Soft tissue sarcomas are an aggressive group of roughly 65 mesenchymal malignancies diagnosed in 200,000 people worldwide annually. The treatment approach for sarcomas has not changed significantly in 25 years. Furthermore, recent sequencing efforts have revealed that consistent oncogenic mutations are rare in muscle cancers. As a result, these tumors are not sensitive to most available targeted therapeutics, which specifically interfere with the functions of mutant oncogenic pathways. We are investigating the possibility that together with p53 or RB1 mutation/loss, alterations in chromatin state and epigenetic regulation rather than oncogenic mutation, are required for sarcoma transformation. Based on our current findings we are testing the hypothesis that epigenetic deregulation of Angiomotin (AMOT), a novel tumor suppressor we have identified, is critical for sarcomagenesis. AMOT is highly expressed in differentiated human muscle tissue but is silenced in muscle-derived sarcomas including undifferentiated pleomorphic sarcoma (UPS), a commonly diagnosed and aggressive subtype. Furthermore, ectopic expression of the p130 isoform of AMOT significantly inhibits UPS proliferation. This finding is consistent with the only known function of AMOT in cancer cells, which is to sequester the Hippo pathway effector YAP1 and facilitate its degradation. YAP1 is a pro-proliferation transcriptional activator and deletion of Yap1 in the KrasG12D/+; Trp53fl/fl autochthonous mouse model of UPS. Importantly, treatment of sarcoma cells and tumors with the epigenetic inhibitors SAHA and JQ1 restores p130 AMOT expression, inhibits Yap1, and dramatically reduces sarcomagenesis in vivo. Interestingly, SAHA/JQ1 treatment also induces expression of muscle markers including MYOD and p57 suggesting that epigenetic modulation promotes differentiation as well as inhibiting proliferation. Gene expression studies of control and Yap1-deficient tumors revealed that Yap1 controls NF-κB signaling. We have performed H3K27Ac ChIP-seq and super-enhancer analysis of human UPS tumors to determine whether NF-κB targets are important in human sarcomas. These analyses revealed that NF-κB is the most transcriptionally active pathway in UPS. Together these data suggest that persistent YAP1-mediated NF-κB signaling promotes sarcomagenesis. In normal undifferentiated muscle cells NF-κB signaling is essential for proliferation and inhibition of differentiation. We have now shown that SAHA/JQ1-mediated YAP1 inhibition subsequently decreases NF-κB activity, underscoring the ability of epigenetic therapies to decrease tumorigenesis and enhance differentiation. Our findings suggest that pharmacological modulation of chromatin modifiers will allow us to regain epigenetic control over the Hippo pathway and its downstream targets. Thus we have an opportunity to finally change the treatment paradigm for these patients with novel targeted therapeutics. Note: This abstract was not presented at the meeting. Citation Format: T. S. Karin Eisinger, Shuai Ye. Epigenetic deregulation of the Hippo pathway in muscle-derived sarcomas [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 3341. doi:10.1158/1538-7445.AM2017-3341

Soft tissue sarcomas are an aggressive group of roughly 65 mesenchymal malignancies diagnosed in 200,000 people worldwide annually. Unlike in epithelial cancers, where novel targeted therapies have had a dramatic effect on patient survival, the treatment approach for sarcomas has not changed significantly in 25 years. Furthermore, recent sequencing efforts have revealed that consistent oncogenic mutations are rare in muscle cancers. Though an individual sarcoma tumor may express mutant CDK4 or PTEN, these mutations are not widely or uniformly found in sarcomas and are not unifying molecular factors associated with a given subtype. As a result, these tumors are not sensitive to most available targeted therapeutics, which specifically interfere with the functions of mutant oncogenic pathways. Therefore, we investigated alternate genetic/epigenetic mechanisms underlying sarcomagenesis to facilitate development of novel therapeutics. Our previous work showed that copy number loss of upstream regulators deactivates the Hippo pathway leading to stabilization of the Hippo effector, Yes-Associated Protein 1 (YAP1). YAP1 stabilization promotes tumorigenesis in multiple