Abstract IA12: Epigenetic deregulation of the Hippo pathway mediates NF-κB driven sarcomagenesis
Authors: Eisinger T. S. Karin 
Published: August 2018

Abstract
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.