SS18-SSX-Dependent YAP/TAZ Signaling in Synovial Sarcoma

Author Manuscript Published OnlineFirst on February 27, 2019; DOI: 10.1158/1078-0432.CCR-17-3553 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

SS18-SSX-dependent YAP/TAZ Signaling in Synovial Sarcoma

Ilka Isfort1,2, Magdalene Cyra1,2, Sandra Elges2, Sareetha Kailayangiri3, Bianca Altvater3, Claudia Rossig3,4, Konrad Steinestel2,5, Inga Grünewald1,2, Sebastian Huss2, Eva Eßeling6, Jan-Henrik Mikesch6, Susanne Hafner7, Thomas Simmet7, Agnieszka Wozniak8, Patrick Schöffski8, Olle Larsson9, Eva Wardelmann2, Marcel Trautmann1,2§, and Wolfgang Hartmann1,2#§

1 Division of Translational Pathology, Gerhard-Domagk-Institute of Pathology, Münster University Hospital, Germany 2 Gerhard-Domagk-Institute of Pathology, Münster University Hospital, Germany 3 Department of Pediatric Hematology and Oncology, University Children´s Hospital Münster, Germany 4 Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster, Germany 5 Institute of Pathology and Molecular Pathology, Bundeswehrkrankenhaus Ulm, Germany 6 Department of Medicine A, Hematology, Oncology and Respiratory Medicine, University Hospital Münster, Germany 7 Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University, Germany 8 Laboratory of Experimental Oncology, Department of Oncology, KU Leuven, and Department of General Medical Oncology, University Hospitals Leuven, Leuven Cancer Institute, Leuven, Belgium 9 Departments of Oncology & Pathology, The Karolinska Institute, Stockholm, Sweden

 Authors contributed equally

Running title: YAP/TAZ signals in synovial sarcoma Keywords: Synovial sarcoma, SS18-SSX, YAP, TAZ, verteporfin

§Correspondence: Wolfgang Hartmann & Marcel Trautmann; Division of Translational Pathology, Gerhard-Domagk-Institute of Pathology, Münster University Hospital, 48149 Münster, Germany; Phone: +49 (0) 251-83-58479 and -57623; Fax: +49 (0) 251-83-57559 E-mail: [email protected]; [email protected]

The authors declare no potential conflicts of interest.

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 27, 2019; DOI: 10.1158/1078-0432.CCR-17-3553 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

YAP/TAZ signals in synovial sarcoma

STATEMENT OF TRANSLATIONAL RELEVANCE

Acting as a powerful transcriptional dysregulator, the chimeric SS18-SSX fusion protein constitutes the major oncogenic driver of synovial sarcoma. Given the notorious difficulty to target the fusion protein itself, functional insights into SS18-SSX-shaped tumor biology are essential to decipher druggable tumor vulnerabilities. We here describe a molecularly based mechanism of YAP/TAZ activation in synovial sarcoma effected by the SS18-SSX fusion protein that involves an IGF-II/IGF-IR signaling loop, leading to dysregulation of the Hippo effectors LATS1 and MOB1 and provide evidence of high efficacy of a YAP/TAZ-directed therapeutic approach in synovial sarcoma in vitro and in vivo. Our study highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis and refines the concept of biologically founded molecular strategies to inhibit SS18-SSX-driven tumorigenesis.

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YAP/TAZ signals in synovial sarcoma

ABSTRACT Purpose: Synovial sarcoma is a soft tissue malignancy characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein acts as a transcriptional dysregulator representing the major driver of the disease; however, the signaling pathways activated by SS18-SSX remain to be elucidated in order to define innovative therapeutic strategies.

Experimental Design: Immunohistochemical evaluation of the Hippo signaling pathway effectors YAP/TAZ was performed in a large cohort of synovial sarcoma tissue specimens. SS18-SSX dependency and biological function of the YAP/TAZ Hippo signaling cascade were analyzed in five synovial sarcoma cell lines and a mesenchymal stem cell model in vitro. YAP/TAZ-TEAD-mediated transcriptional activity was modulated by RNAi-mediated knockdown and the small molecule inhibitor verteporfin. The effects of verteporfin were finally tested in vivo in synovial sarcoma cell line-based avian chorioallantoic membrane and murine xenograft models and a patient-derived xenograft.

Results: A significant subset of synovial sarcoma showed nuclear positivity for YAP/TAZ and their transcriptional targets FOXM1 and PLK1. In synovial sarcoma cells, RNAi-mediated knockdown of SS18-SSX led to significant reduction of YAP/TAZ-TEAD transcriptional activity. Conversely, SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ-dependent signals, mechanistically mediated by an IGF-II/IGF-IR loop leading to dysregulation of the Hippo effectors LATS1 and MOB1. Modulation of YAP/TAZ-TEAD-mediated transcriptional activity by RNAi or verteporfin treatment resulted in significant growth inhibitory effects in vitro and in vivo.

Conclusions: Our preclinical study identifies an elementary role of SS18-SSX-driven YAP/TAZ signals, highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis and provides evidence for innovative therapeutic approaches.

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YAP/TAZ signals in synovial sarcoma

INTRODUCTION Synovial sarcoma (SySa) is an aggressive malignancy comprising approximately 2% of all sarcomas with a predominance in adolescents and young adults (1, 2). Wide surgical resection, radiotherapy and chemotherapy with ifosfamide and doxorubicin represent established therapeutic options; however, prognosis in the metastatic situation is poor (3-5). Specific molecularly targeted therapeutic approaches are currently limited. The molecular hallmark of SySa is a pathognomonic reciprocal t(X;18)(p11;q11) translocation, leading to the fusion of SS18 (SYT) and one of the homologues SSX genes (most frequently SSX1 or SSX2, in rare cases SSX4), generating chimeric SS18-SSX fusion proteins (6-8). Although the SS18-SSX protein is known to play a crucial role in SySa tumorigenesis, its specific biological function and its mechanism of action remain to be elucidated. Neither SS18 and the SSX proteins, nor the chimeric SS18-SSX oncoproteins feature known DNA-binding motifs; however, they have been reported to contribute to the dysregulation of gene expression through association with SWI/SNF and Polycomb chromatin remodeling complexes (8-12). The YAP/TAZ Hippo signaling pathway is an evolutionarily conserved pathway essential in the control of tissue homeostasis and organ size through the regulation of cell proliferation, apoptosis, and stem cell self-renewal (13-15). The central component is a kinase module comprising the serine-threonine kinases MST1/2 and LATS1/2, complemented by the adaptor proteins SAV1 and MOB1 to control the transcriptional co-activators YAP and TAZ. While in their non-phosphorylated state YAP and TAZ translocate to the nucleus and interact with TEAD1-4 transcription factors to induce expression of target genes such as CTGF, CYR61, PLK1 and FOXM1, LATS1/2-mediated phosphorylation of YAP/TAZ results in their cytoplasmic retention and proteasomal degradation (14, 16). Though convincing data on the oncogenic function of YAP/TAZ signals in several epithelial tumors is available (13), only little is known about their role in malignant soft tissue tumors. First evidence for a function of YAP/TAZ in mesenchymal tumorigenesis was gathered by St John and colleagues who demonstrated that 15% of LATS1/2-deficient mice develop metastasizing spindle-cell sarcomas (17). Analyzing genetic data from The Cancer Genome Atlas (TCGA) dataset, Eisinger-Mathason et al. detected frequent copy number losses in the Hippo pathway components and activators LATS2, NF2, and SAV1 in several high-grade sarcomas with complex karyotypes, leading to an activation of YAP signals (16). Interestingly, they did not report significant genomic alterations within the group of soft tissue tumors driven by genomic translocations. In contrast, Fullenkamp and colleagues reported high nuclear expression levels of YAP (50%) and TAZ (66%) among 159 different soft tissue malignancies, including a (minor) set of 12 SySa specimens (18). This raises the question if activation of YAP/TAZ in SySa might