sarcoma subtypes including muscle-derived tumors like human undifferentiated pleomorphic sarcoma (UPS), which are the focus of our studies. Deletion of Yap1 in the LSL-KrasG12D/+; Trp53fl/fl (KP) autochthonous mouse model of UPS resulted in significantly decreased tumorigenesis. Though much work has been done to establish YAP1 as a major regulator of tumorigenesis, most of the related mechanistic studies have been done in epithelial cancer models. Thus, the functional role of the Hippo pathway in mesenchymal tumors is still being debated. We have now identified a critical mechanism of YAP1-mediated sarcomagenesis in UPS using ChIP-seq (H3K27Ac) and super-enhancer (SE) analysis. H3K27Ac ChIP-seq and super-enhancer analysis of human UPS tumors revealed that NF-κB, a major regulator of proliferation and differentiation in muscle progenitor myoblasts, is the most transcriptionally active pathway in UPS. Using gene set enrichment analysis (GSEA) of microarray gene expression studies comparing control (KP) and Yap1-deficient (KPY) murine UPS tumors, we determined that Yap1 modulates expression of many NF-κB pathway components. Additionally, we found that YAP1 is constitutively active in these tumors due to epigenetic silencing of its inhibitor Angiomotin (AMOT). Together, these data suggest that persistent YAP1-mediated NF-κB signaling promotes sarcomagenesis. In addition to Hippo pathway deregulation, several studies have recently shown that alterations in epigenetic status can promote sarcomagenesis. Certain pediatric sarcomas are linked to chromosomal translocations of transcription factor loci encoding chromatin remodeling factors. Copy number loss and deletion of chromatin modulators have also been found in some subtypes. These proteins influence chromatin structure and coordinate regulation of normal developmental transcriptional pathways, indicating that disruption of chromatin architecture may be a common event in sarcomagenesis. Our recent work showed that treatment with the epigenetic modulating drug Vorinostat (SAHA), a pan-HDAC inhibitor, leads to reexpression of HIF2α and a 50% reduction in UPS sarcomagenesis in vivo. These studies support the hypothesis that epigenetic modulation can reexert control over malfunctioning pathways and return key transcription factors like YAP1 and HIF2α to the expression and activity levels found in quiescent cells. Treatment with the epigenetic modulators (SAHA and JQ1) inhibited YAP1 transcription and restored expression of AMOT, resulting in near ablation of YAP1 expression. Interestingly, SAHA/JQ1 treatment initiated a muscle differentiation program and reduced tumorigenesis in vivo. Unexpectedly, we also found that YAP1 promotes NF-κB activity by suppressing transcriptional oscillation of USP31, a newly identified upstream regulator of NF-κB. Both YAP1 shRNA and SAHA/JQ1 treatment induce USP31 mRNA oscillation over multiple days of treatment. Virtually nothing is known about USP31 beyond its structure and ability to inhibit NF-κB signaling. These findings suggest that Usp31, as well as other NF-κB components, might oscillate in quiescent or differentiating cells. Furthermore, we hypothesize that this oscillation plays a role in normal myoblast differentiation and is deregulated in YAP1-driven muscle tumors. Such oscillations are generally linked with the peripheral circadian clock, and disruptions in the clock are known to promote tumorigenesis. Furthermore, the link between an oscillating transcriptional program, circadian clock function, and normal muscle development has been established. However, our work is the first to suggest that perturbations in oscillating NF-κB targets and regulators are linked to sarcomagenesis. These data indicate that inappropriate expression of YAP1 may interfere with a temporally constricted NF-κB transcriptional program. This work establishes NF-κB, a key regulator of normal muscle development, as a pathway that becomes misregulated during sarcomagenesis through the aberrant activity of YAP1. Collectively, our data implicate multiple targets of YAP1 that could serve as useful biomarkers in UPS and provide the mechanistic rationale for epigenetic therapy for the treatment of this disease. Citation Format: T. S. Karin Eisinger. Epigenetic deregulation of the Hippo pathway mediates NF-κB driven 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 IA12.

