and assembly, leading to neuron-like differentiation.    EPZ-6438 induces SCCOHT cell cycle arrest and apoptosis  Next, we used flow cytometry to validate the effect of EPZ-6438 on cell cycle distribution of SCCOHT cells. FACS analysis demonstrated that 1 µM EPZ-6438 treatment induced cell cycle arrest at G1 in a time-dependent manner in BIN67 and COV434 cells (Fig. 5A), and also triggered cell death (sub-G1 population) beginning at 7 days after drug exposure in both BIN67 and COV434 cell lines, consistent with a mild induction of apoptosis predicted by proteomic data (Supplemental Table S4). In contrast, 1 µM EPZ-6438 induced cell death more rapidly (3 days) and potently in SCCOHT-1 cells (Fig. 5A), consistent with the lower IC50 in this cell line than others (Fig. 2E). To precisely monitor the induction of apoptosis, we measured the activation of caspase-3/7 through fluorescent microscopy coupled with live cell imaging (Fig. 5B). This revealed that EPZ-6438 rapidly induced cell apoptosis in SCCOHT-1 cells after 48 hours of EPZ-6438 treatment at either 0.3 or 1 µM, whereas apoptosis in BIN67 and COV434 cells increased 		 13	gradually beginning at about 120 hours of exposure to 1 µM EPZ-6438 (Fig. 5C). Next, we employed western blotting to determine the expression of some cell cycle and apoptosis regulating genes that were significantly altered by EPZ-6438 in BIN67 cells by proteomic profiling. We confirmed that the expression of CDKN1A (p21) and BAD (Log2FC =0 .7 and 1.4, respectively, Supplemental Table S1) was gradually induced by EPZ-6438, while that of Myc (Log2 FC = -0.9, Supplemental Table S2) was potently down-regulated by EPZ-6438 in all three SCCOHT cell lines (Fig. 5D). In contrast, the expression of CDKN2A (p16) (Log2 FC = 0.2, Supplemental Table S1), which is known to play an important role in EZH2 inhibitor-triggered growth arrest [31], was only significantly induced upon EPZ-6438 treatment in SCCOHT-1 cells (Fig. 5D).   EPZ-6438 induces neuron-like differentiation in SCCOHT cells In agreement with the prediction of induced neuron development in BIN67 cells from proteomic profiling, the surviving SCCOHT cells displayed a neuron-like morphology after prolonged exposure to 1 µM EPZ-6438 (Fig. 6A). In contrast, SCCOHT cells exposed to cytotoxic agents, such as cisplatin, etoposide, and paclitaxel, displayed no morphology change other than fragmentation during cell death (Supplemental Fig. S7). Immunofluorescence confirmed that the expression of MAP2, a specific marker of neuronal differentiation [32], was increased upon EPZ-6438 treatment in a time-dependent manner in BIN67 cells (Fig. 6B), consistent with increased expression with treatment observed in the proteomic data (Log2 FC = 2.2, Supplementary Table S2).  Western blot analysis further confirmed that the expression levels of two neuronal markers (MAP2 and TUBB3 (βIII tubulin), Log2FC=1.2, Supplementary Table S2) were induced by EPZ-6438 treatment (Fig. 6C). In agreement with the induction of differentiation, the expression 		 14	of EZH2 (Log2FC=-0.4, Supplementary Table S1) also dropped significantly upon EPZ-6438 treatment in a time-dependent manner (Fig. 6C). Therefore, our data show that unlike cytotoxic agents, EZH2 inhibitors can induce expression of markers of neuronal differentiation of SCCOHT cells.    Discussion Transformation of normal cells requires acquisition of hallmark characteristics of cancer including survival and proliferation even in the presence of counteracting signals. These features usually include extensive rewiring of cellular signaling networks driven by mutations and deregulation of oncogenes and tumor suppressors [33], leading to a strict reliance on either an oncogenic driver event (oncogene addiction) or the activity of certain gene products that are not essential in normal cells (non-oncogene addiction or synthetic lethality) [34]. Our findings suggest that inactivation of SMARCA4 in SCCOHT’s unknown precursor cells may rewire their cellular signaling network to be dependent on the catalytic activity of the histone methyltransferase EZH2 in transcriptional repression. In support of this notion, previous studies have shown that SMARCA4 and the associated SWI/SNF chromatin-remodeling complex can suppress EZH2 either directly [21] or through repression of E2F transcription factors [35-37].  Concordantly, re-expression of SMARCA4 in SCCOHT cell lines lowered the expression of EZH2 and reduced the global level of histone H3K27me3, suggesting that SMARCA4 loss may promote SCCOHT tumorigenesis, at least partially, through the direct up-regulation of EZH2 expression.  In addition to this work, several other studies have demonstrated the requirement for the methyltransferase activity of EZH2 in cancers with a defective subunit of the SWI/SNF complex.  		 15	However, two ARID1A-deficient ovarian clear cell carcinoma cell lines (OVISE and OVTOKO) and several lung cancer cell lines with SMARCA4 deficiency or SMARCA4/SMARCA2 dual deficiency did not respond or responded poorly to EPZ-6438 treatment in our study (Fig. 2C and Supplemental Fig. S3). Therefore, the absence of subunits of the SWI/SNF complex alone does not predict sensitivity to inhibition of EZH2 catalytic activity. Whether the efficacy of EZH2 inhibitors depends upon additional genetic or epigenetic features of the tumor remains unclear.  Of note, both SCCOHT and MRT cell lines [28] that respond to EZH2 inhibitors lack the core subunits of SWI/SNF complex (either SMARCA4/A2 or SMARCA2/SMARCB1 deficiency), while ARID1A-deficient ovarian clear cell carcinoma cells usually retain an active complex that includes ARID1B [38].  Similarly, most SMARCA4-deficient ovarian and lung carcinomas retain SMARCA2 expression and its associated chromatin remodeling activity. Accordingly, it has been reported that ARID1A-deficient ovarian cancer and SMARCA4-deficient lung cancer depend on ARID1B and SMARCA2, respectively, for maintaining their oncogenic property [17,18,38]. Furthermore, most tumors with SMARCA4 or ARID1A loss usually display a higher mutation burden in comparison to the minimally disturbed genomes of either SCCOHT or MRT [8,39-41].  The lack of additional mutations in the latter tumors implicates a dependence upon other epigenetic changes such as EZH2 overexpression.  Thus, SMARCA4 inactivation in the absence of SMARCA2 may rewire the cellular signaling network to be dependent on EZH2-mediated oncogenesis while additional mutations possibly negate this requirement in other cancers.  Although the potency of EZH2 inhibitors has been demonstrated in several cancer types, the molecular mechanisms are still not well understood. EZH2 is the catalytic component of the PRC2 complex that mediates the transcriptional repression of targets by trimethylation of histone H3 at lysine residue 27 in their promoters. Therefore, the PRC2 complex could promote 		 16	tumorigenesis by specifically repressing tumor-suppressor genes, including the major tumor suppressor locus CDKN2A, through recruitment of DNA methyltransferases. Accordingly, treatment with EZH2 inhibitors can rescue the expression of p16 in cancer cells, such as MRT cells and leukemia stem cells [28,42]. Unlike MRT cells, in only one of the three SCCOHT cell lines (SCCOHT-1) was p16 expression altered upon EPZ-6438 treatment. The other two cell lines expressed substantial amounts of p16, which were not further induced upon the EPZ-6438 treatment. Therefore, a different mechanism may underlie the efficacy of EPZ-6438 in the BIN67 and COV434 cell lines.  Proteome analysis revealed that EPZ-6438 altered about 15% of the proteome in BIN67 cells with a significant enrichment of proteins involved in cell cycle control and development of neurons. These results implicate these pathways as the major biological events behind the cellular response to EZH2 inhibitors in SCCOHT cells. Particularly, we discovered that the expression of several cell cycle control genes, such as Myc, were repressed by EPZ-6438. Although the PRC2 complex may upregulate the expression of these genes indirectly through suppressing the negative regulators of these genes, it has been suggested that the transcription of Myc can be activated directly by the PRC2 complex in glioblastoma cancer stem cells [43]. Given that Myc activation is a hallmark of tumor initiation and maintenance, suppression of Myc may have a significant contribution to the efficacy of EZH2 inhibitors in SCCOHT and other cancers, such as MRT and glioblastoma [43].  