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YAP/TAZ signals in synovial sarcoma be based on a functional fusion protein-dependent mechanism in place of genomic alterations affecting regulatory elements of the Hippo pathway. The current study was performed to explore the prevalence and functional relevance of YAP/TAZ signals in a large set of SySa, including its molecular dependence on the pathognomonic SS18-SSX fusion protein, and to test a molecularly targeted approach employing a small molecule YAP/TAZ inhibitor in a preclinical setting.

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YAP/TAZ signals in synovial sarcoma

MATERIALS AND METHODS

Tumor specimens and tissue microarray (TMA) SySa TMAs were prepared from formalin-fixed, paraffin-embedded (FFPE; with two representative 1 mm cores) tumor specimens selected from the archive of the Gerhard-Domagk-Institute of Pathology, Münster University Hospital, essentially as previously described (19). Two areas within each tumor were selected by experienced pathologists (E.W., W.H.) in order to represent potential heterogeneity. Necrobiotic areas and their neighborhood were excluded from TMA sampling to avoid the detection of secondary (e.g. hypoxia-induced) alterations. All diagnoses were reviewed by three experienced pathologists (S.H., S.E. and W.H.) according to the current WHO classification of tumours of Soft Tissue and Bone (2), based on morphological and immunohistochemical criteria, fluorescence in situ hybridization (FISH) or reverse transcription PCR (RT-PCR) analysis. In total, 65 SySa tissue specimens were included in the study (30/46.2% female, 35/53.8% male; median age at diagnosis was 45 years, range 8- 78 years). Forty-eight tumors belong to the monophasic subtype, 14 to the biphasic subtype, and 3 tumors were classified as poorly differentiated SySa. Median tumor size was 5 cm (range 0.3- 20 cm). In all cases, FISH or RT-PCR analysis confirmed the diagnosis of SySa, revealing the pathognomonic t(X;18) translocation as described (20). Clinicopathological characteristics of the comprehensive cohort are summarized in Table 1. The study was approved by the Ethics Committee of the University of Münster (2015-548-f-S) and conducted in accordance with current ethical standards (Declaration of Helsinki, 1975).

Immunohistochemistry (IHC) Following primary antibodies were applied: YAP (monoclonal rabbit, D8H1X, 1:100, #14074 Cell Signaling Technology), TAZ (polyclonal rabbit, 1:200, #HPA007415 Sigma-Aldrich), FOXM1 (monoclonal mouse, G-5; 1:1000, #376471 Santa Cruz), PLK1 (monoclonal rabbit, 208G4, 1:25, #4513 Cell Signaling Technology). IHC staining was performed with a BenchMark ULTRA Autostainer (VENTANA/Roche) on 3 μm TMA sections. In brief, the staining procedure included heat-induced epitope retrieval (HIER) pretreatment using Tris-Borate-EDTA buffer (pH 8.4; 95-100°C, 32-72 min) followed by incubation with respective primary antibodies for 16-120 min and employment of the OptiView DAB IHC Detection Kit (VENTANA/Roche), essentially as previously described (19). Positive and negative control stainings using an appropriate IgG subtype (DCS) were included. Immunoreactivity was assessed using a semi-quantitative score (0, negative; 1, weak; 2, moderate; and 3, strong) defining the staining intensity in the positive control (hepatocellular carcinoma) as strong. Only TMA tissue cores with at least moderate

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YAP/TAZ signals in synovial sarcoma staining (semi-quantitative score ≥2) and 30% (YAP, TAZ and FOXM1) or 5% (PLK1) positive cells were considered positive for the purposes of the study (Supplementary Table S1). The IHC readers were blinded to outcome data, the score cut point (positive = semiquantitative score ≥2) was pre-specified without prior analyses of the clinical course.

Cell culture and cell lines The human SySa cell lines HS-SY-II (expressing SS18-SSX1), FUJI, 1273/99, CME-1 and SYO- 1 (all expressing SS18-SSX2) were cultured as described previously (21). For the purpose of cell line authentication, presence of the pathognomonic SS18-SSX translocation was confirmed by RT-PCR and subsequent Sanger sequencing using specific primers for the t(X;18) translocation subtypes. The human mesenchymal stem cell line SCP-1 was kindly provided by Dr. Attila Aszodi, Munich, Germany (22). The human rhabdomyosarcoma cell line RD was obtained from the American Type Culture Collection (ATCC) and authenticated by STR-PCR. All monolayer cell cultures were grown under standard incubation condition (37°C, humidified atmosphere, 5% CO2) and maintained in Dulbecco's Modified Eagle medium (DMEM; HS-SY-II, SYO-1), Roswell Park Memorial Institute medium 1640 (RPMI; FUJI, CME-1 and RD), F-12 (1273/99) or Minimum Essential Media (MEM; SCP-1) supplemented with 10–15% fetal bovine serum (FBS; Life Technologies). Mycoplasma testing was performed quarterly by standardized PCR, and cells were passaged for a maximum of 20 to 30 culturing cycles between thawing and use in the described experiments. To study the effects of the YAP/TAZ-TEAD inhibitor verteporfin (23, 24) and the specific IGF-IR inhibitor BMS-754807 (25), SySa cells were grown in medium supplemented with 2% FBS. Short-term and long-term treatments with BMS-754807 were performed for 10 min and 36 h, respectively. Treatments with verteporfin (0.25-1 µM) to analyze effects on the protein level were performed for 14 h. Prior to stimulation with human recombinant insulin-like growth factor (IGF)-II (#50342 Biomol, 200 ng/ml IGF-II for 30 min), cells were incubated with medium devoid of FBS supplement for 4 h. Cell lysis, protein extraction and immunoblotting were performed as previously described (26). Subcellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Reverse transcription polymerase chain reaction (RT-PCR) The AllPrep DNA/RNA Mini Kit (Qiagen) was applied for RNA isolation followed by cDNA synthesis with the ProtoScript II First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions. PCR primer sequences are given in Supplementary Table S2.

