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S1. MAPK dependence in UPS and HT-1080 cells

S7. Modeling UPS extravasation with zebrafish xenografts

S6. The role of RHAMM in UPS migration

S5. The role of RHAMM in UPS in proliferation and TGFB signaling

Supplementary Figure Legends 1-7

S3. YAP1 target expression in human and mouse UPS

S2. Colon carcinomas are insensitive to TGFB inhibition relative to sarcomas

Supplemental patient information from tissue microarray and additional quantitations.

Supplemental in vitro rescue assays and qRT-PCR of SAHA/JQ1 treated cells

Supplemental Ingenuity Pathway Analysis and cleaved caspase 3 findings

Supplemental ChIP-Seq and RNA-seq information

The role of AMOT in sarcomagenesis

The effects of SAHA/JQ1 on phospho-YAP1

The role of myc in UPS and the effects of SAHA/JQ1 on YAP1 levels in sarcoma cell lines

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705–20. ©2018 AACR.

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFβ signaling. Therefore, we investigated crosstalk between YAP1 and TGFβ resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFβ and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFβ–YAP1–RHAMM axis prevents vascular migration of tumor cells to distant sites. Implications: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFβ cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment. Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705–20. ©2018 AACR.

Nearly 11,000 individuals in the United States and 200,000 individuals worldwide are diagnosed with a form of soft-tissue sarcoma (STS) every year. STS is a rare heterogeneous and complex family of disease associated with no uniform underlying genetic abnormality. However, sarcoma treatment has not changed significantly in 25 years. Patients are limited to surgery, radiotherapy, and chemotherapy. Therefore, the discovery of novel targets and mechanisms is critical. The Hippo pathway functions as a signaling hub facilitating cellular responses to multiple stimuli during tumorigenesis. The most important downstream effector of the Hippo pathway is Yes-activated protein (YAP), a transcriptional coactivator that promotes pro-proliferation and suppresses apoptosis. Inactivation of the Hippo pathway promotes nuclear localization of YAP. Whereas many studies have defined YAP1's transcriptional targets in epithelial tumors and normal tissues, its role in mesenchymal tumors is unclear. Here, we confirm that increased YAP1 mRNA expression correlates with worse overall survival in undifferentiated pleomorphic sarcoma (UPS) patients and that YAP1 protein levels are frequently elevated in human sarcomas. Furthermore, immunohistochemical (IHC) analysis of human sarcoma tissue microarray (TMA) showed that YAP levels are dramatically increased in the nuclei of high-grade UPS tumor cells, compared with normal skeletal, adipose, and arterial tissue. UPS is one of the more commonly diagnosed and aggressive subtypes of sarcomas found in adults. To define mechanisms of YAP1-mediated sarcomagenesis, we developed a novel genetic mouse model in which YAP1 is conditionally deleted from KrasG12D+; Trp53fl/fl (KP) UPS tumors (KPY). Microarray analysis of both tumors revealed that YAP1 loss inhibits expression of NF-κB signaling components and transcriptional targets. Consistent with these findings, ChIP-seq and super-enhancer analysis of human UPS tumors (n=3) revealed that many of the 900 identified UPS super enhancers regulate expression of NF-κB targets dependent on YAP1 in our system. This finding is particularly relevant as NF-κB is a key regulator of muscle cell proliferation and suppresses differentiation by inhibiting expression of the myogenic transcription factor, MYOD1. Together, these data suggest that upregulation of YAP1 activity promotes NF-κB-mediated proliferation and inhibits differentiation. Importantly, we have discovered that a combination of JQ1, a BET family inhibitor, and vorinostat (SAHA), a histone deacetylase (HDAC) inhibitor, decreases YAP1 expression, YAP1 protein stability, and sarcoma cell proliferation. Mechanistically, SAHA/JQ1 treatment significantly increases expression of the tumor suppressor angiomotin (AMOT), which binds YAP1, sequesters it in the cytoplasm, and facilitates its degradation. We have also found that AMOT expression is lost in sarcomas compared with normal muscle tissue, likely due to epigenetic suppression. SAHA/JQ1 treatment also decreased gene expression of YAP1 targets and caused reexpression of the muscle differentiation markers p57, MEF2C, and MYOD1. Based on these data we will investigate the efficacy of SAHA/JQ1 against UPS and other sarcomas in vivo using subcutaneous and autochthonous mouse models. We will probe the ability of SAHA/JQ1 treatment to inhibit YAP1 in vivo, to suppress NF-κB dependent mechanisms of cell proliferation and tumorigenesis, and to promote terminal differentiation of sarcoma cells. Ultimately, we will determine whether this approach represents a novel course of targeted treatment for sarcoma patients. Citation Format: Shuai Ye, Koreana Pak, Jennifer Shah, Susan Chor, Shaun Egolf, Gloria Marino, T.S. Karin Eisinger-Mathason. Regaining epigenetic control of the Hippo pathway to inhibit sarcomagenesis [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B07.