Furthermore, we also observed a time-dependent induction of BAD pro-apoptotic protein in three SCCOHT cells following EPZ-6438 treatment. By inactivating the function of the anti-apoptotic proteins Bcl-2 and Bcl-xl, BAD can promote apoptosis [44]. Our data together with the previous study showing that BAD was up-regulated upon EZH2 depletion by shRNA in lung cancer cell lines [45] suggest that it may play a key role in EZH2 inhibitor-		 17	triggered apoptosis. In addition to causing cell cycle arrest and apoptosis, prolonged exposure to EZH2 inhibitors also drove the differentiation of SCCOHT cells towards the neuronal lineage. Consistent with the neuron-like differentiation, EPZ-6438 caused a late induction of p21 (Fig. 4F), a cyclin kinase inhibitor that may drive neural precursor cells into cycle exit and differentiation [46]. It will be interesting to determine whether depletion of p21 can prevent the neuron-like differentiation caused by EZH2 inhibitors.  A neuron-like morphology change has also been reported in G401 cells upon EPZ-6438 treatment [28]. Both genomic analysis and mouse transgenic models have provided evidence that some MRT may arise from neural precursor cells [41,47]. Interestingly, some studies suggest that SCCOHT is the MRT of ovary. Therefore, our present finding, that EZH2 inhibition induced markers of neuronal differentiation of SCCOHT, suggest that SCCOHT may develop from multi-potent stem cells inside the ovary with the capability of undergoing neuronal differentiation.  In support of this model, extensive IHC analysis of multiple sections of two SCCOHT cases previously revealed rare foci of immature teratoma [11], which mainly contain primitive neuroepithelium in their immature region [48]. Future studies are therefore needed to explore the possible cellular origin of SCCOHT from neural precursor cells.  SCCOHT is a rare but extremely aggressive disease for young women.  No effective treatment strategies have been developed for fighting this lethal disease. In the present study, we provide support that the methyltransferase EZH2 is universally expressed in SCCOHT and may serve as a potential therapeutic option for young women with this deadly disease.  Importantly, Epizyme pharmaceutics has recently launched a phase 1 clinical trail for evaluating the toxicity of EPZ-6438 in solid tumors with SMARCB1 or SMARCA4 deficiency (clinicaltrials.gov).  Their interim results showed that EPZ-6438 (Tazemetostat) was well tolerated and elicited a 		 18	partial response by RECIST criteria in two SCCOHT patients previously treated with chemotherapy (www.epizyme.com).  Therefore, identifying other targeted therapy strategies and determining the efficacy of combining EZH2 inhibitors with other putative targeted therapies for SCCOHT treatment remains a high priority.  Acknowledgements We acknowledge for the technical support from Sarah Maines-Bandiera, Winnie Yang, Christine Chow, Nicole Wretham, Dana Masin, Hong Yan, Jenna Rawji and Chris Ke-dong Wang. We thank Drs. Barbara Vanderhyden and Ralf Hass for providing BIN67 and SCCOHT-1 cells, respectively. This work was supported by research funds from the Canadian Cancer Society Research Institute (#703458, to D.G.H.) and the National Institute of Health (1R01CA195670-01, to D.G.H., J.T. and B.W.), the Terry Fox Research Institute Initiative New Frontiers Program in Cancer (D.G.H.), the British Columbia Cancer Foundation, the Marsha Rivkin Center for Ovarian Cancer Research, the Ovarian Cancer Alliance of Arizona, the Small Cell Ovarian Cancer Foundation, and philanthropic support to the TGen Foundation.  Statement of author contributions Y.W. designed and performed experiments, analyzed data and wrote the manuscript. S.Y.C. performed experiments and analyzed data. A.N.K. performed IHC, analyzed data and edited the manuscript. S.C. performed mass spectrometry experiments, analyzed data and wrote the manuscript. N.D.S helped the design of xenograft studies and analyzed data. J.L., W.P.D.H., K.A.O., B.E.W., G.B.M. and J.F.T. provided thoughtful discussion and edited the manuscript. 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(A) The expression of PRC2 complex proteins in three SCCOHT versus two normal ovaries were extracted out from our previous Agilent microarray analyses (GE access No.: GSE49887 and GSE66434). (B) The expression of EZH2 protein in multiple cell lines was analyzed by Western blotting analysis. (C) The expression of EZH2 protein in primary SCCOHT samples was determined using immunohistochemistry on TMA. Representative images of strong or variable staining are shown. (D) SCCOHT cell lines were infected with lentivirus expressing either GFP or SMARCA4. Cells were harvested 72 hours post infection for determining the effect of SMARCA4 re-expression on EZH2 and histone H3 K27me3 by Western blotting. * P<0.001.  Figure 2. SCCOHT cells are sensitive to EZH2 depletion and suppression. (A, B) Cells were infected with lentivirus expressing control scramble shRNA or EZH2 shRNA followed by puromycin selection for 48 hours. Cells were then reseeded in 24-well plates for 6 days before being fixed and quantitated by crystal violet staining assay. (C, D) Cells were treated as indicated for 3 days for Western blot analysis of histone H3K27me3 level. (E) Cells were seeded in 96-well plates, treated with EZH2 inhibitors GSK126 or EPZ-6438 at indicated doses and incubated for 6 days and measured for cell survival by crystal violet assay. (F) The IC50s of cell lines to EZH2 inhibitors in (E) were compared between SCCOHT lines and other ovarian lines. Note: 20µM was assigned to the cell lines that were not responsive to EZH2 inhibitors at 10µM. * P<0.01, ** P<0.001  		 24	Figure 3. In vivo efficacy of EPZ-6438 in BIN67 mouse xenograft model. (A-B) The efficacy of EPZ-6438 was evaluated in BIN67-derived mouse subcutaneous xenograft model. Tumor volume and the overall survival of each treated group until the study endpoint (see Material and Methods for details) were plotted against the days post cell inoculation, respectively. (C, D) The efficacy of EPZ-6438 was evaluated in SCCOHT-1-derived mouse subcutaneous xenograft model. Tumor volume of either vehicle or 200 mg/kg EPZ-6438-treated group was plotted against the days post cell inoculation (C). Final tumor weight was determined and compared between two treated groups (D). * P<0.05, ** P<0.01, *** P<0.001  Figure 4. The effect of EPZ-6438 on the proteome of BIN67 cells. (A) Volcano plot of the proteome of BIN67 cells exposed to EPZ-6438 or vehicle. Cells were treated with either DMSO or 1µM EPZ-6438 for 7 days and then processed for proteomic profiling. Peptide data were subjected to PECA analysis for identification of significantly altered proteins (p.fdr<0.05 and Log2FC>mean+SD or <mean-SD). (B) IPA analysis of significantly altered proteins caused by EPZ-6438 identified top affected biological functions by EPZ-6438 treatment. Any biological function with an activation z-score greater than 2 or less than -2 was predicted to be significantly increased or decreased by IPA analysis. (C) IPA analysis identified significantly increased cellular activities related to neuronal development. (D) Clustering analysis of proteins in significantly altered biological functions.  Figure 5. EPZ-6438 induces SCCOHT cell cycle arrest and apoptosis. (A) FACS analysis of cell cycle profiles in SCCOHT cells after DMSO or 1µM EPZ-6438 treatment for the shown times. (B, C) SCCOHT cell lines were treated with 1µM EPZ-6438 and cultured in the presence 		 25	of a cell-permeable fluorescent dye for monitoring activated caspase-3/7 activity with IncuCyte live cell imaging system (see Materials and Methods). Apoptotic index was calculated by dividing the overall fluorescent object counts to cell numbers under each condition and plotted over incubation time. (D) The effects of EPZ-6438 (BIN67/COV434: 1µM; SCCOHT-1: 0.25µM) on the expression of Myc, BAD, p16 and p21 proteins was determined by western blotting.   