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YAP/TAZ signals in synovial sarcoma

Immunoblotting Following primary antibodies were used according to the manufacturer’s instructions: CTGF (#6992) obtained from Abcam, β-Actin (#A5441) from Sigma-Aldrich, SS18/SYT (#8819) and FOXM1 (#500) both obtained from Santa Cruz Biotechnology, AKT (#9272), phospho-AKT (Ser473) (#4060), GAPDH (#5174), Histone H3 (#4499), phospho-Histone H3 (Ser10) (#9701), IGF-I Receptor β (#9750), phospho-IGF-I Receptor β (Tyr1135/1136) / phospho-Insulin Receptor β (Tyr1150/1151) (#3024), LATS1 (#3477), phospho-LATS1 (Thr1079) (#8654), MOB1 (#13730), phospho-MOB1 (Thr35) (#8699), PLK1 (#4513), SS18 (#21792), phospho-TAZ (Ser89) (#59971), YAP (#14074), phospho-YAP (Ser127) (#13008) and YAP/TAZ (#8418) all obtained from Cell Signaling Technology. The SS18-SSX fusion protein was detected with an antibody targeting the N-terminus of SS18 (which is retained in the SS18-SSX fusion oncoprotein). Secondary antibody labeling (Bio-Rad Laboratories) as well as immunoblot development was performed using an enhanced chemiluminescence detection kit (SignalFire ECL Reagent; Cell Signaling Technology) and the Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad Laboratories), as previously described (19).

Immunofluorescence (IF) IF staining procedure was as follows: Cells were grown on poly-L-lysine (0.001%) coated chamber slides (both Sigma-Aldrich), fixed in 4% paraformaldehyde (15 min), pre-treated in blocking buffer (5% BSA, 0.3% Triton X-100 in 1x PBS) and incubated with primary YAP (monoclonal rabbit, D8H1X, 1:100, #14074 Cell Signaling Technology) or TAZ (polyclonal rabbit, 1:200, #HPA007415 Sigma-Aldrich) antibody solution (4°C, overnight). After secondary antibody incubation (2 h), cells were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). To detect YAP/TAZ localization upon BMS-754807 (or DMSO) treatment (4-12 h), cells were grown in medium supplemented with 2% FBS. IF analyses were performed with a Leica DM5500 B microscope.

Stable lentiviral transduction Lentiviral transductions were performed in SCP-1 cells essentially as described previously (27) using pLenti6.2/V5-DEST_SS18-SSX1 or pLenti6.2-GW/EmGFP (Gateway cloning system, Life Technologies) and blasticidin (10 µg/ml; Life Technologies) for cell selection. EmGFP expression was verified by fluorescence microscopy and SS18-SSX1 expression by immunoblotting.

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YAP/TAZ signals in synovial sarcoma

RNA interference (RNAi) To exclude unspecific off-target effects, a set of pre-validated siRNAs for YAP (Set of 3): #1 = HSS115942, #2 = HSS115944, #3 = HSS173621, TAZ (Set of 3): #1 = HSS119545, #2 = HSS119546, #3 = HSS119547, IGF-IR: #1 = s7211, #2 = 74 and a non-targeting negative control siRNA (BLOCK-iT Alexa Fluor Red Fluorescent Control; all purchased by Life Technologies) were applied. To target the SS18-SSX fusion transcript, a set of published and pre-validated duplex oligos (28, 29) was employed (#1 sense: 5′-AAC CAA CUA CCU CUG AGA AGA-3′; antisense: 5′-UCU UCU CAG AGG UAG UUG GUU-3′, #2 sense: 5′-CAA GAA GCC AGC AGA GGA ATT-3′; antisense: 5′-UUC CUC UGC UGG CUU CUU GTT-3′ and #3 sense: 5′-AUA UGA CCA GAU CAU GCC CAA GAU U-3′; antisense: 5′-UCU UGG GCA UGA UCU GGU CAU AUU U-3′). CME-1 and FUJI cells were transfected with the indicated siRNA using Lipofectamine RNAiMAX (Life Technologies). Medium containing transfection reagent was replaced after 3-6 h with medium supplemented with 2% FBS. After incubation for 24-72 h, siRNA-transfected cells were lysed and knockdown efficiency was documented by immunoblotting. Experiments were repeated at least three times.

Luciferase reporter assay To assess YAP/TAZ-mediated transcriptional activity, SySa cells were transfected with a YAP/TAZ-responsive TEAD luciferase reporter plasmid (8xGTIIC; Addgene #34615) (30) using either Lipofectamine 2000 (Life Technologies) or Viromer RED (Lipocalyx) transfection reagents. Luciferase reporter assays were performed using the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. For experimental activation, cells were co-transfected with mutant YAP S127A (Addgene #27370) or TAZ S89A (Addgene #32840) plasmid DNA (31, 32). The amount of plasmid DNA in each transfection was kept constant by addition of the non-coding backbone plasmid. After incubation for 24 h, cells were lysed and luciferase activity was measured in quintuplicates (GloMax-Multi Detection System, Promega). Firefly luciferase activity was normalized to Renilla luciferase activity (co-transfected pRL-TK control plasmid; Promega) to account for potential differences in transfection efficiency. Cells were transfected with the 8xGTIIC TEAD luciferase reporter plasmid to evaluate the effects of YAP/TAZ nuclear activation and to determine the suppressive effects of verteporfin (17-24 h) and BMS-754807 (17-24 h) on YAP/TAZ-TEAD-mediated transcriptional activity. SySa cells co- transfected with S127A YAP or S89A TAZ expression plasmids were incubated with verteporfin (0.075 µM) for 72 h. For experiments including cytokine stimulation, prior to IGF-II incubation (200 ng/ml IGF-II for 5 min), cells were grown in medium devoid of FBS supplement for 19 h. At least three independent experiments were performed (each in quintuplicates).

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YAP/TAZ signals in synovial sarcoma

Cell viability assay (MTT) To document cell viability after drug treatment or RNAi-mediated knockdown, the MTT cell proliferation kit (Roche) was applied according to the manufacturer’s instructions, as previously described (19). In brief, SYO-1 (8×103), FUJI (8×103), 1273/99 (7×103), CME-1 (6×103), HS-SY- II (15×103), and RD rhabdomyosarcoma (6×103) cells (33) were seeded in 96-well cell culture plates (medium supplemented with 2% FBS) and exposed to increasing concentrations of verteporfin (0.1875-3 µM) for 72 h. An appropriate DMSO vehicle control was included. In CME- 1 and FUJI cells YAP and TAZ siRNA transfection was performed 72 h prior to the MTT cell viability assay. At least three independent experiments were performed (each in quintuplicates).

