Synovial Sarcoma: Recent Discoveries As a Roadmap to New Avenues for Therapy

Published OnlineFirst January 22, 2015; DOI: 10.1158/2159-8290.CD-14-1246

REVIEW

Synovial Sarcoma: Recent Discoveries as a Roadmap to New Avenues for Therapy

Torsten O. Nielsen 1 , Neal M. Poulin 1 , and Marc Ladanyi 2

ABSTRACT Oncogenesis in synovial sarcoma is driven by the chromosomal translocation t(X,18; p11,q11), which generates an in-frame fusion of the SWI/SNF subunit SS18 to the C-terminal repression domains of SSX1 or SSX2. Proteomic studies have identiﬁ ed an integral role of SS18–SSX in the SWI/SNF complex, and provide new evidence for mistargeting of polycomb repression in synovial sarcoma. Two recent in vivo studies are highlighted, providing additional support for the importance of WNT signaling in synovial sarcoma: One used a conditional mouse model in which knock- out of β-catenin prevents tumor formation, and the other used a small-molecule inhibitor of β-catenin in xenograft models.

Signiﬁ cance: Synovial sarcoma appears to arise from still poorly characterized immature mesenchymal progenitor cells through the action of its primary oncogenic driver, the SS18–SSX fusion gene, which encodes a multifaceted disruptor of epigenetic control. The effects of SS18–SSX on polycomb-mediated gene repression and SWI/SNF chromatin remodeling have recently come into focus and may offer new insights into the basic function of these processes. A central role for deregulation of WNT–β-catenin signaling in synovial sarcoma has also been strengthened by recent in vivo studies. These new insights into the the biology of synovial sarcoma are guiding novel preclinical and clinical studies in this aggressive cancer. Cancer Discov; 5(2); 124–34. ©2015 AACR.

CLINICAL FEATURES THE SS18 – SSX FUSION ONCOGENE Synovial sarcoma is an aggressive neoplasm that accounts Synovial sarcoma is uniquely characterized by the balanced for 10% to 20% of soft-tissue sarcomas in the adolescent chromosomal translocation t(X,18; p11,q11), demonstrable and young adult population ( 1 ). Although it is typically in virtually all cases ( 2 ), not found in any other human neo- diagnosed in young adults (median age 35), the age range is plasms. This translocation creates an in-frame fusion of the between 5 and 85 years ( 2 ). There is a slight male predeliction SS18 gene to SSX1 or SSX2 (6 ), whereby all but the carboxy (M:F ratio 1.13); 70% of cases present in the extremities, and terminal (C-terminal) 8 amino acids of SS18 become fused the most common pattern of metastatic spread is to the lung to the C-terminal 78 amino acids of the SSX partner (Fig. 1 ). (3 ). The mainstay of treatment is wide surgical excision with An analogous translocation of SSX4 is detected in less than adjuvant or neoadjuvant radiotherapy, which provides a good 1% of cases (7 ). chance of cure for localized disease. However, the disease is Multiple lines of evidence implicate SS18 – SSX as the cen- prone to early and late recurrences, and 10-year disease-free tral genetic “driver” in this cancer: (i) its presence as the sole survival remains on the order of 50% (3 ). Synovial sarcoma is cytogenetic anomaly in up to a third of cases (8 ), (ii) the low moderately sensitive to cytotoxic chemotherapy with agents frequency of additional mutations ( 9 ), (iii) its preservation such as ifosfamide and anthracyclines ( 4, 5 ). in metastatic and advanced lesions ( 8 ), (iv) the death of synovial sarcoma cells upon SS18 – SSX knockdown (10 ), and (v) its ability to induce tumors in conditional mouse models with appropriate histology, gene expression, and immu- 1Department of Pathology and Laboratory Medicine, University of British nophenotype with 100% penetrance ( 11 ). Columbia, Vancouver, British Columbia, Canada. 2 Department of Pathology and Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Functional Studies of SS18–SSX Cancer Center, New York, New York. Corresponding Author: Marc Ladanyi, Molecular Diagnostics Service, Initial functional studies of SS18–SSX used yeast two Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, hybrids and GAL4 fusion constructs, in which the relevant NY 10065. Phone: 212-639-6369; Fax: 212-717-3515; E-mail: ladanyim@ protein domains are fused to the DNA-binding domain of mskcc.org GAL4. These studies showed that SS18 is a transcriptional doi: 10.1158/2159-8290.CD-14-1246 coactivator and that C-terminal SSX domains mediate repres- ©2015 American Association for Cancer Research. sion ( 12, 13 ). The fusion oncoprotein thus contains both

124 | CANCER DISCOVERYFEBRUARY 2015 www.aacrjournals.org

Advances in Synovial Sarcoma REVIEW

SS18 SSX 188 K13Ub K124 ∗ ∗ SNH QPGY KRAB RDDD

SIN3A 418 β-catenin ATF2 LHX4 BRG1, BRM Histone EP300 NLS MLLT10 (AF10) TLE1 SS18 RBM14 (COAA, SIP)

Figure 1. Protein interaction domains involved in synovial sarcoma translocations, where SS18 is fused to one of the highly paralogous SSX1 or SSX2 genes. Translocation breakpoints (vertical arrowheads) result in the fusion of almost all SS18 sequence to the C-terminal region of SSX protein. Protein domains: SNH, SS18 N-terminal homology; QPGY, glutamine/proline/glycine/tyrosine–enriched domain; KRAB, Kruppel-associated box domain; DD, diver- gent domain; RD, repression domain; NLS nuclear localization signal. A conserved site for ubiquitin modiﬁ cation at lysine 13 of SS18 is shown (K13Ub), as well as a site of ubiquitin/acetyl modiﬁ cation at lysine 124 of SSX1 (K124).

activating and repressing domains, although neither partner nately, SMARCB1 may be stabilized in SWI/SNF complexes has a DNA-binding domain. Several other putative bind- by unknown binding partners in different experimental con- ing partners were also identifi ed as shown in Fig. 1 , but the ditions (e.g., transduced vs. transfected HEK293 cells, or dif- mechanisms targeting the fusion protein to specifi c DNA ferences in the parent HEK293 cell lines, which have complex regions were elusive until recently. genomes and known clonal heterogeneity). HEK293 cells, although convenient for transfection and at least partially SS18 and SS18–SSX Incorporate into permissive for (short-term) SS18–SSX expression, may not the SWI/SNF Complex represent the most relevant cell line model as discussed in Thaete and colleagues (14 ) showed association of both the Cellular Background for SS18–SSX Oncogenesis section, SS18 and SS18–SSX with the DNA-dependent ATPase BRM, below. Resolution of these confl icting results will have impor- the catalytic subunit of SWI/SNF chromatin remodeling tant implications because SMARCB1 (aka SNF5, INI1) is a complexes. Subsequently, Kato and colleagues ( 15 ) showed known tumor suppressor, with homozygous loss in 98% of that SS18 is a stable and integral component of SWI/SNF rhabdoid tumors (18 ), 90% of epithelioid sarcomas, and more complexes using coimmunoprecipitation and mass spectros- than half of myoepithelial carcinomas (19 ). If SMARCB1 loss copy in nuclear extracts of HeLa cells. contributes to oncogenesis in synovial sarcoma, advances in Middeljans and colleagues (16 ) extended these results the study of SWI/SNF–directed therapies in rhabdoid tumors by showing that the fusion oncoprotein is similarly incor- may have direct translational implications. Of potential porated into stable SWI/SNF complexes. All commonly importance, synthetic lethalities may be explored by analogy observed subunits were recovered in reciprocal purifi cations to rhabdoid tumors, where, for example, tumorigenesis was between tandem affi nity purifi cation-tagged SS18–SSX1 and found to depend on functional BRG1 in the residual SWI/ other subunits, indicating minimal perturbation of the core SNF complex (20 ). Although caution is currently indicated complex when the fusion oncogene is stably expressed in in drawing simple parallels with rhabdoid tumors, these HEK293 cells. Kadoch and Crabtree ( 17 ) observed high- proteomic studies imply that the mechanisms of SS18–SSX- affi nity binding of both SS18 and SS18–SSX to the core sub- mediated oncogenesis are intimately related to dysregulation units of SWI/SNF, and immunodepletion of nuclear extracts of SWI/SNF chromatin remodeling. showed undetectable levels outside of this association. In SWI/SNF functions to reposition nucleosomes on genomic contrast with the results of Middeljans and colleagues ( 16 ), DNA, and can also promote nucleosome disassembly and his- these authors observed that expression of the fusion onco- tone exchange (reviewed in ref. 21 ). The canonical activity of gene induced depletion of the BAF47 (SMARCB1) subunit SWI/SNF is to create nucleosome-depleted regions at core from the SWI/SNF complex in experiments using transiently promoters and regulatory regions, facilitating transcription expressed GFP-tagged SS18–SSX in an HEK293 background. factor access to DNA. SWI/SNF activity is broadly recruited The authors also reported SMARCB1 loss in synovial sar- across the genome to effect switches in chromatin state and coma cell lines, noting that siRNA knockdown of SS18–SSX has been associated with the induction of a wide range of restored SMARCB1 inclusion in SWI/SNF complexes. transcriptional programs, including cell-cycle control, stem Both studies suggest that SMARCB1’s association with cell maintenance, and differentiation. Importantly, recent SWI/SNF may be more labile than for other subunits, and exome sequencing studies have found mutations in SWI/SNF it is conceivable that SMARCB1 is displaced from SWI/SNF complex members in 20% of cases across a broad spectrum of by aberrant protein interactions involving SS18–SSX. Alter- tumor types, surveyed by Kadoch and colleagues (22 ), who

