SS18-SSX IS A CELL-CONTEXT-DEPEDENT EPIGENETIC REGULATOR: IMPLICATION FOR CELL-OF-ORIGIN OF SYNOVIAL SARCOMAS Sakura Tamaki1,2, Makoto Fukuta1,2,3, Kazuo Hayakawa1,2,3, Tomohisa Kato Jr.1, and Junya Toguchida1,2,4 (No introduction of title, authors and affiliations) 1Dept. Tissue Regeneration, Inst. Frontier Med. Sci., Kyoto Univ. 2Dept. Growth and Differentiation, CiRA, Kyoto Univ. 3Dept. Orthop. Surg., Grad. School Med. Sci, Nagoya City Univ. 4Dept. Orthop, Surg., Grad. School Med., Kyoto Univ.

Synovial Sarcoma (SS) SS18 SSX SS18-SSX 18q11 Xp11 18q11-qter (SSX1, SSX2, SSX4) 18q11-qter Xp11-qter SS18-SSX Among soft tissues sarcomas, we have been working on synovial sarcoma, hereafter called SS. Key genetic feature of SS is the presence of SS18-SSX fusion gene generated by reciprocal translocation between chromosome X and 18. 2

Function of SS18-SSX protein BRM p300 Transcriptional activator PcG Transcriptional repressor SS18-SSX DD SNH RD COOH NH2 QPGY ◆ No DNA binding motif The function of fusion protein is regarded to regulate gene expression, but (click) it has no DNA binding motif. Therefore, it is believed that SS18-SSX associates with (click) transcriptional activators or (click) repressors and (click) dysregultate the gene expression profiles of target cells. Chromatin modulator Up- or down-regulate target genes

Search for genes up-regulated by SS18-SSX ◆ FZD10 is specifically up-regulated in SS, but not in other types of soft tissue sarcomas (STS). MSC MFH LMS LS SS MPNST SS cell lines 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 FZD10 b2MG The FZD10 gene is a candidate as gene up-regulated by SS18-SSX [Nagayama et.al., Cancer Res., 2002] ◆ FZD10 is expressed only in placenta among adult normal tissues. (Sited from LSBM) We previously performed genome-wide gene expression analysis of STSs and identified FZD10 gene as an SS-specifically up-regulated gene. The expression level is very high in almost all SS tumors, (click) but absent in normal tissues except placenta. (click) These observations suggested that FZD10 is a suitable gene to investigate the function of SS18-SSX.

Understand how SS18-SSX regulates the downstream genes using the Objective Understand how SS18-SSX regulates the downstream genes using the FZD10 gene as a representative Based on these backgrounds, the purpose of our study is to understand how SS18-SSX up-regulates the downstream genes using the FZD10 as representative.

SS18-SSX binds core promoter region of FZD10 Relative Luc activity FZD10 (-65) FZD10 (-118) FZD10 (-1332) FZD10 (-3029) FZD10 (-2715) FZD10 (-780) FZD10 (-360) Luc SYO-1 U2OS/EV U2OS/HA-SS18-SSX2 Relative to Input (%) ChIP on FZD10 core promoter rIgG HA IP: Relative expression to SYO-1 EV SS18- SSX2 SS18-SSX2 FZD10 ** U2OS ( ** p<0.01) First, to identify the regulatory region of FZD10 gene, we performed the reporter assay using SS cell line and found that (click) the 100 bp upstream region is critical for the transcriptional activity. (click) And we overexpressed SS18-SSX in FZD10-negative osteosarcoma cell line and confirmed (click) the binding of SS18-SSX to the core promoter region and (click) the induction of FZD10 expression.