Flow cytometry Effects of verteporfin on the apoptotic rate were assessed by flow cytometric analyses. In brief, SySa cells were grown in 175 cm2 cell culture flasks (medium supplemented with 2% FBS) and treated with verteporfin (0.25-0.5 µM; 72 h). Adherent cells were detached using 0.025% Trypsin (Life Technologies), fixed in 2% paraformaldehyde (10 min on ice), washed in 1 x PBS, collected by centrifugation and incubated in 1 x PBS (supplemented with 0.25% Triton X-100) for 5 min on ice. After an additional washing step, cells were incubated with the cleaved Poly-(ADP-ribose)-polymerase (PARP) (Asp214) (BD Biosciences; phycoerythrin-labelled) antibody for 60 min at room temperature. Fluorescence intensity was measured using a FACSCanto II analytical flow cytometer and cytometric data were analyzed using the FACSDiva software (both BD Biosciences). Each experiment was performed at least in duplicates.

Chicken chorioallantoic membrane (CAM) studies CAM assays were performed as previously described (34). In brief, SySa cells (1.5x106 cells/egg; dissolved in medium/matrigel 1:1, v/v) were xenografted onto the egg CAM (7 days after fertilization) and incubated at 37°C with 60% relative humidity. On day 8 of incubation, verteporfin (1-2 µM) or control vehicle (0.2% DMSO in NaCl 0.9%) were topically applied (≥ 6/group). The identical treatment protocol was recapitulated on two consecutive days. Three days after treatment initiation, tumor-containing CAM xenografts were explanted and fixed in 5% paraformaldehyde. Tumor volume (mm3) was calculated according to the formula: 휋 푇푢푚표푟 푣표푙푢푚푒 = [(small diameter)² × large diameter × ] (35). 6

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YAP/TAZ signals in synovial sarcoma

SySa cell line and patient-derived xenograft (PDX) studies To establish SySa cell line xenografts, SYO-1 cells (5×106 cells/100 µl 1 x PBS) were subcutaneously injected into the lower flanks of NOD scid gamma (NSG) mice. Once tumors reached a volume of approximately 100 mm3 (11 days after cell injection), tumor-bearing mice were homogeneously distributed across four treatment groups (≥ 3 mice/group), receiving: (i) verteporfin (75 mg/kg, every other day), (ii) doxorubicin (3 mg/kg, once a week), (iii) combination of verteporfin (75 mg/kg, every other day) and doxorubicin (3 mg/kg, once a week) or (iv) DMSO vehicle control (every other day) by intraperitoneal (i.p.) administration. Due to its established role in conventional therapeutic regimens applied for SySa treatment, doxorubicin was included as positive control; drug combinations were tested to screen for potential additive or complementary effects. Tumor volumes were calculated according to the same formula as described above and normalized to the individual volume at treatment initiation. The SySa PDX model (UZLX-STS7) was described previously (36). In brief, human SySa tissue was subcutaneously transplanted into the lower flanks of NMRI nu/nu mice. On day 27 after transplantation, the identical treatment protocol was initiated (≥ 4 mice/group) as described for the SYO-1 xenografts. For all in vivo mouse studies permission was obtained from the local authorities, and all experiments were performed in accordance with the standards of the National and European Union guidelines.

Compounds

Verteporfin (VP; C41H42N4O8; CAS#: 129497-78-5; Targetmol) (23, 24), and BMS-754807

(C23H24FN9O; CAS#: 1001350-96-4, Biomol) (25) were dissolved in dimethyl sulfoxide (DMSO;

Sigma-Aldrich). Doxorubicin hydrochloride (Doxo; C27H29NO11·HCl; CAS#: 25316-40-9; Actavis) was dissolved in Aqua ad iniectabilia (Braun).

Statistical analysis Two-group comparisons were analyzed by unpaired, two-tailed Student’s t-test (GraphPad Software). Statistical differences were considered significant at P<0.05 (*), P<0.01 (**) and P<0.001(***). Outlier testing was performed for every measurement of quintuplicates and for the xenograft data set according to the formula (n= count of values; SD= standard deviation): 2 × 푆퐷 2 × 푆퐷 푀푒푎푛 − > 푂푢푡푙푖푒푟 > 푀푒푎푛 + √푛 √푛

The concentration of verteporfin required for 50% growth inhibition (IC50 value), was calculated by non-linear regression analysis.

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YAP/TAZ signals in synovial sarcoma

RESULTS

Expression of YAP/TAZ in human SySa tumor tissue and cell lines To determine the involvement of YAP/TAZ-mediated signal transduction in SySa tumorigenesis, expression levels of nuclear YAP and TAZ (corresponding to the transcriptionally active pool) along with the downstream targets FOXM1 and PLK1 were examined in a set of 65 SySa tissue specimens using immunohistochemistry (Table 1, Figure 1A and Supplementary Table S1). Moderate to strong nuclear staining of YAP and TAZ was detected in 80% (52/65) and 35% (23/65) of SySa tissue specimens, respectively. Overall, 11 out of 65 tumors (17%) showed neither YAP nor TAZ nuclear staining positivity. In total, 100% (65/65) of SySa showed a moderate to strong staining intensity for FOXM1 and 72% (47/65) for PLK1 (Figure 1B and Supplementary Figure S1A). Concordance of nuclear YAP and/or TAZ immunoreactivity and expression of FOXM1 and/or PLK1 was demonstrated in 83% (54/65) of SySa (Figure 1C). Strong YAP/TAZ and downstream target (FOXM1, CTGF and PLK1) expression levels were detectable in total protein extracts of all five SySa cell lines (Figure 1D, upper panel). Expression of the pathognomonic SS18-SSX fusion transcript was confirmed by RT-PCR (Figure 1D, lower panel). The transcriptionally active pool of nuclear YAP/TAZ was demonstrated by immunofluorescence staining (Figure 1E and Supplementary Figure S1B) and subcellular fractionation (Figure 1F).

Nuclear localization of YAP/TAZ and TEAD-associated transcriptional activity is driven by the chimeric SS18-SSX fusion protein To evaluate whether YAP/TAZ-mediated transcriptional activity is dependent on the presence of the chimeric SS18-SSX fusion protein, RNAi-mediated SS18-SSX loss-of-function analyses were performed in two SySa cell lines. Nuclear localization of YAP/TAZ was reduced upon RNAi-mediated SS18-SSX knockdown as demonstrated by immunofluorescence (Figure 2A, upper panel) and immunoblotting after subcellular protein fractionation (Figure 2A, lower panel). Depletion of SS18-SSX additionally resulted in significant suppression of YAP/TAZ-responsive TEAD luciferase reporter activity (Figure 2B) and reduced protein levels of YAP, TAZ, FOXM1, CTGF and PLK1 (Figure 2C). Overexpression of SS18-SSX in mesenchymal SCP-1 stem cells resulted in nuclear accumulation of YAP/TAZ protein levels (Figure 2D), significantly induced TEAD luciferase reporter activity (Figure 2E) and consistently increased FOXM1, CTGF and PLK1 expression levels (Figure 2F).