FEBRUARY 2015CANCER DISCOVERY | 125

REVIEW Nielsen et al. review the importance of this complex in tumor suppres- component BRM and the polycomb component EZH2 were sion. However, the specifi c mechanisms of tumor suppression observed to coelute with the SS18–SSX oncoprotein. Recip- remain elusive, due to the diverse and tissue-specifi c roles of rocal immunoprecipitations showed copurifi cation of core the complex in transcriptional regulation. PRC2 subunits EZH2, SUZ12, and EED with TLE1, SS18– SMARCB1 and BRG1 inactivation lead to increased prolifera- SSX, and ATF2, indicating the presence of a stable repressive tion ( 23 ), at least in part due to impaired transcriptional activa- complex nucleated by the fusion oncoprotein. ChIP and tion of the CDKN2A / B tumor suppressors ( 24 ). This result has electrophoretic mobility shift assays confi rmed the presence been related to the requirement of SWI/SNF activity for the of this repressive complex at conserved CRE elements in ATF2 eviction of repressive polycomb complexes from chromatin, target gene promoters. Treatment with HDAC inhibitors dis- and highlights the general principle of antagonism between rupts this complex ( 32 ). SWI/SNF and polycomb activities. In this regard, the domi- TLE family proteins are corepressors of WNT target genes nance of polycomb repression would have profound implica- ( 33 ) and effectors of HES-mediated NOTCH repression ( 34 ), tions for the regulation of stemness and differentiation, and and are highly expressed in a number of embryonic progeni- may prove to be a central aspect of oncogenesis in SWI/SNF– tor fi elds where these pathways are known to regulate self- mutated tumors. Other mechanisms of SWI/SNF oncogenesis renewal and stemness. The identifi cation of the corepressor have been elaborated, including mutations in ARID1A , where TLE1 as an SS18–SSX-interacting protein is also signifi cant it has been suggested that functional SWI/SNF is required to because expression profi ling experiments have identifi ed execute the p53 transcriptional response (25 ). Although not TLE1 as among the most consistently highly expressed genes shown in synovial sarcoma, such an effect may explain the in primary synovial sarcoma specimens ( 29 , 35 ). Incidentally, lack of TP53 mutation in the majority of these lesions. SWI/ nuclear expression of TLE1 is readily detectable by immuno- SNF dysfunction also compromises DNA double-strand break histochemistry on formalin-fi xed paraffi n-embedded tissues, repair, and may confer sensitivity to DNA-damaging agents and its diagnostic value for synovial sarcoma has been con- ( 26 ); this may contribute to the sensitivity of synovial sarcoma fi rmed ( 36, 37 ), leading to its adoption into clinical use. to radio- and anthracycline therapies. SS18–SSX and Epigenetic THE CELLULAR BACKGROUND Transcriptional Repression FOR SS18–SSX ONCOGENESIS Whereas SS18, through its interactions with the SWI/SNF The above fi ndings contribute to knowledge about SS18– complex, might be expected to have a role in transcriptional SSX target genes and/or the cellular background in which the activation, its fusion partner SSX associates with the poly- oncogene operates, but do not clear up enduring mysteries comb repressor complex, which has opposing effects. An about the true cell of origin for synovial sarcoma or its line early observation was that SS18–SSX localizes at discrete of differentiation. Despite the name, synovial sarcoma is not nuclear foci within BMI1-labeled polycomb bodies (27 ). More derived from synovium, nor does it differentiate into synovial- recently chromatin immunoprecipitation sequencing (ChIP- type tissue. The term synovial sarcoma is actually a misnomer Seq) results from HA-FLAG–tagged SS18–SSX, expressed in carried over from older literature that postulated synovial dif- transfected C2C12 mouse myoblasts ( 28 ), correlated SS18– ferentiation based on the propensity for this malignancy to SSX binding with polycomb-marked nucleosomes (trimethyl- originate in periarticular regions, and the presence (especially ated histone H3K27) at a subset of genomic H3K27me3 sites. in biphasic cases) of some reminiscent histology. Synovial sar- In studies investigating possible target genes for SS18– comas do indeed display unusual morphologic features that SSX-mediated repression, expression profi ling ( 29 ) showed are suggestive of a capacity for pluripotential differentiation. the tumor suppressor EGR1 to be among the most consist- Whereas approximately 75% of cases are “monophasic” spin- ently downregulated genes in synovial sarcoma. ChIP studies dle cell tumors, the less common biphasic variant constitutes revealed the presence of SS18–SSX at a cyclic AMP response a pathognomonic example of true mesenchymal–epithelial element (CRE ) in the EGR1 promoter (30 ). Furthermore, transition, with formation of internal epithelial surfaces and inhibitors of histone deacetylases (HDAC)—whose antican- even glandular structures. This epiphenomenon is driven by cer activity had been shown previously in synovial sarcoma variable repression of E-cadherin by SS18–SSX interactions xenografts (31 )—were found to reverse the repression of EGR1 , with the SNAIL and SLUG transcription factors ( 38 ). The accompanied by loss of polycomb residency at this locus. predominant mesenchymal component in synovial sarcoma Subsequent studies detailed proteomic and biochemical can grow as a pure sheet of plump spindle cells reminiscent characterizations of SS18–SSX-binding partners, and identi- of embryologic mesenchyme, but can alternatively display fi ed Activating Transcription Factor 2 (ATF2) as the transcrip- variable degrees of multilineage mesenchymal differentiation tion factor responsible for targeting the fusion oncoprotein ( 39 ), with some cases producing loosely myxoid, densely col- to CRE sites (32 ). Moreover, these studies identifi ed a robust lagenized, or even osteoid-type extracellular matrix. interaction of the transcriptional corepressor Transducin- An intriguing observation whose relevance to the cell of Like Enhancer of Split 1 (TLE1) with the SSX portion of origin of synovial sarcoma remains unclear came from the the fusion oncoprotein in human and mouse model syno- discovery that a mouse model of monophasic synovial sar- vial sarcoma cells, confi rmed in surgically excised patient coma can be generated by conditional expression of human tumor tissue. SS18–SSX serves as a bridge between ATF2 SS18– SSX2 in a MYF5 early skeletal muscle precursor (myo- and TLE1, mediating repression of ATF2 targets, including blast) population ( 11 ). These tumors arose with 100% pen- among others EGR1 , ATF3 , and CDKN2A . Both the SWI/SNF etrance, with no other cooperating background transgenes

