Abstract 3898: Loss of polycomb repressive complex 2 activates HOXB8 and remodels super enhancers in malignant peripheral nerve sheath tumors
Authors: Zhang Xiyuan , Lou Hannah E. , Lei Haiyan , Liu Zhihui , Yohe Marielle , Thiele Carol , Widemann Brigitte , Shern Jack F. 
Published: September 2020

Abstract
Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive and highly metastatic soft tissue sarcomas that lack effective treatment. Loss of the functional polycomb repressive complex 2 (PRC2) through genetic alterations in its core components EED and/or SUZ12 is prevalent in this devastating disease. Consequently, the product of PRC2, trimethylation of histone H3 lysine 27 (H3K27me3), a transcriptional repression marker, was found absent in MPNST samples. However, the epigenetic consequences of PRC2 loss remain unclear.
Methods: Using an inducible expression vector, we restored a functional PRC2 in three MPNST cell lines that are PRC2 deficient (PRC2-null). We then profiled the accompanied changes in histone modifications by chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) and transcriptional changes by RNA sequencing (RNAseq). To test the clinical relevance of our findings, we performed single-cell RNAseq (scRNAseq) on one PRC2-null patient MPNST sample. Finally, to target the epigenetic abnormity caused by PRC2 loss, we tested the efficacy of a histone deacetylase inhibitor (HDACi) in treating MPNST cells.
Results: We confirmed the tumor suppressor role of a functional PRC2 in MPNSTs by showing that PRC2 restoration caused growth inhibition in PRC2-null MPNST cells but not in those with a wildtype PRC2. Using ChIP-seq, we identified 6135 H3K27me3 peaks, which are associated with 2285 genes, that were commonly gained in all three PRC2-null MPNST cell lines with PRC2 restoration. Results of RNAseq show that more than 50% of these genes were kept silenced even without H3K27me3 and about 10% of the highly expressed genes were re-repressed by PRC2 (fold change &lt; 0.7). Interestingly, among the 145 genes that are transcriptionally regulated by polycomb, 91 were found to be co-occupied by H3K4me3 at their promoters. Since co-existence of H3K27me3 and H3K4me3 at the promoter define a bivalent gene, we further investigated the epigenetic changes associated with PRC2 restoration there. Analysis of ChIP-seq results revealed that, accompanied with gains of H3K27me3, there was loss of acetylated H3K27, a marker of super enhancer (SE), at the bivalent sites. Particularly, we identified a group of oncogenic transcription factors (TFs), including homeobox B8 (HOXB8), that were activated due to the loss of PRC2 and their activation was driven by SEs. Furthermore, the analysis of scRNAseq of our patient MPNST sample confirmed that these essential SE-driven TFs can be used to separate malignant cells from the tumor microenvironment. Finally, in vitro studies show that HDACi was highly effective in treating MPNST cells by de-activating these SE-driven TFs.
Conclusion: Herein, we report a novel mechanism underlying the polycomb-regulated transcriptional repression. In MPNSTs, the loss of PRC2 caused activation of a group of oncogenic transcription factors through remodeling the SE landscape, which resulted their vulnerabilities to epigenetic treatments by HDACi.
Citation Format: Xiyuan Zhang, Hannah E. Lou, Haiyan Lei, Zhihui Liu, Marielle Yohe, Carol Thiele, Brigitte Widemann, Jack F. Shern. Loss of polycomb repressive complex 2 activates HOXB8 and remodels super enhancers in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3898.