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YAP/TAZ signals in synovial sarcoma

IGF-II/IGF-IR signaling links the SS18-SSX fusion protein to YAP/TAZ activation Given several kinase signaling pathways being activated by SS18-SSX fusion oncoprotein in SySa (20, 26, 37-39) and complex signaling networks modulating YAP/TAZ activation (13), we set out to analyze whether a functional connection between SS18-SSX driven IGF-IR/PI3K/AKT signals might be involved in dysregulation of the Hippo signaling pathway. In agreement with our previous findings in T-REx-293 (26), stable expression of SS18-SSX in mesenchymal SCP-1 stem cells resulted in promoter-specific induction of IGF2 expression (Figure 3A, Supplementary Figure S2A). In two SySa cell lines, IGF-II stimulation significantly enhanced TEAD luciferase reporter activation, indicating elevated YAP/TAZ-mediated transcriptional activity (Figure 3B). On the protein level, IGF-II stimulation of SySa cells (expectedly) induced phosphorylation of IGF-IR (Tyr1135/6) and AKT (Ser473) while phosphorylation of LATS1 (Thr1079), MOB1 (Thr35), YAP (Ser127) and TAZ (Ser89) was reduced. The cytoplasmic localization of phosphorylated YAP (Ser127) and TAZ (Ser89) was confirmed by subcellular fractionation (Supplementary Figure S2B), demonstrating that these phosphorylation events are associated with YAP/TAZ transcriptional inactivation and that a decrease in phosphorylation levels is associated YAP/TAZ activation. These data imply an IGF-II-dependent modulation of YAP/TAZ phosphorylation levels through dysregulation of the Hippo effectors LATS1 and MOB1, resulting in YAP/TAZ activation (Figure 3C). Accordingly, RNAi-mediated knockdown of IGF-IR reduced YAP/TAZ, FOXM1 and CTGF protein levels in CME-1 cells (Figure 3D). In order to substantiate these results with a pharmacological approach allowing shorter incubation periods, we performed additional experiments applying the specific IGF-IR inhibitor BMS-754807 (25) (Supplementary Figure S2C) in three SySa cell lines. Inhibition of IGF-IR led to inactivation of YAP/TAZ as mirrored by significantly reduced TEAD luciferase reporter activity (Figure 3E), accompanied by elevated phosphorylation levels of LATS1 (Thr1079), MOB1 (Thr35), YAP (Ser127) and TAZ (Ser89) (Figure 3F) in short-term incubations as well as consistently reduced YAP/TAZ, FOXM1, CTGF and PLK1 protein levels in long-term treatments (Figure 3G) going along with diminished nuclear localization of transcriptional active YAP/TAZ (Figure 3H and Supplementary Figure S2D).

Knockdown of YAP and/or TAZ affects target gene expression and SySa cell viability To document the functional role of YAP and TAZ in SySa in a non-pharmacological approach, two different SySa cell lines (CME-1 and FUJI) were transfected with pre-validated siRNAs-directed against human YAP and TAZ. RNAi-mediated depletion resulted in decreased FOXM1, CTGF and PLK1 protein levels and reduced phosphorylation of Histone H3 (Ser10) (Figure 4A). To exclude unspecific off-target effects, two additional siRNAs were tested

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YAP/TAZ signals in synovial sarcoma

(Supplementary Figure S3A). In both SySa cell lines, YAP/TAZ knockdown significantly reduced TEAD luciferase reporter activity (Figure 4B) and significantly suppressed SySa cell viability in MTT assays (Figure 4C), pointing to an essential role of YAP/TAZ-mediated signals in SySa.

Verteporfin reduces cell viability, inhibits YAP/TAZ-mediated transcriptional activity and induces apoptosis in SySa cells in vitro To investigate the growth-suppressive effects of YAP/TAZ-TEAD inhibition, five SySa cells were exposed to increasing concentrations of verteporfin. In MTT assays, verteporfin was effective in suppressing SySa cell viability with IC50 values ranging from 0.13-0.51 µM, showing a dose-dependent mode of action (Figure 4D and Table 2). YAP/TAZ-driven rhabdomyosarcoma RD cells included as positive control cells (33) were less sensitive to verteporfin treatment compared to SySa cells. As demonstrated in epithelial cells before (40, 41), in all five analyzed SySa cell lines, verteporfin led to a significant and dose-dependent reduction of YAP/TAZ and their downstream targets (Figure 4E and Supplementary Figure S3B). Consistently, TEAD luciferase reporter assays indicated a dose-dependent suppression of YAP/TAZ-TEAD transcriptional activity (Figure 4F and Supplementary Figure S3C). In flow cytometric analyses, treatment with verteporfin induced a significant and dose-dependent increase of poly-adenosine diphosphate (ADP)-ribose polymerase (PARP; Asp214) cleavage in three SySa cell lines (Figure 4G and Supplementary Figure S3D). TEAD luciferase reporter activity induced through overexpression of constitutively active S127A YAP or S89A TAZ in SySa cells decreased upon verteporfin treatment, indicating specific suppression of YAP/TAZ-mediated transcriptional activity (Figure 4H).

In vivo efficacy of verteporfin in SySa cell line-based CAM, mouse xenografts and a SySa PDX model As a final step of our preclinical evaluation of the efficacy of YAP/TAZ-TEAD inhibition on SySa tumor growth, we applied several in vivo models. First, we performed chick CAM assays xenografting SYO-1 cells to initiate SySa tumor formation. Topical verteporfin administration resulted in a significant and dose-dependent reduction of tumor volumes compared to the DMSO vehicle control group (Figure 5A). In the next step, SYO-1 cells were subcutaneously xenografted into the lower flank region of NSG mice, receiving: (i) mono verteporfin, (ii) mono doxorubicin (iii) combination of verteporfin and doxorubicin or (iv) DMSO vehicle control. Intraperitoneal administration of verteporfin and doxorubicin resulted in a significant reduction of tumor volumes compared to the DMSO vehicle control group. Highest growth-suppressive effects were observed under combined administration of verteporfin and doxorubicin (Figure 5B-

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YAP/TAZ signals in synovial sarcoma

C). In the final step, human SySa tissue was subcutaneously transplanted into the lower flanks of NMRI nu/nu mice, receiving the identical treatment protocol. Consistent with the SYO-1 xenografts, PDX mice treated with verteporfin and doxorubicin showed a significant reduction of SySa tumor growth (Figure 5D-E). As expected, and consistent with the in vitro data, treatments with mono verteporfin and the combination of verteporfin and doxorubicin resulted in a reduction of YAP, TAZ, FOXM1, CTGF and PLK1 protein levels compared to the control and mono doxorubicin treatment (Supplementary Figure 4).