Figure 6. EPZ-6438 induces neuronal differentiation in SCCOHT cells. (A) Morphology of SCCOHT cells after prolonged exposure to EPZ-6438. Cells were treated with EPZ-6438 at either 1µM (BIN67/COV434) or 0.25µM (SCCOHT-1) for 12 days and characterized by phase contrast microscopy. (B) BIN67 cells were fixed and immunostained for MAP2, a selective neuronal marker. (C) Western blotting for neuronal proteins from SCCOHT cells treated with EPZ-6438 at either 1µM (BIN67/COV434) or 0.25µM (SCCOHT-1) for the days shown in the panel. Vinculin serves as a loading control.      A	C	 Strong	staining	of	EZH2	Fig	1.	EZH2	is	abundantly	expressed	in	SCCOHT	0	2	4	6	8	10	12	EZH2 EZH1 EED SUZ12 Relative gene expression Normal ovary SCCOHT *	D	SMARCA4								-													+																		-														+																-											+		BIN67	 SCCOHT1	H3K27Me3	Vinculin	EZH2	SMARCA4	Total	H3	B	NOY1	SVOG3e	OVTOKO	OVISE	JHOC5	G401	BIN67	SCCOHT1	COV434	OVCAR8	EZH2	Vinculin	19/24	(79%)	Variable	staining	of	EZH2	5/24	(21%)	COV434	Fig.	1	(Wang	et	al,	2016)	Fig.2	SCCOHT	cells	are	sensiMve	to	EZH2	suppression	EZH2 shctrl     shEZH2_1 shEZH2_2 H3K27Me3 H3 BIN67 A	 B	C	 D	0				0.1			0.5								0					0.1			0.5						0				0.1			0.5	EPZ-6438	(μM	)	BIN67	 SCCOHT1	H3K27Me3	0						1							5						0					0.1				1									0							1						5	H3	GSK126	(μM)	0	20	40	60	80	100	120	140	% Growth shCtrl	shEZH2_1	shEZH2_2	**	*	**	**	**	**	EPZ-6438	-2 -1 0 1050100Log10(conc)% SurvivalEPZ-6438BIN67SCCOHT1COV434G401ES-2RMG1OVCAR-8OVISEOVTOKONOY1F	-2 -1 0 1050100Log10(conc)% SurvivalGSK126BIN67SCCOHT1COV434G401ES-2RMG1OVCAR-8NOY1GSK126	SCCOHT		lines	Other	ovarian		lines	SCCOHT		lines	Other	ovarian		lines	**	*	GSK126	 EPZ-6438	IC50 (µM) E	Fig.	2	(Wang	et	al,	2016)	H3K27Me3	H3	COV434	 BIN67	 SCCOHT1	 COV434	BIN67	 COV434	SCCOHT-1	 ES-2	SCCOHT	lines	Fig.3		In	vivo	efficacy	of	EPZ-6438	in	mouse	xenogra\	models	0	200	400	600	800	1000	1200	13	 18	 23	 28	 33	 38	 43	 48	Tumor volume (mm3 ) Days after cell inoculation vechicle	100mg/Kg	EPZ-6438	200mg/Kg	EPZ-6438	Treatment	started	(BID)	Treatment halted Treatment		re-started	(QD)	A	0	20	40	60	80	100	120	0	 10	 20	 30	 40	 50	Percentage	Survival		Days	aYer	cell	inoculaZon	vechicle	100mg/Kg	EPZ-6438	200mg/Kg	EPZ-6438	B	C	BIN67	xenogra\	model	 BIN67	xenogra\	model	D	SCCOHT-1	xenogra\	model	 SCCOHT-1	xenogra\	model	0	200	400	600	800	1000	1200	10	 15	 20	 25	 30	 35	Tumor Volume (mm3 ) Days	post	inoculaZon	Vehicle	200mg/kg	EPZ-6438	0.0	0.5	1.0	1.5	2.0	Vehicle	 EPZ-6438	Average	final	tumor	weight	(g)	Treatment	started	(QD)	*	**	 **	**	**	*	*	*	**	***	***	**	**	ss	ve i le ss	hic  Fig.	3	(Wang	et	al,	2016)	Fig.	4	Effect	of	EPZ-6438	on	the	proteome	of	BIN67	cells	B	C	0	 2	 4	 6	 8	formaZon	of	cellular	protrusions	microtubule	dynamics	neuritogenesis	development	of	neurons	-Log2P-value	Log2(FC)	A	-4	 -2	 0	 2	 4	AcZvaZon	z-Score	Cell	Cycle	Cellular	Assembly	and	OrganizaZon	Cellular	Development	Cell-To-Cell	Signaling	and	InteracZon	Cellular	Movement	Cellular	FuncZon	and	Maintenance	Cell	Death	and	Survival	Cellular	Growth	and	ProliferaZon	DNA	ReplicaZon,	RecombinaZon,	and	Repair	1.00E-12	1.00E-10	1.00E-08	1.00E-06	1.00E-04	P-value	Fig.	4	(Wang	et	al,	2016)	Fig.	5	EPZ-6438	induces	cell	cycle	arrest	and	apoptosis	in	SCCOHT	cells	Phase Activated caspase3/7 EPZ-6438	DMSO	B	A	C	Myc	BIN67	 SCCOHT-1	EPZ-6438			0					3					7				10							0						3				7			10						0						4						7	days	p21	COV434	BAD	AcZn	p16	BIN67	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	120	 144	 168	 192	 216	ApoptoZc	Index	Time	aYer	EPZ-6438	treatment	(hours)	DMSO	0.3μM	EPZ-6438	1	μM	EPZ-6438	COV434	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	120	 144	 168	 192	 216	ApoptoZc	index	Time	aYer	EPZ-6438	treatment	(hours)	DMSO	0.3μM	EPZ-6438	1	μM	EPZ-6438	Fig.	5	(Wang	et	al,	2016)	0	20	40	60	80	Control Day 3 Day 7 Day 12 %	Cells	COV434	0	20	40	60	80	Control	 Day	3	 Day	7	 Day	12	%	cells	BIN67	0	20	40	60	80	Control	 Day	3	 Day	7	%	Cells	SCCOHT1	subG1	G1	S	G2/M	D	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0	 24	 48	 72	 96	ApoptoZc	index	Tim	aYer	EPZ-6438	treatment	(hours)	DMSO	0.3μM	EPZ-6438	1μM	EPZ-6438	SCCOHT-1	Fig.6	EPZ-6438	induces	neuron-like	differenMaMon	in	SCCOHT	cells	B	AMAP2	 DAPI	 Merged	DMSO	EPZ-6438		day	7	EPZ-6438		day	12	BIN67	 COV434	MAP2	EPZ-6438										0							3							7							12												0							3								7						12									0									4									7			days	TUBB3	EZH2	Vinculin	C	BIN67	 SCCOHT1	 COV434	SCCOHT-1	Fig.	6	(Wang	et	al,	2016)	 		 1	The histone methyltransferase EZH2 is a therapeutic target in small cell carcinoma of the ovary, hypercalcemic type  Running title: Targeting EZH2 in SCCOHT  Yemin Wang1, Shary Yuting Chen1, Anthony N. Karnezis1, Shane Colborne2, Nancy Dos Santos3, Jessica D. Lang4, William P.D. Hendricks4, Krystal A. Orlando5, Damian Yap1, Friedrich Kommoss1, Marcel B. Bally3, Gregg B. Morin3,6, Jeffrey M. Trent4, Bernard E. Weissman5, David G. Huntsman1,7,#   1Department of Pathology and Laboratory Medicine, University of British Columbia and Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, BC, Canada.  2Michael Smith Genome Science Centre, British Columbia Cancer Agency, Vancouver, BC, Canada.  3Department of Experimental Therapeutics, British Columbia Cancer Research Centre, Vancouver, BC, Canada. 4Division of Integrated Cancer Genomics, Translational Genomics Research Institute (TGen), Phoenix, AZ, USA.  5Department of Pathology and Laboratory Medicine and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA. . 6Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 7Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, 		 2	Canada  Corresponding author  David Huntsman, MD, FRCPC, FCCMG Dr. Chew Wei Memorial Professor of Gynaecologic Oncology, UBC Professor, Departments of Pathology and Lab Medicine and Obstetrics and Gynaecology, UBC Distinguished Scientist, Department of Molecular Oncology, BC Cancer Research Centre  #4111-675 West 10th Avenue, Vancouver, BC V5Z 1L3 Canada  Phone: 604.675.8205; Email: dhuntsma@bccancer.bc.ca  Conflict of interest: None of the authors report any conflicts of interest. Word Count for main text (limit 4000): 3984 		 3	Abstract Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodeling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodeling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumor samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells.  In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis and neuron-like differentiation. EPZ-6438 suppressed tumor growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease.   Keywords: SCCOHT, SWI/SNF, chromatin remodeling complex, differentiation, EZH2, SMARCA4, ovarian cancer  		 4	Introduction Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), a rare but highly aggressive ovarian malignancy with unknown cellular origin, occurs both sporadically and in families [1,2]. Unlike most common ovarian cancers, SCCOHT primarily affects women in their teens and twenties [3-5]. Surgical debulking followed by adjuvant chemotherapy is the mainstay of therapy for SCCOHT. However, recurrence is generally rapid, and the prognosis is dismal – about two-thirds of patients with advanced stage disease die within two years of diagnosis [5,6]. Therefore, more effective treatment options are urgently needed. Despite that Otte et al. have recently discovered c-Met inhibitors as potential targeted therapy agents for a subset of SCCOHT [7], the biology-driven therapeutic options for SCCOHT remain to be explored.  