SS18-SSX directly regulates the FZD10 gene SS cell lines SYO-1 1273/99 We also knock-downed SS18-SSX in FZD10-positive SS cell lines. (click) Depletion of the fusion gene down-regulated the FZD10. These results suggest that SS18-SSX directly regulates the FZD10 gene. SS18-SSX2 FZD10 ( * P<0.05, ** p<0.01)

SS18-SSX failed to induce FZD10 in hDF YaFu-SS SS-18SSX1 SS18-SSX2 SYO-1 SS18-SSX EV SS18-SSX1 SS18-SSX2 FZD10 Human dermal fibroblast However, when we over-expressed this fusion protein in FZD10-negative human dermal fibroblast, (click) FZD10 was not induced. (click) These results let us to think what kind of cells are appropriate for our study. ACTB (FZD10-negative) → Are hDFs not appropriate cells? → If so, what kind of cells are appropriate?

SS+MPNST SS shares the gene expression profile with MPNST 1204 genes ↓ Create “Cluster Trees” Expressed in >90% of 47 STS cases Those expression ratio varied by SD>1 ↓ 1,204 Genes (Nagayama et al., Cancer Res., 2002) 606 391 712 446 298 623 357 697 406 679 734 508 577 330 407 551 181 327 543 341 416 580 602 325 456 370 267 473 403 350 53 188 97 646 487 582 334 190 737 44 558 259 248 213 438 316 397 By our genome-wide gene expression profiling, we previously found that SS shared the expression profile with MPNST (malignant peripheral nerve sheath tumor). SS+MPNST MFH LMS MPNST SS PLS or DLS 9

SS shares the protein expression profile with CCS (Suehara et al., Proteomics 6: 4402-9, 2006) MPNST Clear cell sarcoma (CCS) Synovial sarcoma And Dr. Kondo’s group in NCRC in Tokyo demonstrated that (click) SS shares the global protein expression profile with CCS (Clear cell sarcoma) and MPNST.

SS is also a neural crest-derive tumor? SS precursor ? Melanocyte Schwann cell SS CCS MPNST Since CCS and MPNST were thought to be neural crest-derived tumors, (click) we hypothesize that the cell-of-origin of SS is also a derivative of neural crest cells. 11

Application of iPS cells for sarcoma research : investigation for the cell-of-origin of synovial sarcoma Makoto Fukuta1,2,3, Kazuo Hayakawa1,2,3, Sakura Tamaki1,2, Kazuya Sekiguchi1,2, Shingo Sato1, Makoto Ikeya2, Takanobu Otsuka3, Knut Woltjen4, Junya Toguchida1,2 Pluripotent stem cell (PSC) SS18-SSX2 + Dox SS18-SSX2 + Dox Bone Fat Cartilage Muscle Skin fibroblast NCC Neural crest cell NC-derived Mesenchymal stem cell NC-MSC To identify the cell-of-origin of SS, (click) we established pluripotent stem cells harboring DOX-inducible SS18-SSX, (click) and our colleague, Makoto has been developing induction method from the pluripotent stage to neural crest cell (NCC) and NC-derived mesenchymal stem cell (NC-MSC). (click) Now we are able to induce the SS18-SSX in these cells, on which we hypothesize the cell-of-origin of SS should exist. 12 12 12 12

Property of NCC CD271 (p75) PAX6/Ap2α/DAPI Relative expression After the initial induction, more than 75% of cells were positive for p75, one of the neural crest markers, and FACS selected cells were also positive for other neural crest marker genes such as PAX3, SOX10 and FOXD3. Relative expression

Property of NC-MSC Osteogenic differentiation Chondrogenic differentiation As for the property of NCMSC, we examined osteogenic and chondrogenic differentiation properties of NCMSC using standard protocols and as shown in this slide, we successfully induce both lineages. Alizarin Red staining Alucian Blue staining 14

Induction of the FZD10 in ESC and NCC Stuffer SS18-SSX2 (** p<0.01) KhES1 SS18-SSX2 FZD10 ** 0.03 1.0 0.3 0.1 Relative expression to SYO-1 DOX (ug/ml) 0.03 0.1 0.3 1.0 NCC-KhES1 SS18-SSX2 Then we treated cells by DOX to induce the SS18-SSX at different stages. In ESC and NCC, DOX treatment successfully induced SS18-SSX in a dose-dependent manner and (click) the ectopic expression of this fusion protein significantly induced the FZD10 expression. Relative expression to SYO-1 0.1 0.3 1.0