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YAP/TAZ signals in synovial sarcoma

DISCUSSION

Synovial sarcoma (SySa) is a malignant soft tissue tumor of adolescence and young adulthood with a particular propensity to develop local relapse and distant metastases (2, 8). Prognosis in the metastasized situation is poor (4). SySa is molecularly characterized by a SS18-SSX gene fusion encoding an aberrant transcriptional regulator that has been shown to be sufficient to drive tumorigenesis in a conditional MYF5-CRE transgenic mouse model with 100% penetrance (42). While it has been convincingly shown that SS18-SSX associates with SWI/SNF and Polycomb chromatin complexes to dysregulate gene expression in a genome-wide fashion through the disruption of epigenetic regulation (9, 11), knowledge about the actual oncogenic signals effected by SS18-SSX is still limited. We and others have previously shown that Insulin-like growth factor-dependent signals, the subsequent PI3K/AKT cascade, the SRC kinase as well as WNT/-catenin signals contribute to SySa tumorigenesis (20, 21, 26, 29, 38, 39). Since targeting of the chimeric SS18-SSX fusion protein itself represents a particular challenge, it appears reasonable to therapeutically address the signaling pathways which are known to be functionally dependent on the primary oncogenic driver, i.e. the SS18-SSX fusion protein. In-depth insights into the dysregulated signaling pathways are therefore elementary to design tailored therapeutic concepts. Given first data documenting a role of YAP/TAZ in mesenchymal tumorigenesis (16-18), we set out to analyze in detail the prevalence and functional relevance of YAP/TAZ signals in SySa tumorigenesis. Aiming at a truly biologically motivated therapeutic approach, we put a particular focus on the analysis of the functional dependency of YAP/TAZ signals on the chimeric SS18-SSX fusion protein before testing a small molecule YAP/TAZ-TEAD inhibitor in a preclinical setting in different model systems. In a large cohort of tumor specimens and a representative set of SySa cell lines, we detected a high prevalence of nuclear YAP/TAZ expression, associated with concordant expression of the downstream targets FOXM1 and/or PLK1 in 83% of primary SySa and all SySa cell lines (Figure 1). FOXM1 expression has previously been investigated in SySa and was found to be prognostically relevant (43). Addressing the impact of SS18-SSX on YAP/TAZ signals in two different model systems, we unveiled nuclear translocation of YAP/TAZ and associated YAP/TAZ-TEAD signaling to be functionally dependent on the chimeric SS18-SSX fusion protein, both in RNAi-based experiments in SySa cells and through stable overexpression of SS18-SSX in SCP-1 mesenchymal stem cells (Figure 2). In view of the known example of an “indirect” fusion-dependent mechanism of YAP activation in alveolar rhabdomyosarcoma (exerted via PAX3-FOXO1-dependent upregulation of RASSF4 leading to inhibition of MST1 and MOB1) (33), we wondered if a comparably complex molecular network might be effective in

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YAP/TAZ signals in synovial sarcoma

SySa cells. Integrating the established role of SS18-SSX as a molecular driver of IGF2 expression (26, 37) resulting in an activation of IGF-IR/PI3K/AKT signal transduction (20, 38), we here disclosed that an IGF-II/IGF-IR signaling loop contributes to aberrant YAP/TAZ activation through dysregulation of the Hippo effectors LATS1 and MOB1. Consistently, molecular inhibition of IGF-IR reconstituted the function of the “negative” Hippo effectors, resulting in phosphorylation of YAP and TAZ, thereby contributing to downregulation of YAP/TAZ signals (Figure 3). This molecular circuit represents a novel mechanism to regulate “active” nuclear YAP/TAZ levels; however, it is in good agreement with the concept of regulatory inputs of diverse signaling pathways on the Hippo axis (13, 44, 45). We are aware of the limitations of the signaling concept proposed here, as it naturally presents only an excerpt of a much more complex system - however, we render a conclusive model integrating diverse signaling pathways driven by the SS18-SSX fusion. Yet, it appears probable to us that further signaling inputs beyond IGF-IR-mediated signals exist and even direct effects of SS18-SSX on YAP/TAZ are still conceivable. Targeting the transcriptionally active YAP/TAZ-TEAD complex, either by specific RNAi-mediated functional depletion of YAP/TAZ or through pharmacological inhibition with verteporfin, led to a significant and dose-dependent impact on SySa cell growth, coupled with the expected regulation of YAP/TAZ-TEAD transcriptional activity. Though verteporfin is known to have off- targets effects so that our data need to be interpreted with caution, it may be regarded as a proof of specificity in the context of SySa that we could show that verteporfin is able to revert experimental overactivation of YAP/TAZ-dependent gene transcription, as measured in TEAD luciferase reporter assays. Aiming at a further insight in the concrete mode of action of verteporfin, we demonstrated a significant dose-dependent induction of apoptosis in SySa cells due to YAP/TAZ-TEAD inhibition (Figure 4). Consistent with our in vitro results, topical administration of verteporfin to SYO-1 CAM xenografts led to a significant suppression of SySa tumor growth in vivo. This finding could eventually be translated to SYO-1 NSG mouse xenografts and human SySa PDX model, in which we observed significant therapeutic effects of mono-treatment with verteporfin and in combination with doxorubicin (Figure 5). The enhanced therapeutic efficacy of the combination of verteporfin and doxorubicin observed in the PDX model might imply a role of YAP/TAZ inhibition in counteracting mechanisms of chemoresistance as reported in epithelial tumors (46, 47); however, further systematic experiments are required to evaluate this therapeutic option. In summary, our findings argue in favor of a relevant functional role of YAZ/TAZ signals in the oncogenic translation of SS18-SSX fusion protein-driven effects and disclose a novel, molecularly based therapeutic target in SySa.

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YAP/TAZ signals in synovial sarcoma

Given the described lack of genomic alterations including effectors of the Hippo pathway in SySa (16, 48), the setting of Hippo YAP/TAZ signaling in SySa to some extent parallels what we and others have shown for the activation of the WNT/-catenin pathway in SySa. Both effects seem mainly not to be due to genetic alterations in pathway effectors such as APC and/or -catenin in the case of WNT-signaling but to be rather (indirectly) mediated through the SySa-specific SS18-SSX fusion protein (21, 29). Since there is convincing evidence of a simultaneous activation of several different signaling pathways contributing to tumorigenesis in SySa, future therapeutic concepts need to be based on an integrated signaling network concept. Based on our data, SySa joins alveolar rhabdomyosarcoma, in which the PAX3-FOXO1 fusion protein was also shown to promote tumorigenesis by dysregulation of YAP (33), to make up a subgroup of soft tissue tumors in which a specific chimeric fusion protein effects its oncogenic signal through dysregulation of the Hippo pathway. In another fusion-driven soft tissue tumor, epithelioid hemangioendothelioma, Hippo pathway disruption is realized through oncogenic translocations which directly involve the YAP and WWTR1 (encoding TAZ) genes, leading to an aberrant signaling pathway activation (49, 50). These two oncogenic mechanisms add to what has been reported by Eisinger-Mathason and colleagues in non-fusion-driven, karyotypically complex sarcomas, in which genetic events affecting the tumor suppressive Hippo regulators LATS2, NF2, and SAV1 were found in 39% of all 261 sarcomas deposited in the TCGA database (16). The diversity of activation mechanisms of YAP/TAZ signals in soft tissue sarcomas represents a particular challenge for molecular tumor diagnostics since the identification of an appropriate predictive biomarker is mandatory before entering molecularly based therapeutic studies. With regard to the current state of knowledge, immunohistochemical screening for nuclear YAP/TAZ expression could provide such a biomarker which should be prospectively assessed. In conclusion, the results of our study demonstrate that activation of YAP/TAZ signals is a common pattern in SySa and is functionally dependent on the chimeric SS18-SSX fusion protein. Disruption of YAP/TAZ-mediated signal transduction via a small molecule inhibitor may provide an effective and novel therapeutic approach for the treatment of SySa.