Several research teams, including ourselves, have independently identified inactivating, often homozygous or bi-allelic, mutations of the SMARCA4 gene in over 90% of SCCOHT cases [8-11], which leads to loss of SMARCA4 protein in the majority of SCCOHT tumors and cell lines [8-11]. Unlike common malignancies, no recurrent somatic, non-silent mutations besides those in SMARCA4 have been detected by paired exome or whole-genome sequencing analysis in SCCOHT [8-10,12]. Therefore, the inactivating mutations in SMARCA4 appear to be the primary driver in SCCOHT tumorigenesis and may help inform novel treatment strategies for SCCOHT. SMARCA4 is one of the two mutually exclusive ATPases of the SWI/SNF multi-subunit chromatin-remodeling complex, which uses ATP hydrolysis to destabilize histone-DNA interactions and mobilize nucleosomes. The SWI/SNF complex localizes near transcriptional regulatory elements and regions critical for chromosome organization to regulate the expression of many genes involved in cell cycle control, differentiation and chromosome organization [13,14]. Several subunits of the SWI/SNF complex, such as SMARCA4, SMARCB1, ARID1A, 		 5	PBRM1, are frequently mutated and inactivated in a variety of cancers [14-16]. This highlights the broader potential utility of effective targeted therapies for patients with a defective SWI/SNF complex. Recently, several studies reported that SMARCA4-deficient lung cancer cell lines relied on the activities of SMARCA2, the mutually exclusive ATPase, for proliferation [17,18], raising the possibility of selectively targeting SMARCA2 as therapeutic approaches for these patients. However, all SMARCA4-negative SCCOHT tumors and tumor-derived cell lines also lack the expression of SMARCA2 without apparent mutations in the SMARCA2 gene [19], indicating the need for developing different biologically informed treatment approaches for SCCOHT. The interplay between the SWI/SNF complex and the Polycomb repressive complex 2 (PRC2) was originally demonstrated through genetic studies in Drosophila [20]. Mouse studies revealed that tumorigenesis driven by SMARCB1 loss was ablated by the simultaneous loss of EZH2, the catalytic subunit of PRC2 that trimethylates lysine 27 of histone H3 (H3K27) to promote transcriptional silencing [21]. Therefore, EZH2 has emerged as a putative therapeutic target for SMARCB1-deficient malignant rhabdoid tumors (MRTs), ARID1A-deficient ovarian clear cell carcinomas, SMARCA4-deficient lung cancers and PBRM1-deficient renal cancers, although the non-catalytic activity of EZH2 was likely responsible for the therapeutic potential in some cases [21-23]. Therefore, we set out to address whether targeting EZH2 is a feasible strategy for treating SMARCA4-deficient SCCOHT. We discovered that EZH2 is abundantly expressed in SCCOHT and its inhibition robustly suppressed SCCOHT cell growth, induced apoptosis and neuron-like differentiation, and delayed tumor growth in mouse xenograft models of SCCOHT.  		 6	Materials and methods SCCOHT tissue microarray and immunohistochemistry SCCOHT tissue microarrays (TMA), as described previously [19], were cut at 4 µm thickness onto Superfrost+ glass slides, and stained for EZH2 (#612667, BD Transduction Laboratories, Mississauga, ON, Canada) on a Ventana Discovery XT autostainer (Ventana Medical Systems, Tucson, AZ, USA). The EZH2 staining was scored by a pathologist (A.N.K.) as negative (<1% of tumor cells showing definite, i.e. moderate to strong, nuclear staining), positive variable (1-50% of tumor cells) or positive diffuse (>50% of tumor cells). Cell culture BIN67, SCCOHT-1, COV434, and G401 cells were grown in DMEM/F-12 supplemented with 10% FBS. ES-2, RMG1, OVCAR-8, NOY1, OVISE and OVTOKO cells were cultured in RPMI supplemented with 10% FBS. All the cells were maintained at 37°C in a humidified 5% CO2-containing incubator. All cell lines have been certified by STR analysis, tested regularly for Mycoplasma and used for the study within six months of thawing. Proteomics Cells were lysed in 100mM HEPES buffer (pH 8.5) containing 1% SDS and 1x protease inhibitor cocktail (Roche). After chromatin degradation by benzonase, reduction and alkylation of disulfide bonds by dithiothreitol and iodoacetamide, samples were cleaned up and prepared for trypsin digestion using SP3-CTP method [24]. Briefly, proteins were digested for 14 hours at 37°C followed by SP3 beads removal. Tryptic peptides from each sample were individually labeled with TMT 10-plex labels, pooled and fractionated into 12 fractions by high pH RP-HPLC, desalted, orthogonally separated and analyzed using and Easy-nLC 1000 coupled to a Thermo Scientific Orbitrap Fusion mass spectrometer operating in MS3 mode. Raw MS data 		 7	were processed and peptide sequences were elucidated using Sequest HT in Proteome Discoverer software (v2.1.0.62), searching against the UniProt Human Proteome database.  Mouse xenografts Animal handling, care, and treatment procedures were performed according to guidelines approved by the Animal Care Committee of the University of British Columbia (A14-0290). Briefly, BIN67 (1x107 cells/mouse) or SCCOHT-1 cells (4x106 cells/mouse) were injected with a 1:1 mix of matrigel (Corning) in a final volume of 200 µl subcutaneously into the back of NRG (NOD.Rag1KO.IL2RγcKO) mice. Mice were randomized to treatment arms once the average tumor volume reached 100mm3. EPZ-6438 (ActiveBiochem) was formulated in 0.5% NaCMC with 0.1% Tween-80 in water. For the BIN67 xenograft model, mice were treated by oral administration of vehicle or EPZ-6438 (100 or 200 mg/kg) twice daily (BID, 0800/1600h) for eight days, halted for six days due to body weight loss in all groups, and then resumed with once daily (QD) dosing for additional three weeks or until the humane endpoint (i.e. tumor volume reached 800 mm3). For the SCCOHT-1 xenograft model, mice were treated by oral administration of vehicle or EPZ-6438 (200 mg/kg, QD) for three weeks or until reaching a humane endpoint. Tumor volume and mouse weight were measured thrice weekly. Tumor volume was calculated as length x (width)2 x 0.52. Statistical analysis The student's t test was used to evaluate the significant difference between two groups in all experiments except proteomics. The Peptide Expression Change Averaging (PECA) analysis [25] was performed for comparing the proteomic profiles of BIN67 cells treated with or without EPZ-6438 to generate signal log ratio values (log fold-change), p-values and false discovery rate (fdr) adjusted p-values (p.fdr) using peptide level signal values. The PECA analysis [25] was 		 8	performed for comparing the proteomic profiles of BIN67 cells treated with or without EPZ-6438. Survival curves and IC50 of drug treatment were determined by PRISM software. A p-value or p.fdr-value (for proteomics data) < 0.05 was considered significant.  Additional Material and Methods are available in the Supplemental Information online.  Results EZH2 is abundantly expressed in SCCOHT The poorly differentiated state of SCCOHT and the critical role of EZH2 in the maintenance of embryonic and tissue stem cells [26], together with the recently discovered antagonism between the SWI/SNF complex and EZH2 [23], imply that EZH2 may play a role in SCCOHT tumorigenesis. We first determined the expression levels of EZH2 in SCCOHT primary tumor samples and cell lines. Using gene expression profiles extracted from microarray data of four primary SCCOHT samples and two normal ovaries [19], we observed that only the expression of EZH2, but not other polycomb group (PcG) proteins, was significantly elevated in SCCOHT tumors compared to normal ovaries (Fig. 1A). Western blotting analysis demonstrated that two SCCOHT cell lines, BIN67 and SCCOHT-1, expressed EZH2 protein at a level comparable to or higher than that in several other ovarian cancer cell lines and the MRT cell line G401 (Fig. 1B). COV434, a cell line originally designated as a juvenile granulosa cell tumor cell line [27] but recently redefined as a SCCOHT cell line with dual deficiency of SMARCA4 and SMARCA2 (manuscript in preparation), also abundantly expressed EZH2 protein (Fig. 1B). Next, we performed EZH2 immunohistochemistry (IHC) on TMA of 24 primary tumors. Seventy-nine % (19/24) displayed strong diffuse EZH2 staining with variable staining in the remainder (Fig. 1C). 		 9	To test whether loss of SMARCA4 upregulates EZH2, we re-introduced SMARCA4 into SCCOHT cell lines. Western blotting revealed that EZH2 was substantially downregulated by SMARCA4 re-expression alongside a reduction in histone H3 lysine residue K27 trimethylation (H3K27me3) levels in all three SCCOHT cell lines (Fig. 1D). These data suggest that loss of SMARCA4 leads to EZH2 upregulation in SCCOHT.  