Search

Loss-of-function mutations in the polycomb repressive complex 2 (PRC2) occur frequently in malignant peripheral nerve sheath tumor, an aggressive sarcoma that arises from NF1-deficient Schwann cells. To define the oncogenic mechanisms underlying PRC2 loss, we use engineered cells that dynamically reassemble a competent PRC2 coupled with single-cell sequencing from clinical samples. We discover a two-pronged oncogenic process: first, PRC2 loss leads to remodeling of the bivalent chromatin and enhancer landscape, causing the upregulation of developmentally regulated transcription factors that enforce a transcriptional circuit serving as the cell's core vulnerability. Second, PRC2 loss reduces type I interferon signaling and antigen presentation as downstream consequences of hyperactivated Ras and its cross talk with STAT/IRF transcription factors. Mapping of the transcriptional program of these PRC2-deficient tumor cells onto a constructed developmental trajectory of normal Schwann cells reveals that changes induced by PRC2 loss enforce a cellular profile characteristic of a primitive mesenchymal neural crest stem cell.

Genetic alterations that result in the dysregulation of polycomb repressive complex 2 (PRC2) are common features of multiple cancer types. Loss-of-function mutations in the PRC2 genes, SUZ12 or EED, occur at high frequency in malignant peripheral nerve sheath tumors (MPNST), a clinically aggressive sarcoma that arises from NF1-deficient Schwann cells. To define the oncogenic mechanisms mediated by PRC2 loss, we used engineered MPNST model systems that dynamically reassemble a competent PRC2 coupled with single cell sequencing from clinical samples to evaluate the transcriptomic and epigenetic consequences of PRC2 loss. We discovered a two-pronged oncogenic process; first, PRC2 loss leads to remodeling of bivalent chromatin and the enhancer landscape of the tumor cell. These epigenetic changes result in the upregulation of developmentally regulated transcription factors, including FOXC1, which enforce a transcriptional circuit that is a core vulnerability of the cell. Second, PRC2 loss reduces type I interferon signaling and antigen presentation as a downstream consequence of hyperactivated Ras signaling and its crosstalk with STAT and IRF transcription factors. Mapping of the transcriptional program of these PRC2-deficient tumor cells onto a constructed developmental trajectory of the developing neural crest stem cell revealed that changes induced by PRC2 loss enforced a cellular profile characteristic of a primitive mesenchymal neural crest stem cell.

Objective: Recurrent mutations in the polycomb repressive complex 2 (PRC2) occur in ~80% of malignant peripheral nerve sheath tumors (MPNST), which originate from NF1-deficient Schwann cells. In MPNST, PRC2 loss results in a diverse set of transcriptomic and phenotypic consequences, including gain of acetylated H3K27 (H3K27ac) accompanying the loss of trimethylated H3K27 (H3K27me3), hyperactivated Ras signaling, and tumor escape from immune surveillance. Mechanistic understanding of how PRC2 loss results in these diverse consequences remains unresolved. Methods: To delineate the oncogenic mechanisms mediated by PRC2 loss, we engineered MPNST cell lines to dynamically reassemble a functional PRC2 and evaluated the transcriptomic and epigenetic consequences of PRC2 restoration. We further extended these finding using single cell RNA sequencing (scRNAseq) from human MPNST. Results: Through integrative analysis of RNAseq and H3K27me3 ChIP-seq in PRC2-deficient MPNST cells, we identified 6134 H3K27me3 peaks gained when a functional PRC2 was restored. Of the 876 significantly altered genes, 699 genes were downregulated, and 177 genes were upregulated when a restored PRC2 caused genome-wide redistribution of H3K27me3 peaks. Detailed mechanistic dissection of these PRC2-regulated genes revealed a two-pronged oncogenic process mediated by PRC2 loss and they cooperatively contribute to the tumorigenesis of MPNST. First, PRC2 loss leads to the upregulation of a transcriptional circuit that remodels the enhancer landscape of MPNST cells and establishes an enhancer-driven transcription factor, FOXC1, as a master regulator and thus a core vulnerability of the cell. Second, PRC2 loss reduces type I interferon (IFN) signaling and antigen presentation as a downstream consequence of the upregulated Ras signaling. These data were further integrated with scRNAseq of human MPNST. Importantly, we discovered that the PRC2-deficient tumor cells have a corrupted transcriptional program characteristic of a mesenchymal precursor cell found during the trajectory of normal Schwann cell development. Interestingly, malignant cells expressing high levels of IFN and antigen presentation genes were missing in the metastatic MPNST, potentially allowing the tumor cell to escape the immune surveillance. The transcriptional circuit established by PRC2-regulated FOXC1 and its downstream targets sustains the malignant program in both primary and metastatic MPNST. Conclusion: In summary, we discovered the activation of a lineage specific oncogenic transcription program in PRC2-deficient MPNST, which is characteristic of a neural crest-derived mesenchymal stem cell. Our findings provide mechanistic understanding of the inherent metastatic potential, chemotherapy and radiotherapy resistance, and immune escape that are clinically characteristic of these tumors. Citation Format: Xiyuan Zhang, Hannah E. Lou, Vishaka Gopalan, Zhihui Liu, Hilda Jafarah, Haiyan Lei, Paige Jones, Carly M. Sayers, Marielle E. Yohe, Prashant Chittiboina, Brigitte C. Widemann, Carol J. Thiele, Michael C. Kelly, Sridhar Hannenhalli, Jack F. Shern. Loss of PRC2 enforces a mesenchymal neural crest stem cell phenotype in NF1-deficient cancer through activation of core transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 701.

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 < 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.

Summary Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs). Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue., Highlights • Neonatal tissue is a viable alternative to fetal for mouse humanization • Over 1,000 NeoThy mice can be made from one neonatal thymus donor • The NeoThy enables robust pre-clinical immunogenicity studies of iPSC therapies, Corresponding author William Burlingham and colleagues created a humanized mouse model called the NeoThy. The NeoThy uses human neonatal, rather than fetal, tissue sources for generating a human immune system within immunocompromised mouse hosts. NeoThy mice are an attractive alternative to conventional humanized mouse models, as they enable robust and reproducible iPSC immunogenicity experiments in vivo.