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YAP/TAZ signals in synovial sarcoma

ACKNOWLEDGEMENTS The authors thank Charlotte Sohlbach, Inka Buchroth, Christin Fehmer and Christian Bertling for excellent technical support and Jasmien Wellens for excellent technical help with PDX in vivo experiments. Dr. Attila Aszodi, Experimental Surgery and Regenerative Medicine, Department of General-, Trauma- and Reconstructive Surgery, Ludwig-Maximilians-University of Munich, Munich, Germany, kindly provided SCP-1 cells. Dr. Shinya Tanaka, Laboratory of Molecular & Cellular Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Japan, kindly provided FUJI cells. Dr. Hiroshi Sonobe, Department of Laboratory Medicine, Chungoku Central Hospital, Fukuyama, Hiroshima, Japan, kindly provided HS-SY-II cells. Dr. Akira Kawai, Division of Orthopaedic Surgery, National Cancer Center Hospital, Tokyo, Japan, kindly provided SYO-1 cells.

FINANCIAL SUPPORT The study was supported by grants from the DFG (W.H. and M.T. HA 4441/2-1), the Wilhelm- Sander-Stiftung (W.H., M.T., and K.S.; 2016.099.1), and the "Innovative Medical Research" of the University of Münster Medical School (M.T. and S.H.; HU121421; M.T.; TR121716 and TR221611). The experimental PDX work was partially supported by research grant from Kom op tegen Kanker (Stand up to Cancer), the Flemish Cancer Society (grant to P.S.).

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YAP/TAZ signals in synovial sarcoma

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47. Pan W, Wang Q, Zhang Y, Zhang N, Qin J, Li W, et al. Verteporfin can Reverse the Paclitaxel Resistance Induced by YAP Over-Expression in HCT-8/T Cells without Photoactivation through Inhibiting YAP Expression. Cell Physiol Biochem. 2016;39:481-90.

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50. Tanas MR, Ma S, Jadaan FO, Ng CK, Weigelt B, Reis-Filho JS, et al. Mechanism of action of a WWTR1(TAZ)-CAMTA1 fusion oncoprotein. Oncogene. 2016;35:929-38.

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TABLES

Table 1. Clinicopathological characteristics (n=65) Age (years) mean (±SD) 41 (±17) median (range) 45 (8-78) < 41 28 (43.1%) ≥ 41 37 (59.9%) Type primary tumor 43 (66.2%) metastasis 9 (13.8%) recurrence 7 (10.8%) ND 6 (9.2%) Histology monophasic 48 (73.9%) biphasic 14 (21.5%) poorly differentiated 3 (4.6%) Size (cm) mean (±SD) 7 (±5) median (range) 5 (0.3-20) < 7 32 (49.2%) ≥ 7 18 (27.7%) ND 15 (23.1%) Gender female 30 (46.2%) male 35 (53.8%) FISH SS18 (break-apart) positive 52 (80.0%) ND 13 (20.0%) t(X;18) translocation type SS18-SSX1 28 (43.1%) SS18-SSX2 20 (30.8%) ND 17 (26.1%) Grading (FNCLCC) G2 16 (24.6%) G3 24 (36.9%) ND 25 (38.5%) Abbreviation: SD, standard deviation; ND, not determined; FISH, fluorescence in situ hybridization; FNCLCC, Fédération Nationale des Centres de Lutte Contre le Cancer

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YAP/TAZ signals in synovial sarcoma

Table 2. IC values for verteporfin in synovial sarcoma cell lines 50 IC (µM) 50 Compound SYO-1 FUJI 1273/99 CME-1 HS-SY-II (SS18-SSX2) (SS18-SSX2) (SS18-SSX2) (SS18-SSX2) (SS18-SSX1) verteporfin 0.51 ± 0.14 0.18 ± 0.09 0.27 ± 0.16 0.13 ± 0.03 0.41 ± 0.08 Cytotoxic effects on synovial sarcoma cell viability were assessed in MTT assays (72 h). Results are represented as mean ± SD of at least three independent experiments performed in quintuplicates.

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FIGURE LEGENDS

Figure 1. Nuclear expression and activation of YAP/TAZ signaling in a comprehensive cohort of SySa tissue specimens (n=65) and five SySa cell lines. (A) Immunohistochemical stainings show strong nuclear localization of YAP and TAZ in a representative SySa tissue specimen. Strong expression levels of FOXM1 and PLK1 indicate YAP/TAZ-mediated transcriptional activity (original magnification: x10, inset x20). (B) Immunohistochemical staining intensity of nuclear YAP/TAZ, FOXM1 and PLK1 summarized as bar chart (intensity score). (C) Venn diagram indicating the concordance of nuclear YAP and/or TAZ immunoreactivity in line with FOXM1 and/or PLK1 expression. (D) Immunoblotting demonstrates strong expression levels of YAP, TAZ, FOXM1, CTGF and PLK1 in total protein extracts of five SySa cells lines (β-Actin used as loading reference). Detection of t(X;18) SS18-SSX fusion gene transcripts in all SySa cell lines by RT-PCR (28S rRNA used as loading reference). (E) Immunocytochemical detection of YAP and TAZ (left panel), DAPI-stained nuclei (middle panel) and merged fluorescence images (right panel) (original magnification x40), demonstrating predominant nuclear YAP/TAZ localization, implying transcriptional YAP/TAZ “activity”. (F) Detection of ‘N’ nuclear and ‘C’ cytoplasmic fractions of YAP and TAZ in all five SySa cell lines (Histone H3 and GAPDH used as loading references for the nuclear and cytoplasmic fractions, respectively).