SCCOHT cells are sensitive to EZH2 inhibition To determine whether SCCOHT relies on EZH2 for proliferation, we ablated the expression of EZH2 with two specific EZH2 shRNAs, either of which led to a significant reduction of H3K27me3 levels (Fig. 2A). Depletion of EZH2 significantly inhibited the growth of BIN67, COV434 and SCCOHT-1 cells, but not of ES-2 cells, an ovarian cancer cell line with intact SMARCA4 and SMARCA2 (Fig. 2B), suggesting that EZH2 is a therapeutic target specifically in SMARCA4/A2-deficient SCCOHT cells. Next, we determined whether SCCOHT cell lines are responsive to the inhibition of EZH2 catalytic activity with two pharmaceutical inhibitors, GSK126 and EPZ-6438, both of which are being tested in clinical trials. Western blotting analysis confirmed that each inhibitor potently suppressed histone H3K27Me3 levels in SCCOHT cells at either 0.1 or 0.5 µM (Fig. 2C and 2D). In a six-day drug treatment assay, three SCCOHT cell lines were significantly more sensitive to either GSK126 or EPZ-6438 than other ovarian cancer cell lines tested (Fig. 2E and 2F). Among three SCCOHT cell lines, SCCOHT-1 cells were about 5 or 10 fold more sensitive than the other two lines and the MRT cell line G401 to the treatment of GSK126 or EPZ-6438, respectively (Fig. 2E). Concordant with the drastic anti-proliferative effects observed in MRT lines after a two-week exposure to EPZ-6438 [28], the growth of BIN67 and COV434, which 		 10	displayed lower sensitivity than SCCOHT-1, was strongly suppressed by prolonged exposure to each inhibitor at either 1µM or below (Supplemental Fig. S1). However, neither SMARCA4- nor SMARCA4/SMARCA2-dual-deficient lung tumor cell lines were as sensitive to EZH2 inhibitors as the SCCOHT cell lines (Supplemental Fig. S2).    EPZ-6438 suppresses tumor growth in a mouse SCCOHT xenograft model Next, we employed mouse subcutaneous xenografts of SCCOHT cells to evaluate in vivo efficacy of EPZ-6438, which, of the two inhibitors, displayed more selectivity in SCCOHT cells in vitro (Fig. 2D). First, we dosed BIN67 xenograft-bearing mice with EPZ-6438 (100 mg/kg or 200 mg/kg, n=10) twice a day [28-30]. BIN67 xenografts harvested three hours following the EPZ-6438 treatment displayed a significantly lower amount of histone H3K27me3 than vehicle-treated tumors (Supplemental Fig. S3), demonstrating suppression of EZH2 enzymatic activity by EPZ-6438. Accordingly, EPZ-6438 at either 100 or 200 mg/kg suppressed tumor growth compared to those treated with vehicle (Fig. 3A, day 28, P <0.01, P <0.01, respectively). Unexpectedly, mice from all the treatment groups displayed significant weight loss due to dehydration (Supplemental Fig. S4A). After stopping dosing for 6 days to allow recovery from dehydration, the tumors in mice exposed to EPZ-6438 grew back from about 100 mm3 to 300 mm3 (Fig. 3A, day 34). To further test whether the once daily treatment with EPZ-6438 was effective in suppressing tumor growth, we resumed treatment with a daily single dose schedule until mice reached humane endpoints. This schedule at 100 mg/kg failed to delay tumor growth (Fig. 3A, days 34-48), while single daily dosing with 200 mg/kg EPZ-6438 retarded growth after 10 days (Fig. 3A). Though overall survival did not differ between two dosages, both dosages significantly improved the median survival of mice to humane endpoint from 37 days (vehicle) to 46 days 		 11	(100mg/kg) and 48 days (200mg/kg) mainly due to the benefit from the first week of twice daily dosing (Fig. 3B, P = 0.0036 and 0.0017, respectively).   Next, we tested the efficacy of EPZ-6438 in the SCCOHT-1 xenograft model. Since SCCOHT-1 cells displayed much higher sensitivity to EZH2 inhibitors than any other cell lines (Fig. 2E), we dosed the mice with 200 mg/kg EPZ-6438 once daily (n=6), which produced only mild effect on the mouse body weight (Supplemental Fig. S4B). Tumor growth was effectively suppressed by the once daily treatment of EPZ-6438 at 200 mg/kg wherein the tumor growth doubling time (tumor volume from 150 mm3 to 300 mm3) increased from 5 days (vehicle) to 17 days (EPZ-6438) (Fig. 3C). The average tumor weight was also reduced by 50% in the EPZ-6438-treated group compared to the vehicle-treated group at the end of three weeks of treatment (Fig. 3D, p < 0.05). These data suggest that once daily treatment of EPZ-6438 at 200 mg/kg significantly slowed down the growth of SCCOHT-1 xenografts.  Effects of EPZ-6438 on the proteome of BIN67 cells To identify the molecular mechanisms driving growth inhibition caused by EZH2 inhibitors in SCCOHT cells, we determined the proteomic profiles of BIN67 cells treated with EPZ-6438 or vehicle using mass spectrometry. Unsupervised hierarchical clustering resulted in clustering based on the treatment conditions (Supplemental Fig. S5) with approximately 8.4% (571/6768) and 7.4% (498/6768) being significantly upregulated or downregulated, respectively, in BIN67 cells treated with 1 µM EPZ-6438 for 7 days versus those treated with DMSO (Fig. 4A and Supplemental Table S1 and S2, pfdr<0.05 and log2FC>mean+SD or <mean-SD). Ingenuity pathway analysis (IPA) of the significantly altered proteins by EPZ-6438 revealed a significant enrichment of proteins involved in biological functions, such as “Cell cycle”, “Cellular Assembly 		 12	and Organization” and “Cellular development” (Fig. 4B, Supplemental Table S3). Particularly, both “development of neurons” and several processes related to neuronal development, such as “formation of cellular protrusions”, “microtubule dynamics” and “neuritogeneis”, were predicted to be enhanced significantly in EPZ-6438-treated BIN67 cells (Fig. 4C, Supplemental Table S3, z>2). Clustering of the enriched proteins confirmed that many of the identified proteins involved in microtubule dynamics, formation of celluar protrusions and organization of cytoskeleton or cytoplasm were also involved in development of neurons (Supplemental Fig. S6), supporting their engagement in neuron development. These data suggest that EPZ-6438 not only altered the cell cycle of SCCOHT cells, but also triggered significant changes in cell organization and assembly, leading to neuron-like differentiation.    EPZ-6438 induces SCCOHT cell cycle arrest and apoptosis  Next, we used flow cytometry to validate the effect of EPZ-6438 on cell cycle distribution of SCCOHT cells. FACS analysis demonstrated that 1 µM EPZ-6438 treatment induced cell cycle arrest at G1 in a time-dependent manner in BIN67 and COV434 cells (Fig. 5A), and also triggered cell death (sub-G1 population) beginning at 7 days after drug exposure in both BIN67 and COV434 cell lines, consistent with a mild induction of apoptosis predicted by proteomic data (Supplemental Table S4). In contrast, 1 µM EPZ-6438 induced cell death more rapidly (3 days) and potently in SCCOHT-1 cells (Fig. 5A), consistent with the lower IC50 in this cell line than others (Fig. 2E). To precisely monitor the induction of apoptosis, we measured the activation of caspase-3/7 through fluorescent microscopy coupled with live cell imaging (Fig. 5B). This revealed that EPZ-6438 rapidly induced cell apoptosis in SCCOHT-1 cells after 48 hours of EPZ-6438 treatment at either 0.3 or 1 µM, whereas apoptosis in BIN67 and COV434 cells increased 		 13	gradually beginning at about 120 hours of exposure to 1 µM EPZ-6438 (Fig. 5C). Next, we employed western blotting to determine the expression of some cell cycle and apoptosis regulating genes that were significantly altered by EPZ-6438 in BIN67 cells by proteomic profiling. We confirmed that the expression of CDKN1A (p21) and BAD (Log2FC =0 .7 and 1.4, respectively, Supplemental Table S1) was gradually induced by EPZ-6438, while that of Myc (Log2 FC = -0.9, Supplemental Table S2) was potently down-regulated by EPZ-6438 in all three SCCOHT cell lines (Fig. 5D). In contrast, the expression of CDKN2A (p16) (Log2 FC = 0.2, Supplemental Table S1), which is known to play an important role in EZH2 inhibitor-triggered growth arrest [31], was only significantly induced upon EPZ-6438 treatment in SCCOHT-1 cells (Fig. 5D).   