Soft-tissue sarcomas present a unique challenge to researchers due to their heterogeneity. However, one common mechanism associated with more than 25% of diagnosed sarcomas is deregulation of the Hippo pathway, a critical signaling cascade that responds to cell contact inhibition, among other factors, and negatively regulates cell proliferation. In quiescent cells the pathway remains “on,” which maintains activation of the kinase cascade that phosphorylates and degrades YAP1. However, inactivation of the pathway stabilizes YAP1, allowing it to translocate to the nucleus and activate transcription of pro-proliferative gene targets. Microarray analysis of tumor tissue from the LSLKrasG12D/+;Trp53fl/fl (KP) mouse model of undifferentiated pleomorphic sarcoma (UPS) revealed that YAP1 deletion (KPY) reduces sarcoma cell proliferation and increases expression of circadian rhythm genes. Notably, deletion of YAP1 in KP tumors resulted in a 2.5-fold increase in expression of PER1. Although PER1 has primarily been characterized as a negative regulator of the circadian clock, upregulation of PER1 is known to modulate the G2/M cell cycle checkpoint at both the protein and mRNA levels. Our findings represent a novel link between the circadian clock and Hippo pathway. Our studies also indicate that Hippo signaling is epigenetically regulated in sarcomas. Thus we employed the epigenetic modulating drugs JQ1, a BET inhibitor, and SAHA, an HDAC inhibitor, to regain control of this pathway. Treatment with these inhibitors significantly reduced YAP1 expression, decreased sarcoma growth in vivo and in vitro, and surprisingly increased muscle cell differentiation markers, including the master muscle regulator MyoD. Under JQ1 and SAHA drug treatment, as well as YAP1 shRNA knockdown conditions, we demonstrated that PER1 is significantly increased at both the protein and transcriptional levels in KP tumor derived cells. Together, these results suggest that YAP1 represses PER1 expression in sarcoma, and that epigenetic therapies can cause reexpression of PER1. Another key cellular response to SAHA/JQ1 exposure in sarcoma cells is alteration in NF-κB signaling. NF-κB, a critical signaling cascade for muscle cell progenitor proliferation, oscillates temporally in sarcoma cells treated with SAHA and JQ1. We hypothesize that this oscillation may be disrupted when PER1 is suppressed due to high YAP1 levels in sarcoma leading to uncontrolled proliferation. NF-κB activation enhances proliferation and suppresses differentiation in normal muscle progenitor cells and myoblasts by preventing MyoD upregulation. Importantly, it has been reported that MyoD is a clock-controlled gene. Given this relationship between MyoD and PER1, it is possible that PER1 functions to regulate MyoD through NF-κB in sarcoma and thus alters the balance between cell proliferation and differentiation. Citation Format: Gloria Marino, Shaun Egolf, Shuai Ye, Koreana Pak, Jenn Shah, Adrian Rivera-Reyes, Susan Chor, T. S. Karin Eisinger-Mathason. YAP1-mediated circadian oscillation in sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B21.