126 | CANCER DISCOVERYFEBRUARY 2015 www.aacrjournals.org

Advances in Synovial Sarcoma REVIEW

required. Expression in later stage MYF6+ myoblasts caused Secondary Mutations in Synovial Sarcoma myopathy but no tumors, whereas cleavage-stage embryo In keeping with the observed chromosomal stability, the expression or conditional expression in earlier (PAX3+ or + most commonly mutated gene in human cancer, TP53 , is PAX7 ) myoblast populations was embryonic lethal, as was rarely mutated in synovial sarcoma, occurring in 11 of 92 expression of SS18 – SSX2 in early ectoderm (AP2 +), bone/carti- + + + + cases across three published studies ( 46–48 ); in these studies, lage (SOX9 ), endothelial (FLK1 ; TIE2 ), or neural (Nestin1 ) copy-number gains in the p53-suppressing oncogene MDM2 precursor populations (40 ). However, a tamoxifen-induced were somewhat more frequent. Overall, wild-type p53 appears conditional expression model leads to development of less to be retained in most synovial sarcomas, although its func- aggressive synovial sarcomas in a somewhat different ana- tion may be impaired through upstream regulatory events, as tomical distribution (including paraspinal and facial pri- + in several well-described prosurvival interactions within the mary sites), suggesting that backgrounds other than MYF5 AKT–PTEN pathway. myoblasts may also be permissive for SS18–SSX oncogenesis. Otherwise, targeted sequencing approaches have high- Notably, neither in mouse models nor in human tissues do lighted mutations in PTEN , CTNNB1 , and APC in 8% to 14% the synovial sarcoma tumors express biomarkers typical of of cases (49–51 ), providing some support for oncogenic acti- muscle differentiation. vation of the AKT–mTOR and WNT signaling pathways in Whereas expression of SS18–SSX in most cellular back- this sarcoma (see below). grounds appears to precipitate cell death, pluripotential stem As of this writing, only one whole-exome sequencing study cells as well as human mesenchymal stem cells are permis- had been published on synovial sarcoma, on tumors from 7 sive—although importantly the expression profi les induced patients ( 9 ). This work identifi ed an average of eight somatic by SS18–SSX in these two precursor populations are very mutations per tumor exome, and no genomic losses in the different (41 ). HEK293 cells can also be successfully engi- majority of cases. Solitary mutations in cancer pathway genes neered to express SS18–SSX, but as mentioned above, differ- were identifi ed for TP53 , SETD2 , and FBXW7 . ent oncoprotein complexes appear to be formed in transient On the whole, these results suggest an encouraging setting transfection versus stably transfected models. In this regard, for targeted therapy of synovial sarcoma—if treated early in its the high levels of TLE1 observed in synovial sarcoma may natural history, when the majority of tumors do not exhibit be crucial, considering its possibly central role in recruiting genomic instability or extensive genomic rearrangements. Cell repressive activities to target promoters. Expression of TLE1 death pathways and growth controls may be largely intact and its interacting transcription factors in stem/progenitor and responsive to effective targeting of SS18–SSX. Compared cells may help defi ne permissive lineages for the develop- with most common cancer types, there may be fewer escape ment of tumors in transgenic animals. These observations mechanisms for emergence of therapy-resistant subclones in highlight a major limitation of most engineered models of synovial sarcoma. synovial sarcoma—the cellular background is probably not correct. Therefore, it is critical that experimental fi ndings attributed to synovial sarcoma biology from experimental Gene and Protein Expression in Synovial Sarcoma systems are verifi ed in models derived from primary human A major theme from gene-expression profi ling studies of tumor tissue expressing SS18–SSX under its endogenous synovial sarcoma relates to high expression of mediators promoter. These include several published cell lines grown involved in the patterning systems of early embryogenesis, as monolayers, 3D spheroids (42 ) or xenografts (43 ). Results including WNT (LEF1 , AXIN2 , WIF1 , WNT5A , and FZD10 ), ultimately will have to be verifi ed on patient tissue samples. NOTCH (HES1 , JAG1 , JAG2 , and NRARP ), Hedgehog (PTCH1 , Methodologically, verifi cation of SS18 – SSX fusion transcript GLI1 , and GLI2 ), FGF (FGFR2 , FGFR3 , FGF18 , and FGF9 ), expression is an important way to confi rm model or tissue and BMP pathways (BMP7 , BMP5 , BMPR2 , and SOSTDC1 ). integrity in synovial sarcoma research. These results and expression of other markers of embryonic primordia (SALL2 , TLE1 , SIX1 , SIX4 , and DLX2) support the idea that synovial sarcoma cells have a stem-like or early pro- ADDITIONAL MOLECULAR CHANGES FOUND genitor phenotype. Of note, primary synovial sarcomas show IN SYNOVIAL SARCOMA TUMORS a remarkable correspondence with the expression profi les of the conditional MYF5–CRE mouse model (11 ) and SS18 – SSX - Chromosomal Alterations in Synovial Sarcoma transduced embryonic stem cells ( 41 ). Synovial sarcoma differs from most common forms of Synovial sarcomas commonly express relatively high levels cancer by virtue of having much lower genetic complex- of mRNA for cancer–testis antigens, a group of potentially ity. Almost half of primary tumors have no chromosomal immunogenic proteins expressed in many tumor types, but aberrations other than t(X;18), and the remainder display not in normal adult tissues outside the germline: PRAME , only small numbers of changes (median 2–4; ref. 44 ). Nota- CTAG1A (encoding NY-ESO-1), and MAGEE1 are promi- bly, tumors with a balanced t(X;18) and no other genomic nent examples. The possible relevance of this aberrant anti- changes can show a completely “fl at” genomic copy-number gen expression in terms of response in recently developed profi le. Additional genomic copy-number changes, which immuno therapy approaches remains to be determined. are more common in adults (>18 years) than in pediatric High expression of neural ( NPTX2, NEFH , and NTNG2) patients, are strongly associated with metastatic spread and and chondrocyte (COL2A1 , COL9A3 , SOX9 , and TRPS1) line- poor outcomes. Not surprisingly, metastatic and recurrent age markers has been interpreted as consistent with differen- tumors display increased chromosomal complexity ( 45 ). tiation from neural crest progenitors. High mRNA expression