EPZ-6438 induces neuron-like differentiation in SCCOHT cells In agreement with the prediction of induced neuron development in BIN67 cells from proteomic profiling, the surviving SCCOHT cells displayed a neuron-like morphology after prolonged exposure to 1 µM EPZ-6438 (Fig. 6A). In contrast, SCCOHT cells exposed to cytotoxic agents, such as cisplatin, etoposide, and paclitaxel, displayed no morphology change other than fragmentation during cell death (Supplemental Fig. S7). Immunofluorescence confirmed that the expression of MAP2, a specific marker of neuronal differentiation [32], was increased upon EPZ-6438 treatment in a time-dependent manner in BIN67 cells (Fig. 6B), consistent with increased expression with treatment observed in the proteomic data (Log2 FC = 2.2, Supplementary Table S2).  Western blot analysis further confirmed that the expression levels of two neuronal markers (MAP2 and TUBB3 (βIII tubulin), Log2FC=1.2, Supplementary Table S2) were induced by EPZ-6438 treatment (Fig. 6C). In agreement with the induction of differentiation, the expression 		 14	of EZH2 (Log2FC=-0.4, Supplementary Table S1) also dropped significantly upon EPZ-6438 treatment in a time-dependent manner (Fig. 6C). Therefore, our data show that unlike cytotoxic agents, EZH2 inhibitors can induce expression of markers of neuronal differentiation of SCCOHT cells.    Discussion Transformation of normal cells requires acquisition of hallmark characteristics of cancer including survival and proliferation even in the presence of counteracting signals. These features usually include extensive rewiring of cellular signaling networks driven by mutations and deregulation of oncogenes and tumor suppressors [33], leading to a strict reliance on either an oncogenic driver event (oncogene addiction) or the activity of certain gene products that are not essential in normal cells (non-oncogene addiction or synthetic lethality) [34]. Our findings suggest that inactivation of SMARCA4 in SCCOHT’s unknown precursor cells may rewire their cellular signaling network to be dependent on the catalytic activity of the histone methyltransferase EZH2 in transcriptional repression. In support of this notion, previous studies have shown that SMARCA4 and the associated SWI/SNF chromatin-remodeling complex can suppress EZH2 either directly [21] or through repression of E2F transcription factors [35-37].  Concordantly, re-expression of SMARCA4 in SCCOHT cell lines lowered the expression of EZH2 and reduced the global level of histone H3K27me3, suggesting that SMARCA4 loss may promote SCCOHT tumorigenesis, at least partially, through the direct up-regulation of EZH2 expression.  In addition to this work, several other studies have demonstrated the requirement for the methyltransferase activity of EZH2 in cancers with a defective subunit of the SWI/SNF complex.  		 15	However, two ARID1A-deficient ovarian clear cell carcinoma cell lines (OVISE and OVTOKO) and several lung cancer cell lines with SMARCA4 deficiency or SMARCA4/SMARCA2 dual deficiency did not respond or responded poorly to EPZ-6438 treatment in our study (Fig. 2C and Supplemental Fig. S3). Therefore, the absence of subunits of the SWI/SNF complex alone does not predict sensitivity to inhibition of EZH2 catalytic activity. Whether the efficacy of EZH2 inhibitors depends upon additional genetic or epigenetic features of the tumor remains unclear.  Of note, both SCCOHT and MRT cell lines [28] that respond to EZH2 inhibitors lack the core subunits of SWI/SNF complex (either SMARCA4/A2 or SMARCA2/SMARCB1 deficiency), while ARID1A-deficient ovarian clear cell carcinoma cells usually retain an active complex that includes ARID1B [38].  Similarly, most SMARCA4-deficient ovarian and lung carcinomas retain SMARCA2 expression and its associated chromatin remodeling activity. Accordingly, it has been reported that ARID1A-deficient ovarian cancer and SMARCA4-deficient lung cancer depend on ARID1B and SMARCA2, respectively, for maintaining their oncogenic property [17,18,38]. Furthermore, most tumors with SMARCA4 or ARID1A loss usually display a higher mutation burden in comparison to the minimally disturbed genomes of either SCCOHT or MRT [8,39-41].  The lack of additional mutations in the latter tumors implicates a dependence upon other epigenetic changes such as EZH2 overexpression.  Thus, SMARCA4 inactivation in the absence of SMARCA2 may rewire the cellular signaling network to be dependent on EZH2-mediated oncogenesis while additional mutations possibly negate this requirement in other cancers.  Although the potency of EZH2 inhibitors has been demonstrated in several cancer types, the molecular mechanisms are still not well understood. EZH2 is the catalytic component of the PRC2 complex that mediates the transcriptional repression of targets by trimethylation of histone H3 at lysine residue 27 in their promoters. Therefore, the PRC2 complex could promote 		 16	tumorigenesis by specifically repressing tumor-suppressor genes, including the major tumor suppressor locus CDKN2A, through recruitment of DNA methyltransferases. Accordingly, treatment with EZH2 inhibitors can rescue the expression of p16 in cancer cells, such as MRT cells and leukemia stem cells [28,42]. Unlike MRT cells, in only one of the three SCCOHT cell lines (SCCOHT-1) was p16 expression altered upon EPZ-6438 treatment. The other two cell lines expressed substantial amounts of p16, which were not further induced upon the EPZ-6438 treatment. Therefore, a different mechanism may underlie the efficacy of EPZ-6438 in the BIN67 and COV434 cell lines.  Proteome analysis revealed that EPZ-6438 altered about 15% of the proteome in BIN67 cells with a significant enrichment of proteins involved in cell cycle control and development of neurons. These results implicate these pathways as the major biological events behind the cellular response to EZH2 inhibitors in SCCOHT cells. Particularly, we discovered that the expression of several cell cycle control genes, such as Myc, were repressed by EPZ-6438. Although the PRC2 complex may upregulate the expression of these genes indirectly through suppressing the negative regulators of these genes, it has been suggested that the transcription of Myc can be activated directly by the PRC2 complex in glioblastoma cancer stem cells [43]. Given that Myc activation is a hallmark of tumor initiation and maintenance, suppression of Myc may have a significant contribution to the efficacy of EZH2 inhibitors in SCCOHT and other cancers, such as MRT and glioblastoma [43].  Furthermore, we also observed a time-dependent induction of BAD pro-apoptotic protein in three SCCOHT cells following EPZ-6438 treatment. By inactivating the function of the anti-apoptotic proteins Bcl-2 and Bcl-xl, BAD can promote apoptosis [44]. Our data together with the previous study showing that BAD was up-regulated upon EZH2 depletion by shRNA in lung cancer cell lines [45] suggest that it may play a key role in EZH2 inhibitor-		 17	triggered apoptosis. In addition to causing cell cycle arrest and apoptosis, prolonged exposure to EZH2 inhibitors also drove the differentiation of SCCOHT cells towards the neuronal lineage. Consistent with the neuron-like differentiation, EPZ-6438 caused a late induction of p21 (Fig. 4F), a cyclin kinase inhibitor that may drive neural precursor cells into cycle exit and differentiation [46]. It will be interesting to determine whether depletion of p21 can prevent the neuron-like differentiation caused by EZH2 inhibitors.  A neuron-like morphology change has also been reported in G401 cells upon EPZ-6438 treatment [28]. Both genomic analysis and mouse transgenic models have provided evidence that some MRT may arise from neural precursor cells [41,47]. Interestingly, some studies suggest that SCCOHT is the MRT of ovary. Therefore, our present finding, that EZH2 inhibition induced markers of neuronal differentiation of SCCOHT, suggest that SCCOHT may develop from multi-potent stem cells inside the ovary with the capability of undergoing neuronal differentiation.  In support of this model, extensive IHC analysis of multiple sections of two SCCOHT cases previously revealed rare foci of immature teratoma [11], which mainly contain primitive neuroepithelium in their immature region [48]. Future studies are therefore needed to explore the possible cellular origin of SCCOHT from neural precursor cells.  