Abstract A10: Integrated proteome and epigenetic analysis reveals crosstalk between chromatin and cancer pathways in malignant peripheral nerve sheath tumors
Authors: Wojcik John B. , Sidoli Simone , Cooper Kumarasen , Garcia Benjamin A. 
Published: August 2018

Malignant peripheral nerve sheath tumors (MPNST) are rare, aggressive sarcomas. Recent work has identified loss-of-function mutations in PRC2 components in a subset of these tumors. PRC2 mutation leads to a loss of trimethylation at lysine 27 of histone H3 (H3K27me3), and is linked to transcriptional upregulation of the Ras pathway in cell culture models of MPNST. We hypothesized that PRC2 loss would result in global alterations in histone post-translational modifications (PTMs), and such changes would be associated with altered protein expression in oncogenic pathways. We thus implemented protein and histone extraction methods directly on formalin-fixed, paraffin-embedded human tumor samples and performed parallel histone PTM and proteome analysis. Data were merged using our in-house developed chromatin and proteomics bioinformatics tools.

Methods: Human tumors were obtained from pathology archives. Total protein was isolated from 3-mm tissue cores, following deparaffinization, antigen retrieval, lysis, and homogenization. Histone bands were excised following SDS-PAGE of total lysate. For cell lines, total protein was obtained following lysis in urea and histones were obtained via nuclear isolation and acid precipitation. Cell proteome and histone PTMs were analyzed using nano-liquid chromatography and tandem mass spectrometry. The proteome was characterized using MaxQuant. Histone PTMs were identified and quantified using lab-developed software. Statistical analysis was performed using Student's t-test, Gene Ontology enrichment was assessed using hypergeometric distribution annotations, and GSEA analysis was used to identify signatures of pathway activation.

Results: PRC2 loss was associated with decreased H3K27me3 and increased H3K27 acetylation (p=0.02 and p=0.01). This was accompanied by increased hyperacetylation of H4, a marker of open chromatin. We also observed an increase in overall H3K36me2 (p=0.0001). By quantifying combinatorial PTMs, we observed that tandem H3K27me2K36me2 and H3K27me3K36me2 marks were markedly decreased in MPNST with PRC2 loss (p=0.04 and p=0.004). These alterations result in chromatin in which the active H3K36me2 mark is not flanked by repressive PTMs.

At the level of the proteome, PRC2 loss was associated with increased expression of chromatin remodelers, particularly members of the SWI/SNF complex. We confirmed increased Ras pathway expression and noted increases in other tumor pathways, and decreases in proteins associated with nerve sheath differentiation and immune surveillance.

To assess whether PRC2 function was directly linked to the proteome changes seen in the human tumors, we restored PRC2 function in PRC2-mutant MPNST cell lines and measured the proteome response. PRC2 reconstitution downregulated pathways that were upregulated with PRC2 loss in human tumors and restored expression of markers of nerve sheath differentiation and immune surveillance. The same effects were observed in PRC2 mutant cell lines upon shRNA silencing of NSD2, the methyltransferase responsible for H3K36me2, suggesting that, for a subset of genes, H3K27me3 and H3K36me2 function in tandem to control expression.

These results link PRC2 loss to increased active chromatin signatures that promote pathways associated with aggressive tumor behavior. Significantly, the effects of PRC2 loss were ameliorated by restoration of PRC2 function, or inhibition of NSD2 function, revealing crosstalk between H3K27me and H3K36me marks, and suggesting that multiple agents targeting components of the active chromatin state may be beneficial in this rare and deadly disease.

Citation Format: John B. Wojcik, Simone Sidoli, Kumarasen Cooper, Benjamin A. Garcia. Integrated proteome and epigenetic analysis reveals crosstalk between chromatin and cancer pathways in malignant peripheral nerve sheath tumors [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 A10.