Purpose: Myxoid liposarcoma is an aggressive disease with particular propensity to develop hematogenic metastases. Over 90% of myxoid liposarcoma are characterized by a reciprocal t(12;16)(q13;p11) translocation. The resulting chimeric FUS–DDIT3 fusion protein plays a crucial role in myxoid liposarcoma pathogenesis; however, its specific impact on oncogenic signaling pathways remains to be substantiated. We here investigate the functional role of FUS–DDIT3 in IGF-IR/PI3K/Akt signaling driving myxoid liposarcoma pathogenesis. Experimental Design: Immunohistochemical evaluation of key effectors of the IGF-IR/PI3K/Akt signaling axis was performed in a comprehensive cohort of myxoid liposarcoma specimens. FUS–DDIT3 dependency and biological function of the IGF-IR/PI3K/Akt signaling cascade were analyzed using a HT1080 fibrosarcoma-based myxoid liposarcoma tumor model and multiple tumor–derived myxoid liposarcoma cell lines. An established myxoid liposarcoma avian chorioallantoic membrane model was used for in vivo confirmation of the preclinical in vitro results. Results: A comprehensive subset of myxoid liposarcoma specimens showed elevated expression and phosphorylation levels of various IGF-IR/PI3K/Akt signaling effectors. In HT1080 fibrosarcoma cells, overexpression of FUS-DDIT3 induced aberrant IGF-IR/PI3K/Akt pathway activity, which was dependent on transcriptional induction of the IGF2 gene. Conversely, RNAi-mediated FUS–DDIT3 knockdown in myxoid liposarcoma cells led to an inactivation of IGF-IR/PI3K/Akt signaling associated with diminished IGF2 mRNA expression. Treatment of myxoid liposarcoma cell lines with several IGF-IR inhibitors resulted in significant growth inhibition in vitro and in vivo. Conclusions: Our preclinical study substantiates the fundamental role of the IGF-IR/PI3K/Akt signaling pathway in myxoid liposarcoma pathogenesis and provides a mechanism-based rationale for molecular- targeted approaches in myxoid liposarcoma cancer therapy. Clin Cancer Res; 23(20); 6227–38. ©2017 AACR.

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 vivoin 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 targetsFOXM1 and PLK1. In synovial sarcoma cells, RNAi‑mediated knockdown of SS18-SSXled 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 vitroand 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 sarcomapathogenesis and provides evidence for innovative therapeutic approaches.

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.

Myxoid liposarcoma (MLS) is an aggressive soft-tissue tumor characterized by a specific reciprocal t(12;16) translocation resulting in expression of the chimeric FUS–DDIT3 fusion protein, an oncogenic transcription factor. Similar to other translocation-associated sarcomas, MLS is characterized by a low frequency of somatic mutations, albeit a subset of MLS has previously been shown to be associated with activating PIK3CA mutations. This study was performed to assess the prevalence of PI3K/Akt signaling alterations in MLS and the potential of PI3K-directed therapeutic concepts. In a large cohort of MLS, key components of the PI3K/Akt signaling cascade were evaluated by next generation seqeuncing (NGS), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). In three MLS cell lines, PI3K activity was inhibited by RNAi and the small-molecule PI3K inhibitor BKM120 (buparlisib) in vitro. An MLS cell line–based avian chorioallantoic membrane model was applied for in vivo confirmation. In total, 26.8% of MLS cases displayed activating alterations in PI3K/Akt signaling components, with PIK3CA gain-of-function mutations representing the most prevalent finding (14.2%). IHC suggested PI3K/Akt activation in a far larger subgroup of MLS, implying alternative mechanisms of pathway activation. PI3K-directed therapeutic interference showed that MLS cell proliferation and viability significantly depended on PI3K-mediated signals in vitro and in vivo. Our preclinical study underlines the elementary role of PI3K/Akt signals in MLS tumorigenesis and provides a molecularly based rationale for a PI3K-targeted therapeutic approach which may be particularly effective in the subgroup of tumors carrying activating genetic alterations in PI3K/Akt signaling components.

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. Synopsis The transcriptional co‐activator YAP1 is essential in myxoid liposarcoma (MLS), an aggressive soft‐tissue tumor. This study reveals a link between aberrant YAP1 signaling and the FUS DDIT3 fusion oncoprotein that drives MLS. Pharmacologic YAP1 inhibition impairs MLS growth in vitro and in vivo. YAP1, encoding a transcriptional co activator that is inhibited by the Hippo pathway, was identified by RNA interference screening as an essential gene in mesenchymal stem cells expressing FUS‐DDIT3. FUS‐DDIT3‐expressing MLS cell lines and MLS patient specimens exhibited increased YAP1 activity, and YAP1 suppression in MLS cells caused proliferation arrest, senescence, and apoptosis. FUS DDIT3 induced the expression and nuclear localization of YAP1 and its downstream effectors. FUS‐DDIT3 and YAP1 physically associated in the nucleus of MLS cells. MLS cells were sensitive to pharmacologic blockade of YAP1 activity.

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.