FEBRUARY 2015CANCER DISCOVERY | 127

REVIEW Nielsen et al. of SOX2 , an important determinant of neural progenitors and viability in vitro in fi ve human synovial sarcoma cell lines and embryonic stem cells, is common in synovial sarcoma and has reduced in vivo growth of xenografted SYO-1 synovial sarcoma been shown to be critical for growth of some cell lines in vitro cells. ( 15 , 39 ). However, its expression is absent in other cell lines Together, these studies nominate the WNT–β-catenin sig- and is not a universal feature of primary tumor specimens. naling pathway as an important new candidate therapeutic Other notable features of the synovial sarcoma profi le target in synovial sarcoma, with putative mechanisms as include differential high expression of BCL2 , as well as of the outlined in Fig. 2 . As new agents effi ciently targeting this receptor tyrosine kinases (RTK) PDGFRA , EGFR , and ERBB2 . pathway are in clinical development, these studies provide The latter group may be important for the stimulation of a critical preclinical rationale for their evaluation in clini- PI3K–AKT signaling, which has been reported as critical to cal trials (68, 69). Beyond the direct targeting of WNT–β- synovial sarcoma viability in several studies ( 52, 53 ), and is catenin signaling pathway components by small molecules, discussed further below. targeting of key interacting proteins also offers therapeutic Many of the above fi ndings have been confi rmed at the pro- opportunities. For instance, β-catenin forms a complex with tein level in patient specimens, including FZD10 ( 54 ), HES1 the transcriptional modulator YAP1 and the YES1 tyrosine (55 ), NY-ESO-1 (56 ), BMPR2 (57 ), SALL2 and EGFR (58 ), kinase, which may explain why dasatinib, which blocks the BCL2 ( 59 ), SOX9 ( 60 ), FGFs and their receptors ( 61 ), PDGFRA latter, also inhibits WNT–β-catenin–dependent prolifera- (52 ), and WNT pathway mediators (discussed further in the tion (69 ). Interestingly, a recent study found that dasatinib next section). caused apoptosis and inhibition of cellular proliferation in synovial sarcoma cells; some data support that this effect TARGETABLE ONCOGENIC PATHWAYS is mediated by inhibition of activated SRC ( 70 ), but it is ACTIVE IN SYNOVIAL SARCOMA tempting to speculate that the potency of the drug may also have been due to concurrent effects on WNT–β-catenin– WNT– b-Catenin Signaling Pathway dependent targets. There has been longstanding evidence for WNT activa- In light of the recent fi nding that SS18–SSX may, under tion in a subset of synovial sarcomas. Sanger sequencing certain circumstances, displace SMARCB1 from SWI/SNF studies found canonical activating mutations in CTNNB1 , chromatin remodeling complexes (17 ), it is also notable that at frequencies of 4% (N = 24; ref. 62 ), 8% (N = 49; ref. 63 ), loss of SMARCB1 results in activation of WNT–β-catenin sig- and 12% (N = 16; ref. 50 ), and APC mutations in 8% ( N = naling in mouse and cell culture models, refl ecting a role for 49) of cases ( 51 ). At the protein level, WNT pathway activa- the SWI/SNF complex in regulating this pathway ( 71 ). tion, as evidenced by nuclear accumulation of β-catenin, has been studied using immunohistochemistry. By this method, AKT–MTOR Signaling Pathway nuclear accumulation of β-catenin is detected in 30% to 60% Various lines of evidence also support an important role of synovial sarcomas ( 64, 65 ), primarily in monophasic cases for the AKT–mTOR pathway in synovial sarcoma, including or in the spindle cell component of biphasic cases. The SYO-1 genetic, protein level, preclinical, and clinical trial data. At the synovial sarcoma cell line harbors a codon 34 mutation in genetic level, PTEN mutations are found in only a minority of CTNNB1 (G34L) with concomitant protein accumulation cases (49 , 62 , 72 ) and canonical PIK3CA mutations are even and shows reduced proliferation and impairment in stand- more uncommon (72 ). Nonetheless, several groups have dem- ard invasion and migration assays when transfected with an onstrated frequent activation of AKT in synovial sarcoma, inhibitory dominant-negative LEF1 construct (T. Saito and and its dependency on upstream RTKs (53 , 73 , 74 ). As noted colleagues; unpublished data). above, apoptosis induction in synovial sarcoma cell lines by Recently, two independent studies have provided impor- HDAC inhibitors is at least partially dependent on derepres- tant functional evidence for a critical role of this signaling sion of EGR1 and subsequent induction of PTEN (30 ), a phe- pathway in synovial sarcoma. Barham and colleagues (66 ) nomenon that may help to identify strategic combinations showed that genetic loss of β-catenin blocks tumor formation involving these promising therapeutic agents. in the MYF5–CRE SS18–SSX2 transgenic model described Notably, fundamental interactions of PI3K–AKT–mTOR and above. To confi rm this observation in the human setting, RAS–MEK–ERK pathways have been identifi ed downstream of they showed that pharmacologic activation of CSNK1A by RTK signaling, and multiple levels of feedback regulation have pyrvinium, known to inhibit β-catenin signaling, or RNAi been identifi ed within and between these pathways, as summa- knockdown of LRP6 could both impair growth of human rized in Fig. 3 . These interactions underlie intrinsic resistance synovial sarcoma cell lines. These investigators also showed to PI3K–AKT–mTORC1-targeted monotherapies in synovial that SS18–SSX can induce nuclear β-catenin accumulation, sarcoma, for example, involving feedback activation of AKT apparently by inducing autocrine signaling through its aber- following mTOR inhibition, which can be mediated by either rant transcriptional effects. These data may explain the fact IFG1R or PDGFRA (43 , 52 ). These data support the combina- that the proportion of human synovial sarcoma tumors with tion of mTOR inhibitors with inhibitors of particular RTKs nuclear β-catenin accumulation is several fold higher than that potentiate feedback AKT activation in a given tumor. Strat- the rate of CTNNB1 or APC mutations. egies that may intercept these signals at critical downstream In a separate recent study, Trautmann and colleagues ( 67 ) nodes are also under investigation, including the combination found that introduction of SS18–SSX into HEK293 cells of PI3K and MEK inhibitors, and the use of pan-mTOR kinase induced activation of WNT–β-catenin signaling. Conversely, inhibitors targeting both mTORC1 and mTORC2 (which are small-molecule TCF–β-catenin complex inhibitors reduced cell not associated with reactivation of AKT; ref. 75 ).

128 | CANCER DISCOVERYFEBRUARY 2015 www.aacrjournals.org

Advances in Synovial Sarcoma REVIEW

WNT FZD LRP6

Destruction complex DVL GSK3B Acyltransferase CSNK1 LGK974 PORCN Cytoplasm *

AXIN * CTNNB1 +Ub

APC WNT

CTNNB1 Proteasome

SWI/SNF PRI-724 Nucleus BRG1 CTNNB1 JAG2, CCND1, LEF1, CBP AXIN2, WNT11 SS18/SSX LEF1 POLII Wnt targets TLE1 HDAC

TLE1 LEF1 GLI CBP SMAD ? ? HES POLII Wnt ligands ETS

Figure 2. Putative mechanisms for autocrine/paracrine activation of WNT signaling in synovial sarcoma. WNT signals through FZD/LRP6, inhibiting the APC–AXIN–GSK3B–CSNK1 destruction complex, leading to release and nuclear transport of β-catenin (CTNNB1). SS18–SSX integrates within SWI/SNF complexes, with potential interactions with developmental pathways, including NOTCH–HES, BMP–SMAD, Hedghog–GLI, MAPK–ETS, and LEF1–TLE1. Extensive cross-regulation between developmental pathways may lead to transcriptional activation of WNT ligands and/or derepression of WNT target genes. The sites of action of WNT inhibitors PRI-724 and LGK974 are indicated.

Antiapoptotic Pathways mouse tumor model ( 78 ). These fi ndings suggest that BCL2 Histologically, synovial sarcomas typically display few inhibitors may be worth evaluating in synovial sarcoma, mitotic fi gures, while also displaying few apoptotic bodies. perhaps in combination with apoptosis-inducing cytotoxic The tumor’s propensity in many cases for slow growth, late chemotherapy, but as yet no such clinical trials have been recurrences, and chemotherapy resistance is consistent with opened. an antiapoptotic phenotype. A striking feature of synovial sarcoma is its consistently very high expression of BCL2 ( 59 , 76 , 77 ), but no evidence has been found that the BCL2 gene CLINICAL TRIALS IN SYNOVIAL SARCOMA is a direct target for transcriptional upregulation by SS18– Table 1 summarizes several trials in which synovial sarcoma SSX; therefore, its high level of expression may be a second- may be included as an eligible diagnosis. Given its low inci- ary oncogenic effect, or may refl ect a prerequisite cellular dence and a patient population split between pediatric and background to be permissive for SS18–SSX. In contrast, the adult institutions, synovial sarcoma disease-specifi c trials have BCL2 family members BCL2A1 and MCL1 are direct targets to date been largely precluded by logistical challenges. Instead, for SS18–SSX-mediated repression. Resistance to new BH3 patients with this well-defi ned and unique sarcoma have gen- mimetic BCL2 inhibitors in lymphomas is typically induced erally been lumped with patients with other soft-tissue sar- by activation of MCL1, but this escape mechanism is not comas, as in current trials of multitargeted TKIs (pazopanib: available to synovial sarcomas, which appear to be highly NCT02180867; regorafenib: NCT01900743). More closely sensitive to such drugs in human cells and an SS18– SSX2 tied to some of the biologic dependencies of synovial sarcoma

FEBRUARY 2015CANCER DISCOVERY | 129

REVIEW Nielsen et al.