Figure 2. The SS18-SSX fusion protein stimulates YAP/TAZ-mediated transcriptional activity. (A) Immunofluorescence staining demonstrates diminished nuclear localization of YAP and TAZ upon RNAi-mediated knockdown of SS18-SSX (upper panel, original magnification x40). Subcellular protein fractionation shows reduced nuclear localization of YAP/TAZ and diminished expression of the downstream targets FOXM1, CTGF and PLK1 upon RNAi- mediated SS18-SSX depletion (lower panel, Histone H3 and GAPDH used as loading references for the nuclear and cytoplasmic fractions, respectively). (B) Significantly decreased YAP/TAZ-responsive TEAD luciferase reporter activity upon RNAi-mediated SS18-SSX knockdown in CME-1 and FUJI SySa cells (mean of quintuplicates + SD; ***P<0.001). (C) Immunoblotting of total protein lysates shows reduced YAP/TAZ levels and diminished downstream target expression of FOXM1, CTGF and PLK1 upon RNAi-mediated SS18-SSX knockdown (β-Actin used as loading reference). (D) Stable expression of SS18-SSX in mesenchymal SCP-1 stem cells increased nuclear protein levels of YAP and TAZ compared to EmGFP-transduced SCP-1 cells as demonstrated by subcellular protein fractionation (Histone H3 and GAPDH used as loading references for the nuclear and cytoplasmic fractions, respectively). (E) Significantly increased TEAD luciferase reporter activity upon stable

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YAP/TAZ signals in synovial sarcoma

SS18-SSX expression in SCP-1 cells (mean of quintuplicates + SD; ***P<0.001). (F) Enhanced YAP/TAZ, FOXM1, CTGF, and PLK1 total protein levels in SS18-SSX-transduced SCP-1 cells (β-Actin used as loading reference).

Figure 3. IGF-II/IGF-IR signaling pathway as a link between SS18-SSX fusion protein expression and YAP/TAZ activation. (A) In RT-PCR analyses, SS18-SSX expressing SCP-1 cells demonstrate a transcriptional induction of IGF2 (promoter P3 transcript, 28S rRNA used as loading reference). (B) Short-term IGF-II stimulation of SySa cells significantly increases TEAD luciferase reporter activity (mean of quintuplicates + SD; ***P<0.001). (C) IGF-II stimulation of SySa cells induces phosphorylation of IGF-IR (Tyr1135/6) and AKT (Ser473) while phosphorylation of LATS1 (Thr1079), MOB1 (Thr35), YAP (Ser127) and TAZ (Ser89) is reduced (β-Actin used as loading reference). (D) RNAi-mediated knockdown of IGF-IR in SySa cells reduces phosphorylation of AKT (Ser473) as well as total YAP/TAZ, FOXM1, and CTGF protein levels (β-Actin used as loading reference). (E) Significant reduction of TEAD luciferase activity upon incubation with increasing concentrations of BMS-754807 in SySa cells (***P<0.001; **P<0.01). (F) Short-term incubation of BMS-754807 in SySa cells leads to decreased phosphorylation levels of IGF-IR (Tyr1135/6) and AKT (Ser473) while phosphorylation of LATS1 (Thr1079), MOB1 (Thr35), YAP (Ser127) and TAZ (Ser89) is increased (β-Actin used as loading reference). (G) Long-term incubation of BMS-754807 in SySa cells leads to decreased phosphorylation levels of IGF-IR (Tyr1135/6) and AKT (Ser473) as well as total YAP/TAZ, FOXM1, CTGF and PLK1 levels (β-Actin used as loading reference). (H) Immunofluorescence of CME-1 cells demonstrates diminished nuclear YAP and TAZ levels upon BMS-754807 treatment (original magnification x63).

Figure 4. Suppressive effects of RNAi-mediated knockdown of YAP and TAZ or verteporfin treatment on YAP/TAZ-TEAD transcriptional activity and SySa cell viability in vitro. (A) RNAi-mediated knockdown of YAP (siRNA#3) and TAZ (siRNA#3) reduces target protein levels of FOXM1, CTGF and PLK1 as well as phospho-Histone H3 (Ser10), serving as a marker of cellular proliferation (β-Actin used as loading reference). (B) Luciferase reporter assays demonstrate a reduction of YAP/TAZ-TEAD activity upon RNAi-mediated YAP/TAZ depletion (mean of quintuplicates + SD; ***P<0.001). (C) Significant reduction of cell viability (MTT assay) upon YAP or TAZ knockdown in SySa cells (mean of quintuplicates + SD; ***P<0.001). (D) Cell viability of five SySa cell lines is significantly decreased upon treatment with increasing concentrations of verteporfin (MTT assay). RD cells are included as verteporfin- sensitive control (mean of three independent experiments ± SD). (E) SySa cells treated with

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YAP/TAZ signals in synovial sarcoma increasing concentrations of verteporfin show a reduction of YAP, TAZ, FOXM1, CTGF and PLK1 protein levels (β-Actin used as loading reference). (F) Verteporfin treatment results in a significant reduction of TEAD luciferase reporter activity in SySa cells (mean of quintuplicates + SD; ***P<0.001). (G) In flow cytometric analyses, significantly increased rates of apoptosis (cleaved PARP) are detected upon treatment with increasing concentrations of verteporfin in SySa cells (mean of two independent experiments ± SD; **P<0.01; *P<0.05) (H) In CME-1 cells, TEAD luciferase reporter activity is significantly increased upon overexpression of constitutively active S127A YAP or S89A TAZ which is reverted by incubation with verteporfin (mean of quintuplicates + SD; ***P<0.001).

Figure 5. In vivo efficacy of verteporfin in SySa cell line-based CAM, mouse xenografts and a SySa PDX model (A) SYO-1 cells were xenografted on the CAM of chick eggs. Tumor-bearing eggs were randomized and topically treated with verteporfin or vehicle control (DMSO). Significantly reduced tumor volumes + SD and representative CAM explants are shown (scale bar: 1 mm, ***P<0.001; *P<0.05). (B) Intraperitoneal mono- or combined administration of verteporfin and doxorubicin (indicated by the ^ arrow) results in a significant reduction of SYO-1 xenograft tumor volumes compared to the DMSO vehicle control group (normalized tumor volumes are indicated as mean ± SEM with ≥ 3 mice/group). (C) Normalized SYO-1 NSG mice xenograft tumor volumes on day 19 (upper panel, mean + SD; **P<0.01; *P<0.05; ns, not significant) and representative tumor explants (lower panel, diameter: 20 mm; scale bar: 10 mm). (D) Intraperitoneal mono- or combined administration of verteporfin and doxorubicin (indicated by the ^ arrow) results in a significant reduction of SySa PDX volumes compared to the DMSO vehicle control group in NMRI nu/nu mice (normalized tumor volumes are indicated as mean ± SEM with ≥ 4 mice/group). (E) Normalized SySa PDX tumor volumes on day 39 (upper panel, mean + SD; ***P<0.001; **P<0.01) and representative PDX tumor explants (lower panel; scale bar: 10 mm).

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SS18-SSX-dependent YAP/TAZ Signaling in Synovial Sarcoma

Ilka Isfort, Magdalene Cyra, Sandra Elges, et al.

Clin Cancer Res Published OnlineFirst February 27, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-3553

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/02/26/1078-0432.CCR-17-3553.DC1

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