Induction of the FZD10 in NC-MSC NC-MSC-KhES1 Stuffer SS18-SSX2 SS18-SSX2 FZD10 SYO-1 0.1 0.3 1.0 5.0 48 72 96 Relative expression to SYO-1 DOX (ug/ml) 0.1 0.3 1.0 5.0 As same as ESC or NCC, DOX treatment could induce SS18-SSX in NC-MSC, (click) but failed to induce the FZD10 expression. Relative expression to SYO-1 DOX (ug/ml) 1.0 1.0 1.0 (hr) 48 72 96

Change in H3K27me3 at the FZD10 locus by SS18-SSX2 ATG -2kb -1kb +457 FZD10 5’UTR ORF 1 2 3 4 H3K27me3 Stuffer SS18-SSX2 KhES1 NCC-KhES1 NC-MSC-KhES1 FZD10; Induced Induced Not induced To investigate this cell-type specific response to SS18-SSX, we focused on epigenetic modification of the FZD10 promoter. At first, we analyzed the level of H3K27me3, which is a key mark for transcriptional repression. After the induction of fusion protein, (click) the level of H3K27me3 was decreased in all types of cells. Relative to Input (%) Region 1 2 3 4 1 2 3 4 1 2 3 4

Change in H3K4me3 at the FZD10 locus by SS18-SSX2 ATG -2kb -1kb +457 FZD10 5’UTR ORF 1 2 3 4 H3K4me3 Stuffer SS18-SSX2 KhES1 NCC-KhES1 NC-MSC-KhES1 FZD10; Induced Induced Not induced Next, we checked the level of H3K4me3, which is one of a mark for transcriptional activation. (click) It was dramatically induced in ESC after the induction of fusion protein, whereas no remarkable changes were found either in NCC or NC-MSC. Relative to Input (%) Region 1 2 3 4 1 2 3 4 1 2 3 4

Change in H3Ac at the FZD10 locus by SS18-SSX2 ATG -2kb -1kb +457 FZD10 5’UTR ORF 1 2 3 4 H3Ac Stuffer SS18-SSX2 KhES1 NCC-KhES1 NC-MSC-KhES1 FZD10; Induced Induced Not induced Finally we analyzed the level of H3Ac, which is also a key mark for transcriptional activation. (click) Significant increase of this modification were observed in ESC and NCC after the induction of fusion protein, but not in NC-MSC. From the analyses of these three modifications, the response of histone acetylation to SS18-SSX might be a critical factor for the induction of FZD10. Relative to Input (%) Region 1 2 3 4 1 2 3 4 1 2 3 4

Importance of H3Ac for the FZD10 induction TSA (HDAC inhibitor) NCC Relative to Input (%) 1 Region 2 3 4 DMSO TSA Occupancy of H3Ac at FZD10 locus Relative expression (to SYO-1) 0.01 0.03 0.1 0.3 1.0 SS18-SSX2 FZD10 TSA (uM); To further confirm our conclusion, we treated NCC with TSA, one of the HDAC inhibitors. (click) TSA treatment successfully increased the level of AcH3 at the FZD10 locus, (click) resulting in the induction of mRNA expression.

Conclusion (+) (-) (-) (-) (-) SS18-SSX2 NC-MSC ESC NCC FZD10 FZD10 H3Ac H3K27me H3K4me3 In this study, we found that (click) the induction of FZD10 by SS18-SSX was cell-type specific event, which (click) was correlated to (click) the up-regulation of H3Ac occupancy in the promoter region of FZD10 gene. These results suggest that cell-context which determines the effect of SS18-SSX on histone acetylation is a key factor, and therefore cells with such context will be an origin of SS.

Toguchida’s Lab. in IFMS & CiRA, Kyoto Univ. Finally, I would like to thank all of the lab members in Toguchida’s laboratory. Thank you for your attention.