Interdependence of SS18-SSX-driven YAP1 and ß-Catenin Activation in Synovial Sarcoma.

Synovial sarcoma, a rare malignant soft tissue tumor, is characterized by a specific chromosomal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein drives synovial sarcoma pathogenesis by integrating into the BAF complex and dysregulating gene transcription. Because previous functional analyses revealed a connection between SS18-SSX and the activity of the transcriptional coregulators YAP1/TAZ and ß-catenin, respectively, this study examined a potential interdependence between these essential effector proteins in synovial sarcoma. In a large cohort of synovial sarcoma tissue specimens, IHC analyses revealed a substantial subset of synovial sarcoma with concurrent nuclear accumulation of YAP1/TAZ and ß-catenin. In vitro, small-molecule inhibitor treatment, RNAi-mediated knockdown, and vector-based overexpression assays demonstrated that YAP1, TAZ, and ß-catenin transcriptional activity is not only stimulated by the SS18-SSX fusion protein, but that they also mutually enhance each other's activation. These analyses showed the highest cooperative effect with overexpression of YAP1 in combination with ß-catenin. Coimmunoprecipitation experiments detected nuclear interactions between YAP1, ß-catenin, and the SS18-SSX fusion protein, the latter being an integral part of the BAF complex. Disruption of BAF complex assembly affected the coregulation of YAP1 and ß-catenin, indicating that this chromatin remodeling complex plays a crucial role for interdependent YAP1 and ß-catenin activation in synovial sarcoma cells. IMPLICATIONS: This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and ß-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

SS18-SSX drives CREB activation in synovial sarcoma.

PURPOSE: Synovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis. METHODS: Immunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo. RESULTS: We show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis in vitro and in vivo. CONCLUSION: In conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.

Fusion protein-driven IGF-IR/PI3K/AKT signals deregulate Hippo pathway promoting oncogenic cooperation of YAP1 and FUS-DDIT3 in myxoid liposarcoma.

Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.

Recurrent CTNNB1 mutations in craniofacial osteomas.

Osteoma is a benign bone forming tumor predominantly arising on the surface of craniofacial bones. While the vast majority of osteomas develops sporadically, a small subset of cases is associated with Gardner syndrome, a phenotypic variant of familial adenomatous polyposis caused by mutations in the APC gene resulting in aberrant activation of WNT/ß-catenin signaling. In a sequencing analysis on a cohort of sporadic, non-syndromal osteomas, we identified hotspot mutations in the CTNNB1 gene (encoding ß-catenin) in 22 of 36 cases (61.1%), harbouring allelic frequencies ranging from 0.04 to 0.53, with the known S45P variant representing the most frequent alteration. Based on NanoString multiplex expression profiling performed in a subset of cases, CTNNB1-mutated osteomas segregated in a defined "WNT-cluster", substantiating functionality of CTNNB1 mutations which are associated with ß-catenin stabilization. Our findings for the first time convincingly show that osteomas represent genetically-driven neoplasms and provide evidence that aberrant WNT/ß-catenin signaling plays a fundamental role in their pathogenesis, in line with the well-known function of WNT/ß-catenin in osteogenesis. Our study contributes to a better understanding of the molecular pathogenesis underlying osteoma development and establishes a helpful diagnostic molecular marker for morphologically challenging cases.

Prevalence of the Hippo Effectors YAP1/TAZ in Tumors of Soft Tissue and Bone.

Tumors of soft tissue and bone represent a heterogeneous group of neoplasias characterized by a wide variety of genetic aberrations. Albeit knowledge on tumorigenesis in mesenchymal tumors is continuously increasing, specific insights on altered signaling pathways as a basis for molecularly targeted therapeutic strategies are still sparse. The aim of this study was to determine the involvement of YAP1/TAZ-mediated signals in tumors of soft tissue and bone. Expression levels of YAP1 and TAZ were analyzed by immunohistochemistry in a large cohort of 486 tumor specimens, comprising angiosarcomas (AS), Ewing sarcomas, leiomyosarcomas, malignant peripheral nerve sheath tumors (MPNST), solitary fibrous tumors, synovial sarcomas (SySa), well-differentiated/dedifferentiated/pleomorphic and myxoid liposarcomas (MLS). Moderate to strong nuclear staining of YAP1 and TAZ was detected in 53% and 33%, respectively. YAP1 nuclear expression was most prevalent in MPNST, SySa and MLS, whereas nuclear TAZ was predominately detected in AS, MLS and MPNST. In a set of sarcoma cell lines, immunoblotting confirmed nuclear localization of YAP1 and TAZ, corresponding to their transcriptionally active pool. Suppression of YAP1/TAZ-TEAD mediated transcriptional activity significantly impaired sarcoma cell viability in vitro and in vivo. Our findings identify nuclear YAP1 and TAZ positivity as a common feature in subsets of sarcomas of soft tissue and bone and provide evidence of YAP1/TAZ-TEAD signaling as a specific liability to be considered as a new target for therapeutic intervention. Nuclear YAP1/TAZ expression may represent a biomarker suited to identify patients that could benefit from YAP1/TAZ-TEAD directed therapeutic approaches within future clinical trials.

Requirement for YAP1 signaling in myxoid liposarcoma.

Myxoid liposarcomas (MLS), malignant tumors of adipocyte origin, are driven by the FUS-DDIT3 fusion gene encoding an aberrant transcription factor. The mechanisms whereby FUS-DDIT3 mediates sarcomagenesis are incompletely understood, and strategies to selectively target MLS cells remain elusive. Here we show, using an unbiased functional genomic approach, that FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines are dependent on YAP1, a transcriptional co-activator and central effector of the Hippo pathway involved in tissue growth and tumorigenesis, and that increased YAP1 activity is a hallmark of human MLS Mechanistically, FUS-DDIT3 promotes YAP1 expression, nuclear localization, and transcriptional activity and physically associates with YAP1 in the nucleus of MLS cells. Pharmacologic inhibition of YAP1 activity impairs the growth of MLS cells in vitro and in vivo These findings identify overactive YAP1 signaling as unifying feature of MLS development that could represent a novel target for therapeutic intervention.