Nearly 200,000 individuals worldwide are diagnosed with a form of soft-tissue sarcoma (STS) every year. Due to a lack of novel targeted therapeutics sarcoma for these malignancies treatment has not changed significantly in 25 years. Therefore, the discovery of novel targets and mechanisms is critical. One promising avenue the Hippo pathway, which we have identified as a key regulator of sarcomagenesis in undifferentiated pleomorphic sarcoma (UPS), a commonly diagnosed and aggressive subtype of sarcomas. Inactivation of the pathway, promotes nuclear localization of YAP1, a transcriptional co-activator that promotes proliferation. Whereas, many studies have defined YAP1’s transcriptional targets in epithelial tumors and normal tissues, its role in mesenchymal tumors is unclear. Here, we confirm that increased YAP1 mRNA expression correlates with worse overall survival in UPS patients. To define mechanisms of YAP1-mediated sarcomagenesis, we developed a novel genetic mouse model in which YAP1 is conditionally deleted in UPS tumors. Microarray analysis of these tumors revealed that YAP1 loss inhibits expression of NF-κB targets. We have performed ChIP-seq and super-enhancer analysis of human UPS tumors and found that many of the 900 identified super enhancers regulate expression of NF-κB targets that are dependent on YAP1 in our system. This finding is particularly relevant as NF-κB is a key regulator of muscle cell proliferation and suppresses myoblast differentiation. Together, these data suggest that upregulation of YAP1 activity promotes NF-κB-mediated proliferation and inhibits differentiation, resulting in sarcomagenesis. Importantly, we have discovered that a combination of JQ1, a BET family inhibitor, and SAHA, a histone deacetylase inhibitor, decreases YAP1 expression, YAP1 protein stability, and sarcoma cell proliferation. SAHA/JQ1 treatment significantly increased expression of Angiomotin (AMOT), which binds YAP1, sequesters it in the cytoplasm, and facilitates it degradation. We have also found that AMOT expression is lost in sarcomas, likely due to epigenetic suppression. Consistent with these observations, SAHA/JQ1 treatment also decreased gene expression of YAP1 targets and caused re-expression of the muscle differentiation markers p57, MEF2C, and MYOD1. We have investigated the effect of of SAHA/JQ1 in vivo and found dramatic inhibition of tumor growth, as well as reduced YAP1 expression, and increased AMOT expression. Importantly SAHA/JQ1 treatment also inhibited NF-κB activity. Our studies have revealed for the first time that YAP1 expression is epigenetically modulated through AMOT de-regulation in sarcomas, resulting in elevated NF-κB activity and sarcomagenesis. SAHA/JQ1 treatment re-establishes epigenetic control of the Hippo pathway reducing proliferation and enhancing differentiation. Ultimately, we will determine whether this approach represents a novel course of targeted treatment for sarcoma patients. Citation Format: Shuai Ye, Koreana Pak, Jennifer Shah, Susan Chor, Shaun Egolf, Gloria Marino, T.S. Karin Eisinger-Mathason. Hippo signaling promotes sarcomagenesis through the NF-kB pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2017-3342

Soft tissue sarcomas have presented a unique challenge to researchers due to their heterogeneity. One common mechanism underlying more than 25% of diagnosed sarcomas is the deregulation of the Hippo pathway, a critical signaling cascade that responds to cell contact inhibition, among other factors, and negatively regulates cell proliferation. In quiescent cells the pathway remains “on,” which maintains activation of a kinase cascade that phosphorylates and degrades YAP1. However, inactivation of the pathway stabilizes YAP1 allowing it to translocate to the nucleus and activate transcription of pro-proliferative gene targets. Microarray analysis of muscle tissue from the LSL-KrasG12D/+;Trp53fl/fl (KP) mouse model of undifferentiated pleomorphic sarcoma (UPS) revealed that YAP1 deletion (KPY) reduces cell proliferation and increases expression of circadian rhythm genes including PER1. UPS is a commonly diagnosed and aggressive type of muscle-derived sarcoma. The KP model recapitulates human UPS morphologically and histologically, as well as by gene expression profiling. Although PER1 has primarily been characterized as a negative regulator of the circadian clock, upregulation of PER1 is known to modulate the G2/M cell cycle checkpoint at both the protein and mRNA level independent of p53. However, our findings represent a novel link between the circadian clock and the hippo pathway. Notably, deletion of YAP1 in KP tumors leads to a statistically significant 2.5 fold increase in expression of PER1. The study has two specific aims: 1) to identify the YAP1-dependent function of PER1 in sarcoma and 2) determine whether YAP1 directly or indirectly regulates PER1. We have validated the microarray results in KP tumor derived cell lines as well as in KP and KPY tumor tissue. I have also confirmed PER1 suppression in KP cells under YAP1 short hairpin RNA conditions. Our lab has previously observed that treatment with epigenetic modulating drugs JQ1, a BET inhibitor, and SAHA, an HDAC inhibitor, significantly decreased sarcoma growth in vivo and in vitro when administered alone and had an additive effect when combined. These effects can be explained in part by the finding that treatment with these inhibitors significantly reduces YAP1 expression. Under these conditions, as well as YAP1 knockdown conditions, I demonstrated via qRT-PCR and western blot that PER1 is significantly increased at both the protein and transcriptional levels in KP cells. Additionally, preliminary evidence from an MTT proliferation assay showed loss of PER1 increased sarcoma cell proliferation. Further supporting the hypothesis that PER1 modulation impacts sarcoma proliferation, it has been reported that MyoD, the master regulator of muscle cell differentiation, is itself a clock-controlled gene. Together, these results suggest that YAP1 represses PER1 expression in sarcoma, and that epigenetic treatments can cause re-expression of PER1 which functions to inhibit cell proliferation and may promote differentiation. Citation Format: Gloria Marino, Shuai Ye, Koreana Pak, Jennifer Shah, Jason Godfrey, Susan Chor, Shaun Egolf, T.S. Karin Eisinger-Mathason. YAP1-mediated circadian oscillation in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2017-3531