FGFR/PDGFR/ IGF1R Plasma membrane

RAS PIP2 PIP3 PDK1 PI3K

IRS1 PTEN

RAF Mitochondrion FBXW8 RICTOR AKT BAD mTOR S6K MEK BH3 BCL2 GRB10 RAPTOR

ERK mTOR BAX

RHEB SAPK DUSP

TSC1/2 FOXO3 GSK3B

Cytoplasm

HDAC PRC2 FOXO3 Nucleus TLE1 BH3 (BIM, PUMA, NOXA) SS18/SSX

ATF2 AP1 DUSP1, DUSP10

ELK1 EGR1 EGR1, MCL1 PTEN

Figure 3. Drug targets downstream of RTKs involved in synovial sarcoma. Extensive cross-talk and feedback regulation are observed in prosurvival signaling through MAPK–ERK and AKT–mTOR pathways. Intrinsic drug resistance to monotherapies can be mediated by activation of prosurvival signaling in either pathway, due to removal of inhibitory feedback (red lines). Drug targets discussed in the text are shown in green. BH3 represents proapoptotic BH3 domain–only proteins, and their activation by stress-activated protein kinases is shown (SAPK: JNK, p38). Dashed lines represent nuclear export and translation of mRNA to protein.

130 | CANCER DISCOVERYFEBRUARY 2015 www.aacrjournals.org

Advances in Synovial Sarcoma REVIEW

Table 1. Summary data for clinical trials referenced in the text

NCI trial Agent 1 Agent 2 Target accrual Start/end Phase NCT02180867 Pazopanib Radiation/chemoradiation 340 2014–2018 II/III NCT01900743 Regorafenib 192 2013–2016 II NCT01879085 Vorinostat Gemcitabine + docetaxel 62 2013–2020 IB/II NCT01294670 Vorinostat Etoposide 50 2011–2015 I/II NCT01614795 Temsirolimus Cixutumumab 45 2012–2013 II NCT01154452 RO4929097 Vismodegib 120 2010–2014 IB/II NCT01302405 PRI-724 54 2011–2014 IA/IB NCT01351103 LGK974 80 2012–2015 I NCT01469975 OTSA101-DTPA-90Y 18 2011–2014 I NCT01477021 Autologous T cells Cyclophosphamide 7 2012–2014 I NCT02122861 ID-LV305 36 2014–2016 I NCT01058707 MLN0128 190 2010–2014 I

described above are efforts to test histone deacetylase inhibi- open trials, using strategies such as adoptive T-cell transfer tors (including vorinostat: NCT01879085, NCT01294670) (NCT01477021) or dendritic cell activation (NCT02122861). and IGF1R/mTOR inhibitor combinations (cixutumumab/ Direct targeting of SS18–SSX oncoprotein has been temsirolimus: NCT01614795). Most of the above studies will attempted using peptide vaccination with an MHC class incorporate preplanned disease-specifi c subgroup analyses, 1–binding epitope of the oncoprotein, in a small trial of 21 but most likely will remain severely underpowered to identify patients, of whom 8 were unable to complete the 12-week vac- signifi cant activity specifi cally against synovial sarcoma. cination schedule due to disease progression (79 ). Four arms Inhibitors of the Hedgehog and NOTCH pathways are in of the trial were defi ned, depending on the use of agretope- earlier stages of development, but are already being tested in modifi ed peptide with increased affi nity for HLA-A24, and on phase II studies that include synovial sarcoma among the eli- the addition of Freund’s incomplete adjuvant with IFNα. No gible diagnoses (vismodegib + RO4929097: NCT01154452). robust immune responses were detected as measured using The fi rst phase I trials of WNT inhibition in advanced solid delayed type hypersensitivity and tetramer assays, designed tumors may constitute important referrals for synovial sar- to show the presence of antigen-specifi c CD8+ T cells. These coma. These include the agent PRI-724 that blocks the inter- results are in line with the limited clinical effi cacy of MHC-I action of β-catenin with CBP (NCT01302405). Another new peptide vaccines, but may also relate to the generally observed trial (NCT01351103) is of LGK974, an inhibitor of PORCN, lack of infl ammatory infi ltrates in synovial sarcoma. The an acyltransferase required for secretion of active WNT lig- basis of immune evasion in synovial sarcoma is unknown, but and; referral to this trial is contingent on demonstration of may be due to limited neoantigen production in these hypo- tumors with WNT ligand dependency, and this may require mutated tumors, low expression of class I MHC, or to other further preclinical studies for synovial sarcoma. immune suppression mechanisms active in mesenchymal Expression of the WNT receptor Frizzled class receptor 10 cells with stem/progenitor phenotypes. (FZD10) is consistently high in synovial sarcoma, and there Chimeric antigen receptor T cells represent another promising is an open synovial sarcoma–specifi c trial using a Ytrium-90– approach, in which T cells are engineered to express a chimeric labeled antibody to this receptor (NCT01469975). FZD10 is T-cell receptor, whose signaling components are fused with an reported to couple to noncanonical (β-catenin–independent) antibody fragment specifi c to a surface antigen on tumor cells. WNT signaling in synovial sarcoma cells, and the unlabeled Activation of costimulatory signals is also engineered, so that antibody has only weak inhibitory activity against growth the chimeric antigen receptor T cells are able to bypass immune of these cells. However, FZD10 is not expressed on normal checkpoints, recognizing and killing tumor cells independent cells of vital organs, and as such it is excellent for targeting of MHC antigen presentation. Given the unique and markedly the radioisotope specifi cally to synovial sarcoma tumor cells. elevated expression of FZD10 in synovial sarcoma, this would Antibody-based approaches may additionally work by seem to represent an attractive target for this technology. inducing antitumor immune responses; other immunotherapy strategies include blockade of immune checkpoints and eliciting responses to neoantigens. Recent successes FUTURE PERSPECTIVES involving inhibition of immune checkpoints have led to a Despite genomic and clinical progression, the SS18–SSX general resurgence of interest in immunotherapy for solid fusion gene is consistently retained in synovial sarcoma, and tumors. Potential immunogenic CT antigens expressed in synovial sarcoma cells depend on continued SS18–SSX expres- synovial sarcoma but not normal tissues have been identifi ed, sion throughout the course of disease. This provides a basis including NY-ESO-1, an antigen that is the basis for several for monitoring disease recurrence, through PCR detection