Only a small percentage of disseminating tumor cells are capable of forming lethal metastatic foci. Though advances in sequencing and genomics have dramatically enhanced our understanding of primary tumor biology and novel target identification, these techniques alone have not proven sufficient to identify the factors that permit metastasis of this small cellular fraction. For example, we observed that in some tumors the major molecular predictor of metastatic potential is HIF1α protein stabilization in response to low intratumoral O2 tension (hypoxia). These observations clearly show that in-depth understanding of environmental signals and subsequent cellular responses is necessary to fully characterize metastatic potential. Consistent with our earlier observations, we discovered that the collagen-modifying enzyme PLOD2, a direct transcriptional target of HIF1α, dramatically enhances both early (cell migration/invasion) and late (extravasation/lung colonization) steps of the metastatic cascade. We previously reported the role of PLOD2 in modulating the primary tumor microenvironment to facilitate cell migration and intravasation. However, the mechanisms by which PLOD2 and tumor associated collagen impact later metastatic stages (i.e., endothelial adherence, extravasation) are unknown- in part because these processes are particularly difficult to simulate in vitro and to visualize in vivo. Therefore, we have developed new models and tools including a zebrafish embryo xenograft system to image migrating and extravasating tumor cells in vivo. This approach allows us to investigate the late metastatic cascade and define the microenvironmental cues that promote tumor cell dissemination. We also observed that PLOD2-modified collagen is secreted into the extracellular milieu during dissemination and weakens endothelial barrier function. We are investigating the role of tumor collagen and PLOD2 in endothelial adherence and extravasation for the purpose of therapeutically targeting the molecular underpinnings of metastases. Our work focuses on soft tissue sarcomas. However, recent studies by other groups have linked PLOD2 and modified collagen to carcinoma metastasis as well, suggesting a broader applicability for our work. Ultimately, we believe these studies will transform our ability to treat and even prevent metastasis, a unique possibility in sarcoma patients due to the relatively long interval between primary tumor diagnosis and metastatic outgrowth in some patients (5-10 years). The tools we have developed and the mechanisms we are pursuing will open new avenues of research that were once inaccessible and lead to novel therapeutic opportunities for the treatment of metastatic disease in multiple cancer contexts. Citation Format: T.S. Karin Eisinger, Ying Liu, Ileana Murazzi. Identification of a “hypoxic secretome” and its role in metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA024.

Undifferentiated pleomorphic sarcoma (UPS), an aggressive subtype of soft-tissue sarcoma (STS), is exceedingly rare in humans and lacks effective, well-tolerated therapies. In contrast, STS are relatively common in canine companion animals; thus, incorporation of veterinary patients into studies of UPS offers an exciting opportunity to develop novel therapeutic strategies for this rare human disease. Genome-wide studies have demonstrated that UPS is characterized by aberrant patterns of DNA methylation. However, the mechanisms and impact of this epigenetic modification on UPS biology and clinical behavior are poorly understood. Leveraging cell lines and tissue specimens derived from human and canine patients, we discovered that the DNA methyltransferase DNMT3B is overexpressed in UPS relative to normal mesenchymal tissues and associated with a poor prognosis. Consistent with these findings, genetic DNMT3B depletion strongly inhibited UPS cell proliferation and tumor progression. However, existing hypomethylating agents, including the clinically approved drug 5-aza-2'-deoxycytidine and the DNMT3B-inhibiting tool compound nanaomycin A, were ineffective in UPS due to cellular uptake and toxicity issues. Thus, further development of DNMT3B-targeting strategies for these patients is critical.