SCCOHT is a rare but extremely aggressive disease for young women.  No effective treatment strategies have been developed for fighting this lethal disease. In the present study, we provide support that the methyltransferase EZH2 is universally expressed in SCCOHT and may serve as a potential therapeutic option for young women with this deadly disease.  Importantly, Epizyme pharmaceutics has recently launched a phase 1 clinical trail for evaluating the toxicity of EPZ-6438 in solid tumors with SMARCB1 or SMARCA4 deficiency (clinicaltrials.gov).  Their interim results showed that EPZ-6438 (Tazemetostat) was well tolerated and elicited a 		 18	partial response by RECIST criteria in two SCCOHT patients previously treated with chemotherapy (www.epizyme.com).  Therefore, identifying other targeted therapy strategies and determining the efficacy of combining EZH2 inhibitors with other putative targeted therapies for SCCOHT treatment remains a high priority.  Acknowledgements We acknowledge for the technical support from Sarah Maines-Bandiera, Winnie Yang, Christine Chow, Nicole Wretham, Dana Masin, Hong Yan, Jenna Rawji and Chris Ke-dong Wang. We thank Drs. Barbara Vanderhyden and Ralf Hass for providing BIN67 and SCCOHT-1 cells, respectively. This work was supported by research funds from the Canadian Cancer Society Research Institute (#703458, to D.G.H.) and the National Institute of Health (1R01CA195670-01, to D.G.H., J.T. and B.W.), the Terry Fox Research Institute Initiative New Frontiers Program in Cancer (D.G.H.), the British Columbia Cancer Foundation, the Marsha Rivkin Center for Ovarian Cancer Research, the Ovarian Cancer Alliance of Arizona, the Small Cell Ovarian Cancer Foundation, and philanthropic support to the TGen Foundation.  Statement of author contributions Y.W. designed and performed experiments, analyzed data and wrote the manuscript. S.Y.C. performed experiments and analyzed data. A.N.K. performed IHC, analyzed data and edited the manuscript. S.C. performed mass spectrometry experiments, analyzed data and wrote the manuscript. N.D.S helped the design of xenograft studies and analyzed data. J.L., W.P.D.H., K.A.O., B.E.W., G.B.M. and J.F.T. provided thoughtful discussion and edited the manuscript. 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(A) The expression of PRC2 complex proteins in three SCCOHT versus two normal ovaries were extracted out from our previous Agilent microarray analyses (GE access No.: GSE49887 and GSE66434). (B) The expression of EZH2 protein in multiple cell lines was analyzed by Western blotting analysis. (C) The expression of EZH2 protein in primary SCCOHT samples was determined using immunohistochemistry on TMA. Representative images of strong or variable staining are shown. (D) SCCOHT cell lines were infected with lentivirus expressing either GFP or SMARCA4. Cells were harvested 72 hours post infection for determining the effect of SMARCA4 re-expression on EZH2 and histone H3 K27me3 by Western blotting. * P<0.001.  Figure 2. SCCOHT cells are sensitive to EZH2 depletion and suppression. (A, B) Cells were infected with lentivirus expressing control scramble shRNA or EZH2 shRNA followed by puromycin selection for 48 hours. Cells were then reseeded in 24-well plates for 6 days before being fixed and quantitated by crystal violet staining assay. (C, D) Cells were treated as indicated for 3 days for Western blot analysis of histone H3K27me3 level. (E) Cells were seeded in 96-well plates, treated with EZH2 inhibitors GSK126 or EPZ-6438 at indicated doses and incubated for 6 days and measured for cell survival by crystal violet assay. (F) The IC50s of cell lines to EZH2 inhibitors in (E) were compared between SCCOHT lines and other ovarian lines. Note: 20µM was assigned to the cell lines that were not responsive to EZH2 inhibitors at 10µM. * P<0.01, ** P<0.001  		 24	Figure 3. In vivo efficacy of EPZ-6438 in BIN67 mouse xenograft model. (A-B) The efficacy of EPZ-6438 was evaluated in BIN67-derived mouse subcutaneous xenograft model. Tumor volume and the overall survival of each treated group until the study endpoint (see Material and Methods for details) were plotted against the days post cell inoculation, respectively. (C, D) The efficacy of EPZ-6438 was evaluated in SCCOHT-1-derived mouse subcutaneous xenograft model. Tumor volume of either vehicle or 200 mg/kg EPZ-6438-treated group was plotted against the days post cell inoculation (C). Final tumor weight was determined and compared between two treated groups (D). * P<0.05, ** P<0.01, *** P<0.001  Figure 4. The effect of EPZ-6438 on the proteome of BIN67 cells. (A) Volcano plot of the proteome of BIN67 cells exposed to EPZ-6438 or vehicle. Cells were treated with either DMSO or 1µM EPZ-6438 for 7 days and then processed for proteomic profiling. Peptide data were subjected to PECA analysis for identification of significantly altered proteins (p.fdr<0.05 and Log2FC>mean+SD or <mean-SD). (B) IPA analysis of significantly altered proteins caused by EPZ-6438 identified top affected biological functions by EPZ-6438 treatment. Any biological function with an activation z-score greater than 2 or less than -2 was predicted to be significantly increased or decreased by IPA analysis. (C) IPA analysis identified significantly increased cellular activities related to neuronal development. (D) Clustering analysis of proteins in significantly altered biological functions.  Figure 5. EPZ-6438 induces SCCOHT cell cycle arrest and apoptosis. (A) FACS analysis of cell cycle profiles in SCCOHT cells after DMSO or 1µM EPZ-6438 treatment for the shown times. (B, C) SCCOHT cell lines were treated with 1µM EPZ-6438 and cultured in the presence 		 25	of a cell-permeable fluorescent dye for monitoring activated caspase-3/7 activity with IncuCyte live cell imaging system (see Materials and Methods). Apoptotic index was calculated by dividing the overall fluorescent object counts to cell numbers under each condition and plotted over incubation time. (D) The effects of EPZ-6438 (BIN67/COV434: 1µM; SCCOHT-1: 0.25µM) on the expression of Myc, BAD, p16 and p21 proteins was determined by western blotting.   Figure 6. EPZ-6438 induces neuronal differentiation in SCCOHT cells. (A) Morphology of SCCOHT cells after prolonged exposure to EPZ-6438. Cells were treated with EPZ-6438 at either 1µM (BIN67/COV434) or 0.25µM (SCCOHT-1) for 12 days and characterized by phase contrast microscopy. (B) BIN67 cells were fixed and immunostained for MAP2, a selective neuronal marker. (C) Western blotting for neuronal proteins from SCCOHT cells treated with EPZ-6438 at either 1µM (BIN67/COV434) or 0.25µM (SCCOHT-1) for the days shown in the panel. Vinculin serves as a loading control.      A	C	 Strong	staining	of	EZH2	Fig	1.	EZH2	is	abundantly	expressed	in	SCCOHT	0	2	4	6	8	10	12	EZH2 EZH1 EED SUZ12 Relative gene expression Normal ovary SCCOHT *	D	SMARCA4								-													+																		-														+																-											+		BIN67	 SCCOHT1	H3K27Me3	Vinculin	EZH2	SMARCA4	Total	H3	B	NOY1	SVOG3e	OVTOKO	OVISE	JHOC5	G401	BIN67	SCCOHT1	COV434	OVCAR8	EZH2	Vinculin	19/24	(79%)	Variable	staining	of	EZH2	5/24	(21%)	COV434	Fig.	1	(Wang	et	al,	2016)	Fig.2	SCCOHT	cells	are	sensiMve	to	EZH2	suppression	EZH2 shctrl     shEZH2_1 shEZH2_2 H3K27Me3 H3 BIN67 A	 B	C	 D	0				0.1			0.5								0					0.1			0.5						0				0.1			0.5	EPZ-6438	(μM	)	BIN67	 SCCOHT1	H3K27Me3	0						1							5						0					0.1				1									0							1						5	H3	GSK126	(μM)	0	20	40	60	80	100	120	140	% Growth shCtrl	shEZH2_1	shEZH2_2	**	*	**	**	**	**	EPZ-6438	-2 -1 0 1050100Log10(conc)% SurvivalEPZ-6438BIN67SCCOHT1COV434G401ES-2RMG1OVCAR-8OVISEOVTOKONOY1F	-2 -1 0 1050100Log10(conc)% SurvivalGSK126BIN67SCCOHT1COV434G401ES-2RMG1OVCAR-8NOY1GSK126	SCCOHT		lines	Other	ovarian		lines	SCCOHT		lines	Other	ovarian		lines	**	*	GSK126	 EPZ-6438	IC50 (µM) E	Fig.	2	(Wang	et	al,	2016)	H3K27Me3	H3	COV434	 BIN67	 SCCOHT1	 COV434	BIN67	 COV434	SCCOHT-1	 ES-2	SCCOHT	lines	Fig.3		In	vivo	efficacy	of	EPZ-6438	in	mouse	xenogra\	models	