FEBRUARY 2015CANCER DISCOVERY | 131

REVIEW Nielsen et al. of the fusion gene in circulating tumor cells or in free tumor adult and pediatric patients with localized high-grade synovial sar- DNA in plasma. As more effective systemic treatments evolve, coma. Sarcoma 2011 ; 2011 : 231789 . the clinical utility of such tests will become more signifi cant. 6. Clark J , Rocques PJ , Crew AJ , Gill S , Shipley J , Chan AM , et al. Identifi cation of novel genes, SYT and SSX, involved in the t(X;18) Agents that directly target the fusion oncoprotein and its (p11.2;q11.2) translocation found in human synovial sarcoma . Nat interactions remain the Holy Grail for the fi eld, and several Genet 1994 ; 7 : 502 – 8 . newer proximity-based assays could, in principle, be adapted 7. Skytting B , Nilsson G , Brodin B , Xie Y , Lundeberg J , Uhlen M , et al. A for high-throughput screening of small-molecule inhibitors. novel fusion gene, SYT-SSX4, in synovial sarcoma . J Natl Cancer Inst Given the broad scope and ubiquity of such fundamental 1999 ; 91 : 974 – 5 . interactions in normal tissues—for example, between SS18 8. Panagopoulos I , Mertens F , Isaksson M , Limon J , Gustafson P , S kyt- ting B , et al. Clinical impact of molecular and cytogenetic fi ndings in domains and BRG1—in the short term, it may be more fea- synovial sarcoma. Genes Chromosomes Cancer 2001 ; 31 : 362 – 72 . sible to target the critical enzymatic cofactors of SS18–SSX 9. Joseph CG , Hwang H , Jiao Y , Wood LD , Kinde I , Wu J , et al. Exomic (such as histone-modifying transcriptional effectors) or the analysis of myxoid liposarcomas, synovial sarcomas, and osteosarco- critical oncogenic pathways it induces. In this regard, EZH2 mas. Genes Chromosomes Cancer 2014 ; 53 : 15 – 24 . inhibitors are under investigation in synovial sarcoma models 10. Carmody Soni EE , Schlottman S , Erkizan HV , Uren A , Toretsky JA . and early-phase clinical trials. Screening experiments identify- Loss of SS18–SSX1 inhibits viability and induces apoptosis in syno- ing additional epigenetic dependencies are expected to yield vial sarcoma. Clin Orthop Relat Res 2014 ; 472 : 874 – 82 . 11. Haldar M , Hancock JD , Coffi n CM , Lessnick SL , Capecchi MR . A con- further mechanistic insights, which may guide additional ditional mouse model of synovial sarcoma: insights into a myogenic therapeutic strategies. Finally, given the challenges involved origin. Cancer Cell 2007 ; 11 : 375 – 88 . in opening, accruing, and executing well-powered synovial 12. Brett D , Whitehouse S , Antonson P , Shipley J , Cooper C , Goodwin G . sarcoma–specifi c trials, it is possible that drug-repurposing The SYT protein involved in the t(X;18) synovial sarcoma transloca- strategies will provide another set of opportunities for thera- tion is a transcriptional activator localised in nuclear bodies . Hum peutic advances. In cases where there is a good match between Mol Genet 1997 ; 6 : 1559 – 64 . 13. Perani M , Antonson P , Hamoudi R , Ingram CJ , Cooper CS , Garrett drug mechanism and oncogenic pathway, drugs developed for MD , et al. The proto-oncoprotein SYT interacts with SYT-interacting more common cancers may in fact work even better in a more protein/co-activator activator (SIP/CoAA), a human nuclear receptor genetically homogeneous malignancy like synovial sarcoma. co-activator with similarity to EWS and TLS/FUS family of proteins. J Biol Chem 2005 ; 280 : 42863 – 76 . Disclosure of Potential Confl icts of Interest 14. Thaete C , Brett D , Monaghan P , Whitehouse S , Rennie G , Rayner E , No potential confl icts of interest were disclosed. et al. Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM Acknowledgments in the nucleus. Hum Mol Genet 1999 ; 8 : 585 – 91 . 15. Kato H , Tjernberg A , Zhang W , Krutchinsky AN , An W , Takeuchi The authors thank Drs. B Brodin and T. Ito for insightful discus- T , et al. SYT associates with human SNF/SWI complexes and the sions and for their review of this article. The authors acknowledge the C-terminal region of its fusion partner SSX1 targets histones . J Biol contributions of the many sarcoma researchers whose work they are Chem 2002 ; 277 : 5498 – 505 . unable to reference due to space limitations. 16. Middeljans E , Wan X , Jansen PW , Sharma V , Stunnenberg HG , Logie C . SS18 together with animal-specifi c factors defi nes human BAF- Grant Support type SWI/SNF complexes. PLoS ONE 2012 ; 7 : e33834 . This work was supported by Terry Fox Research Institute grant no. 17. Kadoch C , Crabtree GR . Reversible disruption of mSWI/SNF (BAF) 1021 (to T.O. Nielsen), Canadian Cancer Society Research Institute complexes by the SS18–SSX oncogenic fusion in synovial sarcoma. grant no. 701582 (to T.O. Nielsen), the Liddy Shriver Sarcoma Initia- Cell 2013 ; 153 : 71 – 85 . tive (to T.O. Nielsen and M. Ladanyi), and National Cancer Institute 18. Jackson EM , Sievert AJ , Gai X , Hakonarson H , Judkins AR , Tooke L , et al. SPORE grant P50 CA140146 (to M. Ladanyi). Genomic analysis using high-density single nucleotide polymorphism- based oligonucleotide arrays and multiplex ligation-dependent probe amplifi cation provides a comprehensive analysis of INI1/SMARCB1 in Received October 23, 2014; revised December 23, 2014; accepted malignant rhabdoid tumors . Clin Cancer Res 2009 ; 15 : 1923 – 30 . December 29, 2014; published OnlineFirst January 22, 2015. 19. Le Loarer F , Zhang L , Fletcher CD , Ribeiro A , Singer S , Italiano A , et al. Consistent SMARCB1 homozygous deletions in epithelioid sarcoma and in a subset of myoepithelial carcinomas can be reliably detected by FISH in archival material. Genes Chromosomes Cancer REFERENCES 2014 ; 53 : 475 – 86 . 1. Herzog CE . Overview of sarcomas in the adolescent and young adult 20. Wang X , Sansam CG , Thom CS , Metzger D , Evans JA , Nguyen P T , population. J Pediatr Hematol Oncol 2005 ; 27 : 215 – 8 . et al. Oncogenesis caused by loss of the SNF5 tumor suppressor is 2. Ladanyi M , Antonescu CR , Leung DH , Woodruff JM , Kawai A , Healey dependent on activity of BRG1, the ATPase of the SWI/SNF chroma- JH , et al. Impact of SYT-SSX fusion type on the clinical behavior of tin remodeling complex . Cancer Res 2009 ; 69 : 8094 – 101 . synovial sarcoma: a multi-institutional retrospective study of 243 21. Mueller-Planitz F , Klinker H , Becker PB . Nucleosome sliding mecha- patients. Cancer Res 2002 ; 62 : 135 – 40 . nisms: new twists in a looped history. Nat Struct Mol Biol 2013 ; 20 : 3. Sultan I , Rodriguez-Galindo C , Saab R , Yasir S , Casanova M , Ferrari 1026 – 32 . A . Comparing children and adults with synovial sarcoma in the Sur- 22. Kadoch C , Hargreaves DC , Hodges C , Elias L , Ho L , Ranish J, et al. veillance, Epidemiology, and End Results program, 1983 to 2005: an Proteomic and bioinformatic analysis of mammalian SWI/SNF com- analysis of 1268 patients. Cancer 2009 ; 115 : 3537 – 47 . plexes identifi es extensive roles in human malignancy . Nat Genet 4. Spurrell EL , Fisher C , Thomas JM , Judson IR . Prognostic factors in 2013 ; 45 : 592 – 601 . advanced synovial sarcoma: an analysis of 104 patients treated at the 23. Tolstorukov MY , Sansam CG , Lu P , Koellhoffer EC , Helming KC , Royal Marsden Hospital. Ann Oncol 2005 ; 16 : 437 – 44 . Alver BH , et al. Swi/Snf chromatin remodeling/tumor suppressor 5. Al-Hussaini H , Hogg D , Blackstein ME , O’Sullivan B , Catton CN , complex establishes nucleosome occupancy at target promoters. Proc Chung PW , et al. Clinical features, treatment, and outcome in 102 Natl Acad Sci U S A 2013 ; 110 : 10165 – 70 .

132 | CANCER DISCOVERYFEBRUARY 2015 www.aacrjournals.org

Advances in Synovial Sarcoma REVIEW

24. Kia SK , Gorski MM , Giannakopoulos S , Verrijzer CP . SWI/SNF medi- kinase pathway analyses predict sensitivity to the mTOR inhibitor ates polycomb eviction and epigenetic reprogramming of the INK4b- RAD001. Cancer Lett 2014 ; 347 : 114 – 22 . ARF-INK4a locus. Mol Cell Biol 2008 ; 28 : 3457 – 64 . 44. Lagarde P , Przybyl J , Brulard C , Perot G , Pierron G , Delattre O , et al. 25. Guan B , Wang TL , Shih Ie M . ARID1A, a factor that promotes forma- Chromosome instability accounts for reverse metastatic outcomes of tion of SWI/SNF-mediated chromatin remodeling, is a tumor sup- pediatric and adult synovial sarcomas. J Clin Oncol 2013 ; 31 : 608 – 15 . pressor in gynecologic cancers. Cancer Res 2011 ; 71 : 6718 – 27 . 45. Przybyl J , Sciot R , Wozniak A , Schoffski P , Vanspauwen V , Samson 26. Watanabe R , Ui A , Kanno S , Ogiwara H , Nagase T , Kohno T , et al. I , et al. Metastatic potential is determined early in synovial sarcoma SWI/SNF factors required for cellular resistance to DNA damage development and refl ected by tumor molecular features. Int J Bio- include ARID1A and ARID1B and show interdependent protein sta- chem Cell Biol 2014 ; 53 : 505 – 13 . bility. Cancer Res 2014 ; 74 : 2465 – 75 . 46. Schneider-Stock R , Onnasch D , Haeckel C , Mellin W , Franke DS , 27. Soulez M , Saurin AJ , Freemont PS , Knight JC . SSX and the synovial- Roessner A . Prognostic signifi cance of p53 gene mutations and p53 sarcoma-specifi c chimaeric protein SYT-SSX co-localize with the protein expression in synovial sarcomas. Virchows Arch 1999 ; 435 : human Polycomb group complex. Oncogene 1999 ; 18 : 2739 – 46 . 407 – 12 . 28. Garcia CB , Shaffer CM , Eid JE . Genome-wide recruitment to Polycomb- 47. Oda Y , Sakamoto A , Satio T , Kawauchi S , Iwamoto Y , Tsuneyoshi modifi ed chromatin and activity regulation of the synovial sarcoma M . Molecular abnormalities of p53, MDM2, and H-ras in synovial oncogene SYT-SSX2. BMC Genomics 2012 ; 13 : 189 . sarcoma. Mod Pathol 2000 ; 13 : 994 – 1004 . 29. Baird K , Davis S , Antonescu CR , Harper UL , Walker RL , Chen Y , et al. 48. Ito M , Barys L , O’Reilly T , Young S , Gorbatcheva B , Monahan J , Gene expression profi ling of human sarcomas: insights into sarcoma et al. Comprehensive mapping of p53 pathway alterations reveals an biology. Cancer Res 2005 ; 65 : 9226 – 35 . apparent role for both SNP309 and MDM2 amplifi cation in sarcom- 30. Lubieniecka JM , de Bruijn DR , Su L , van Dijk AH , Subramanian S , agenesis. Clin Cancer Res 2011 ; 17 : 416 – 26 . van de Rijn M , et al. Histone deacetylase inhibitors reverse SS18–SSX- 49. Saito T , Oda Y , Kawaguchi K , Takahira T , Yamamoto H , Tanaka K , mediated polycomb silencing of the tumor suppressor early growth et al. PTEN and other tumor suppressor gene mutations as secondary response 1 in synovial sarcoma. Cancer Res 2008 ; 68 : 4303 – 10 . genetic alterations in synovial sarcoma. Oncol Rep 2004 ; 11 : 1011 – 5 . 31. Ito T , Ouchida M , Morimoto Y , Yoshida A , Jitsumori Y , Ozaki T , 50. Subramaniam MM , Calabuig-Farinas S , Pellin A , Llombart-Bosch A . et al. Signifi cant growth suppression of synovial sarcomas by the Mutational analysis of E-cadherin, beta-catenin and APC genes in histone deacetylase inhibitor FK228 in vitro and in vivo. Cancer Lett synovial sarcomas. Histopathology 2010 ; 57 : 482 – 6 . 2005 ; 224 : 311 – 9 . 51. Saito T , Oda Y , Sakamoto A , Kawaguchi K , Tanaka K , Matsuda S , 32. Su L , Sampaio AV , Jones KB , Pacheco M , Goytain A , Lin S , et al. Decon- et al. APC mutations in synovial sarcoma. J Pathol 2002 ; 196 : 445 – 9 . struction of the SS18–SSX fusion oncoprotein complex: insights into 52. Ho AL , Vasudeva SD , Lae M , Saito T , Barbashina V , Antonescu CR , disease etiology and therapeutics. Cancer Cell 2012 ; 21 : 333 – 47 . et al. PDGF receptor alpha is an alternative mediator of rapamycin- 33. Levanon D , Goldstein RE , Bernstein Y , Tang H , Goldenberg D , Stifani induced Akt activation: implications for combination targeted ther- S , et al. Transcriptional repression by AML1 and LEF-1 is mediated apy of synovial sarcoma. Cancer Res 2012 ; 72 : 4515 – 25 . by the TLE/Groucho corepressors. Proc Natl Acad Sci U S A 1998 ; 95 : 53. Friedrichs N , Trautmann M , Endl E , Sievers E , Kindler D , Wurs t P , 11590 – 5 . et al. Phosphatidylinositol-3 ′-kinase/AKT signaling is essential in 34. Grbavec D , Stifani S . Molecular interaction between TLE1 and the synovial sarcoma. Int J Cancer 2011 ; 129 : 1564 – 75 . carboxyl-terminal domain of HES-1 containing the WRPW motif . 54. Nagayama S , Fukukawa C , Katagiri T , Okamoto T , Aoyama T , Oyaizu Biochem Biophys Res Commun 1996 ; 223 : 701 – 5 . N , et al. Therapeutic potential of antibodies against FZD 10, a cell- 35. Nakayama R , Mitani S , Nakagawa T , Hasegawa T , Kawai A , Morioka surface protein, for synovial sarcomas. Oncogene 2005 ; 24 : 6201 – 12 . H , et al. Gene expression profi ling of synovial sarcoma: distinct 55. Ishibe T , Nakayama T , Aoyama T , Nakamura T , Toguchida J . Neuro- signature of poorly differentiated type. Am J Surg Pathol 2010 ; 34 : nal differentiation of synovial sarcoma and its therapeutic applica- 1599 – 607 . tion. Clin Orthop Relat Res 2008 ; 466 : 2147 – 55 . 36. Valente AL , Tull J , Zhang S . Specifi city of TLE1 expression in unclassi- 56. Jungbluth AA , Antonescu CR , Busam KJ , Iversen K , Kolb D , Coplan fi ed high-grade sarcomas for the diagnosis of synovial sarcoma. Appl K , et al. Monophasic and biphasic synovial sarcomas abundantly Immunohistochem Mol Morphol 2013 ; 21 : 408 – 13 . express cancer/testis antigen NY-ESO-1 but not MAGE-A1 or CT7 . 37. Jagdis A , Rubin BP , Tubbs RR , Pacheco M , Nielsen TO . Prospectiv e Int J Cancer 2001 ; 94 : 252 – 6 . evaluation of TLE1 as a diagnostic immunohistochemical marker in 57. Guo W , Gorlick R , Ladanyi M , Meyers PA , Huvos AG , Bertino JR , synovial sarcoma. Am J Surg Pathol 2009 ; 33 : 1743 – 51 . et al. Expression of bone morphogenetic proteins and receptors in 38. Saito T , Nagai M , Ladanyi M . SYT-SSX1 and SYT-SSX2 interfere with sarcomas. Clin Orthop Relat Res 1999 ; 365 : 175 – 83 . repression of E-cadherin by snail and slug: a potential mechanism 58. Nielsen TO , Hsu FD , O’Connell JX , Gilks CB , Sorensen PH , Linn S , for aberrant mesenchymal to epithelial transition in human synovial et al. Tissue microarray validation of epidermal growth factor recep- sarcoma. Cancer Res 2006 ; 66 : 6919 – 27 . tor and SALL2 in synovial sarcoma with comparison to tumors of 39. Naka N , Takenaka S , Araki N , Miwa T , Hashimoto N , Yoshioka K , similar histology. Am J Pathol 2003 ; 163 : 1449 – 56 . et al. Synovial sarcoma is a stem cell malignancy. Stem Cells 2010 ; 28 : 59. Pelmus M , Guillou L , Hostein I , Sierankowski G , Lussan C , Coindre 1119 – 31 . JM . Monophasic fi brous and poorly differentiated synovial sarcoma: 40. Haldar M , Hedberg ML , Hockin MF , Capecchi MR . A CreER-based immunohistochemical reassessment of 60 t(X;18)(SYT-SSX)-positive random induction strategy for modeling translocation-associated cases. Am J Surg Pathol 2002 ; 26 : 1434 – 40 . sarcomas in mice. Cancer Res 2009 ; 69 : 3657 – 64 . 60. Cajaiba MM , Jianhua L , Goodman MA , Fuhrer KA , Rao UN . Sox9 41. Hayakawa K , Ikeya M , Fukuta M , Woltjen K , Tamaki S , Takahara N , expression is not limited to chondroid neoplasms: variable occur- et al. Identifi cation of target genes of synovial sarcoma-associated rence in other soft tissue and bone tumors with frequent expression fusion oncoprotein using human pluripotent stem cells . Biochem by synovial sarcomas. Int J Surg Pathol 2010 ; 18 : 319 – 23 . Biophys Res Commun 2013 ; 432 : 713 – 9 . 61. Ishibe T , Nakayama T , Okamoto T , Aoyama T , Nishijo K , Shibata K R, 42. Wakamatsu T , Naka N , Sasagawa S , Tanaka T , Takenaka S , Araki et al. Disruption of fi broblast growth factor signal pathway inhibits N , et al. Defl ection of vascular endothelial growth factor action by the growth of synovial sarcomas: potential application of signal SS18–SSX and composite vascular endothelial growth factor- and inhibitors to molecular target therapy. Clin Cancer Res 2005 ; 11 : chemokine (C-X-C motif) receptor 4-targeted therapy in synovial 2702 – 12 . sarcoma. Cancer Sci 2014 ; 105 : 1124 – 34 . 62. Barretina J , Taylor BS , Banerji S , Ramos AH , Lagos-Quintana M , 43. Yasui H , Naka N , Imura Y , Outani H , Kaneko K , Hamada K, et al. Decarolis PL , et al. Subtype-specifi c genomic alterations defi ne new Tailored therapeutic strategies for synovial sarcoma: receptor tyrosine targets for soft-tissue sarcoma therapy. Nat Genet 2010 ; 42 : 715 – 21 .