0	200	400	600	800	1000	1200	13	 18	 23	 28	 33	 38	 43	 48	Tumor volume (mm3 ) Days after cell inoculation vechicle	100mg/Kg	EPZ-6438	200mg/Kg	EPZ-6438	Treatment	started	(BID)	Treatment halted Treatment		re-started	(QD)	A	0	20	40	60	80	100	120	0	 10	 20	 30	 40	 50	Percentage	Survival		Days	aYer	cell	inoculaZon	vechicle	100mg/Kg	EPZ-6438	200mg/Kg	EPZ-6438	B	C	BIN67	xenogra\	model	 BIN67	xenogra\	model	D	SCCOHT-1	xenogra\	model	 SCCOHT-1	xenogra\	model	0	200	400	600	800	1000	1200	10	 15	 20	 25	 30	 35	Tumor Volume (mm3 ) Days	post	inoculaZon	Vehicle	200mg/kg	EPZ-6438	0.0	0.5	1.0	1.5	2.0	Vehicle	 EPZ-6438	Average	final	tumor	weight	(g)	Treatment	started	(QD)	*	**	 **	**	**	*	*	*	**	***	***	**	**	ss	ve i le ss	hic  Fig.	3	(Wang	et	al,	2016)	Fig.	4	Effect	of	EPZ-6438	on	the	proteome	of	BIN67	cells	B	C	0	 2	 4	 6	 8	formaZon	of	cellular	protrusions	microtubule	dynamics	neuritogenesis	development	of	neurons	-Log2P-value	Log2(FC)	A	-4	 -2	 0	 2	 4	AcZvaZon	z-Score	Cell	Cycle	Cellular	Assembly	and	OrganizaZon	Cellular	Development	Cell-To-Cell	Signaling	and	InteracZon	Cellular	Movement	Cellular	FuncZon	and	Maintenance	Cell	Death	and	Survival	Cellular	Growth	and	ProliferaZon	DNA	ReplicaZon,	RecombinaZon,	and	Repair	1.00E-12	1.00E-10	1.00E-08	1.00E-06	1.00E-04	P-value	Fig.	4	(Wang	et	al,	2016)	Fig.	5	EPZ-6438	induces	cell	cycle	arrest	and	apoptosis	in	SCCOHT	cells	Phase Activated caspase3/7 EPZ-6438	DMSO	B	A	C	Myc	BIN67	 SCCOHT-1	EPZ-6438			0					3					7				10							0						3				7			10						0						4						7	days	p21	COV434	BAD	AcZn	p16	BIN67	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	120	 144	 168	 192	 216	ApoptoZc	Index	Time	aYer	EPZ-6438	treatment	(hours)	DMSO	0.3μM	EPZ-6438	1	μM	EPZ-6438	COV434	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	120	 144	 168	 192	 216	ApoptoZc	index	Time	aYer	EPZ-6438	treatment	(hours)	DMSO	0.3μM	EPZ-6438	1	μM	EPZ-6438	Fig.	5	(Wang	et	al,	2016)	0	20	40	60	80	Control Day 3 Day 7 Day 12 %	Cells	COV434	0	20	40	60	80	Control	 Day	3	 Day	7	 Day	12	%	cells	BIN67	0	20	40	60	80	Control	 Day	3	 Day	7	%	Cells	SCCOHT1	subG1	G1	S	G2/M	D	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0	 24	 48	 72	 96	ApoptoZc	index	Tim	aYer	EPZ-6438	treatment	(hours)	DMSO	0.3μM	EPZ-6438	1μM	EPZ-6438	SCCOHT-1	Fig.6	EPZ-6438	induces	neuron-like	differenMaMon	in	SCCOHT	cells	B	AMAP2	 DAPI	 Merged	DMSO	EPZ-6438		day	7	EPZ-6438		day	12	BIN67	 COV434	MAP2	EPZ-6438										0							3							7							12												0							3								7						12									0									4									7			days	TUBB3	EZH2	Vinculin	C	BIN67	 SCCOHT1	 COV434	SCCOHT-1	Fig.	6	(Wang	et	al,	2016)	 Supplemental Material and Methods   Plasmids and lentivirus/retrovirus packaging The pLDpuro-SMARCA4 plasmid was constructed by introducing the SMARCA4 from entry vectors (Genecopeia) into the pLDpuro-EnVA destination vector (a gift of Dr. Jason Moffat at the University of Toronto) using the Gateway reaction (Life Sciences). Two EZH2 shRNAs were obtained from Sigma (Mission shRNA: TRCN10475 and TRCN286227). To produce lentivirus, pLDpuro-GFP, SMARCA4, plko.1-scramble control or plko.1-shEZH2 plasmids were co-transfected with packaging plasmids psPAX2 and pMD2.G into HEK293T cells. Supernatants were collected at 48 and 72 hours for lentivirus harvesting.   Cell viability assay Cells were seeded in 96-well plates in quadruplicate at a density of 500-2000 cells per well depending on the growth curve of each cell line. Twenty-four hours later, cells were treated with either DMSO, EZH2 inhibitor GSK126 (Selleckchem) or EPZ-6438 (Medkoo) at doses ranging from 0-20 µM or 0-10 µM, respectively (decreasing in two-fold dilution from the highest concentration). Six days after treatment, cells were fixed in 10% methanol-10% acetic acid and then stained with 0.5% crystal violet in methanol. The absorbed dye was resolubilized with 10% acetic acid in water and measured spectrophotometrically at 595 nM in a microplate reader. Cell survival was calculated by normalizing the absorbance to that of DMSO-treated controls.  Cell cycle and apoptosis analysis To determine the cell cycle profile, cells treated with vehicle or EPZ-6438 were fixed with 70% ice-cold ethanol, stained for DNA content with propidium iodide and subjected to flow cytometry analysis to quantitate cell cycle distribution. To monitor apoptosis, the cell-permeable dye (NucViewTM 488 dye) coupled to an activated caspase-3/7 recognition motif (Essen Bioscience) was added to the culture medium of cells treated as indicated. Upon activation through cleavage by the activated caspase-3/7 during apoptosis, the dye was released and bound to DNA fluorescently, which was detected using the IncuCyte live cell imaging system. The apoptotic index was calculated by dividing the overall fluorescent object counts by cell numbers under each condition. Western blotting Whole-cell extracts were obtained for SDS-PAGE electrophoresis as previously described [25]. Primary antibodies used were rabbit anti-SMARCA4 (Abcam, ab110641), H3K27Me3 (Millipore, 07-449), Myc (Abcam, ab32072), mouse anti-EZH2 (BD Bioscience, 612667), vinculin (Sigma, v9264), Actin (Sigma, A5441) , Histone H3 (Abcam, ab1791), MAP2 (Millipore, MAB3418), TUBB3 (Santa Cruz, sc-51670), BAD (Santa Cruz, sc-8044), p16 (Ventana, 725-4713) and p21 (BD Pharmacogen, 556430) and horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (Amersham). Chemiluminescence was used for detection (Perkin–Elmer Life Sciences). Actin or vinculin were used as protein loading controls. Immunofluorescence and microscopy Cells were grown on coverslips, treated with EZH2 inhibitors for indicated time and then fixed with 2% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100 and then blocked with 3% BSA containing 0.1% Triton X-100. Cells were immunostained for chicken anti-MAP2 (ab5392, Abcam) and AlexaFluro488 conjugated anti-chicken secondary antibody (Life Technologies). Coverslips were then mounted on slides with DAPI-containing mounting medium (Vector Laboratories). Fluorescence images were obtained using a Zeiss LSM 780 confocal microscope (Carl Zeiss).    Supplemental Figures  Fig. S1 EZH2 inhibitors suppressed the proliferation of SCCOHT cells. BIN67 (A) or COV434 (B) cells were treated with EPZ-6438 or GSK126 at indicated doses for 6 days and then re-seeded in 96-well plates at 2000 cells/well in the presence of the indicated drugs before splitting. Cell growth (confluence of wells) was determined and plotted by live cell imaging with an IncuCyte live cell imaging system.    Fig. S2 SMARCA4-deficient lung cancer cells and ovarian clear cell carcinoma cells are not sensitive to EPZ6438. Cells were seeded in 96-well plates and treated with EPZ-6438 at doses ranging from 0.02-10 µM for 6 days before being fixed and quantified for the remaining surviving cells.      BIN67 A B Fig. S3 Effect of EPZ-6438 on histone H3K27me3 in xenograft models. BIN67 xenografted tumors were harvested 3 hours after either vehicle or 100 mg/Kg EPZ-6438 treatment on day 28 (n = 3 for each condition). H3K27me3 levels were determined by Western blotting in comparison to total H3 levels.  H3K27Me3 H3 1						2					3						1					2					3		 Control 	EPZ-6438		 Fig.S4 Mouse body weight changes in BIN67 and SCCOHT-1 xenograft models for the EPZ-6438 efficacy study.     19	20	21	22	23	24	10	 15	 20	 25	 30	 35	 40	 45	 50	Body	weight	(g)	Days	post	innoculation	Body	weight	change-BIN67	xenograft	Vehicle	EPZ6438	100mg/kg	EPZ6438	200mg/kg	A	18	19	20	21	22	23	24	10	 15	 20	 25	 30	 35	Mouse	body	weight	(g)	Days	post	inoculation	Body	weight	change-SCCOHT-1	xenograft	Vehicle	EPZ-6438	B	Fig. S5 Unsupervised clustering analysis of proteins identified by mass spectrometry in BIN67 cells treated with DMSO or EPZ-6438 for 7 days (n=3).     Fig. S6 Clustering analysis of proteins involved in each significantly altered biological function predicted IPA analysis.    Fig. S7 Cytotoxic agents do not induce neuron-like morphologies in SCCOHT cells. Representative photomicrographs of BIN67 cells treated with DMSO, 2 uM cisplatin, 3 nM Paclitaxel or 5 uM etoposide for 6 days were shown.      paclitaxel control cisplatin etoposide 

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