FEBRUARY 2015CANCER DISCOVERY | 133

REVIEW Nielsen et al.

63. Saito T , Oda Y , Sakamoto A , Tamiya S , Kinukawa N , Hayashi K , et al. 72. Teng HW , Wang HW , Chen WM , Chao TC , Hsieh YY , Hsih CH , et al. Prognostic value of the preserved expression of the E-cadherin and Prevalence and prognostic inﬂ uence of genomic changes of EGFR catenin families of adhesion molecules and of beta-catenin mutations pathway markers in synovial sarcoma. J Surg Oncol 2011 ; 103 : 773 – 81 . in synovial sarcoma. J Pathol 2000 ; 192 : 342 – 50 . 73. Bozzi F , Ferrari A , Negri T , Conca E , Luca da R , Losa M , et al. Molecu- 64. Horvai AE , Kramer MJ , O’Donnell R . Beta-catenin nuclear expres- lar characterization of synovial sarcoma in children and adolescents: sion correlates with cyclin D1 expression in primary and metastatic evidence of akt activation. Transl Oncol 2008 ; 1 : 95 – 101 . synovial sarcoma: a tissue microarray study . Arch Pathol Lab Med 74. Setsu N , Kohashi K , Fushimi F , Endo M , Yamamoto H , Takahashi 2006 ; 130 : 792 – 8 . Y , et al. Prognostic impact of the activation status of the Akt/mTOR 65. Ng TL , Gown AM , Barry TS , Cheang MC , Chan AK , Turbin DA , pathway in synovial sarcoma. Cancer 2013 ; 119 : 3504 – 13 . et al. Nuclear beta-catenin in mesenchymal tumors. Mod Pathol 75. Kang MH , Reynolds CP , Maris JM , Gorlick R , Kolb EA , Lock R, et al. 2005 ; 18 : 68 – 74 . Initial testing (stage 1) of the investigational mTOR kinase inhibitor 66. Barham W , Frump AL , Sherrill TP , Garcia CB , Saito-Diaz K , VanSaun MLN0128 by the pediatric preclinical testing program . Pediatr Blood MN , et al. Targeting the Wnt pathway in synovial sarcoma models. Cancer 2014 ; 61 : 1486 – 9 . Cancer Discov 2013 ; 3 : 1286 – 301 . 76. Antonescu CR , Kawai A , Leung DH , Lonardo F , Woodruff JM , Healey 67. Trautmann M , Sievers E , Aretz S , Kindler D , Michels S , Friedrichs N , JH , et al. Strong association of SYT-SSX fusion type and morphologic et al. SS18–SSX fusion protein-induced Wnt/beta-catenin signaling is epithelial differentiation in synovial sarcoma. Diagn Mol Pathol a therapeutic target in synovial sarcoma. Oncogene 2013 ; 33 : 5006 – 16 . 2000 ; 9 : 1 – 8 . 68. Profﬁ tt KD , Madan B , Ke Z , Pendharkar V , Ding L , Lee MA , et al. Phar- 77. Krskova L , Kalinova M , Brizova H , Mrhalova M , Sumerauer D , Ko det macological inhibition of the Wnt acyltransferase PORCN prevents R . Molecular and immunohistochemical analyses of BCL2, KI-67, and growth of WNT-driven mammary cancer. Cancer Res 2013 ; 73 : 502 – 7 . cyclin D1 expression in synovial sarcoma . Cancer Genet Cytogenet 69. Rosenbluh J , Wang X , Hahn WC . Genomic insights into WNT/beta- 2009 ; 193 : 1 – 8 . catenin signaling. Trends Pharmacol Sci 2014 ; 35 : 103 – 9 . 78. Jones KB , Su L , Jin H , Lenz C , Randall RL , Underhill TM, et al. 70. Michels S , Trautmann M , Sievers E , Kindler D , Huss S , Renner M , SS18–SSX2 and the mitochondrial apoptosis pathway in mouse and et al. SRC signaling is crucial in the growth of synovial sarcoma cells . human synovial sarcomas. Oncogene 2013 ; 32 : 2365 – 71 . Cancer Res 2013 ; 73 : 2518 – 28 . 79. Kawaguchi S , Tsukahara T , Ida K , Kimura S , Murase M , Kano M , et al. 71. Mora-Blanco EL , Mishina Y , Tillman EJ , Cho YJ , Thom CS , Pomer oy SYT-SSX breakpoint peptide vaccines in patients with synovial sar- SL , et al. Activation of beta-catenin/TCF targets following loss of the coma: a study from the Japanese Musculoskeletal Oncology Group. tumor suppressor SNF5. Oncogene 2014 ; 33 : 933 – 8 . Cancer Sci 2012 ; 103 : 1625 – 30 .

134 | CANCER DISCOVERYFEBRUARY 2015 www.aacrjournals.org

Synovial Sarcoma: Recent Discoveries as a Roadmap to New Avenues for Therapy

Torsten O. Nielsen, Neal M. Poulin and Marc Ladanyi

Cancer Discovery 2015;5:124-134. Published OnlineFirst January 22, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-14-1246

Cited articles This article cites 79 articles, 20 of which you can access for free at: http://cancerdiscovery.aacrjournals.org/content/5/2/124.full#ref-list-1

Citing articles This article has been cited by 11 HighWire-hosted articles. Access the articles at: http://cancerdiscovery.aacrjournals.org/content/5/2/124.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/5/2/124. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.