[14,15,16] consistent with the observation that the PRC2 complex interacts with DNMT1 and DNMT3A and B [17]. However, PcG mediated repression may also constitute an independent mecha- nism of silencing for some cancer genes, in the absence of DNA- methylation [18]. In the present study integrative genomics was used to define the nature of the underexpressed gene expression profile in MM when compared to normal plasma cells. The identified gene signature significantly correlated to defined Polycomb target genes [19] and was more pronounced in advanced stages of the disease. Enrichment of H3K27me3 at genes found in the profile was confirmed by chromatin immunoprecipitation (ChIP) assay in four newly diagnosed MM patients and two MM cell lines. The S- adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobi- nostat) reactivated the genes repressed by H3K27me3, depleted cells of the PRC2 component EZH2, reduced proliferation and increased apoptosis in human MM cell lines. Using the immunocompetent 5T33MM in vivo model we show gene upregulation, reduction of tumor load and increased overall survival by LBH589. Collectively, our results point to a common gene expression signature in MM mediated by gene silencing via the PRC2 complex. Unraveling the underlying molecular mechanisms and the biological significance of reactivating the genes found in this signature may have new therapeutic implications. Results H3K27-tri-methylation is a common denominator for the underexpressed genes in multiple myeloma (MM) Publicly available gene expression data sets [5,7,20] were analyzed in an integrative genomics approach to identify unifying denominators among genes underexpressed in multiple myeloma (MM). Using the data-mining platform Oncomine [21] a strong connection between genes underexpressed in patients with MM (compared to normal bone marrow) and genes previously described as H3K27me3 targets in human embryonic fibroblasts [19] was discovered (Figure 1A, for a complete gene list see Table S1). The H3K27me3 target genes were also overrepresented among genes underexpressed in MGUS patients (Figure 1B) and strongly associated with decreased expression in ISS stage III MM (Figure 1C), compared to stage I and II (Figure 1D). Consistent with this, overexpression of the PRC2 components EZH2, SUZ12 and EED was significantly correlated with the establishment and progression of MM (Figure S1A–C). Polycomb target genes are H3K27 tri-methylated in patient MM cells and MM cell lines Having established that the underexpressed gene profile in MM significantly overlaps with defined Polycomb target genes, the PRC2-mediated tri-methylation of histone H3 lysine 27 was analyzed by chromatin immunoprecipitation (ChIP) in four newly diagnosed MM patients (Figure 2A–D). Considering the limited availability of patient material, enrichment for the negative H3K27-tri-methylation and positive H3K9-acetylation marks was analyzed in the RPMI 8226 (Figure 2E) and U-266-1984 (Figure 2F) MM cell lines. The chromatin modifications were determined at five PRC2 targets commonly underexpressed in MM (CIITA, CXCL12, GATA2, CDH6 and ICSBP/IRF8; Figure S2A–C). The selected genes were then additionally confirmed to be underexpressed in MM compared to normal counterpart plasma cells in an independent data set [22] (GSE6477, Figure S3). The INK4A/p16 gene was also included, Polycomb Silencing in MM as a previously reported target of the Polycomb group (PcG) proteins [23]. The actively transcribed genes RPL30 and GAPDH were used as control genes for the lack of H3K27-tri-methylation and presence of acetylation on histone H3 lysine 9. Consistent with the role for Polycomb-mediated silencing, the analyzed genes were found enriched for H3K27-tri- methylation in the four MM patient samples (Figure 2A–D) and both MM cell lines RPMI 8226 (Figure 2E) and U-266- 1984 (Figure 2F). These genes also lacked the positive chromatin acetylation mark at H3K9 in both MM cell lines. An exception to this was the ICSBP/IRF8 gene, which was clearly enriched for H3K27-tri-methylation in two of the four patients and the U-266-1984 cell line, whereas in the RPMI 8226 cells carried both the positive and negative mark. The CIITA gene lacked clear enrichment in one of the patients and the CXCL12 gene carried the positive H3K9-acetylation mark in the RPMI 8226 cells and the negative H3K27-tri-methyl- ation mark in the U-266-1984 cells. The S-adenosylhomocysteine hydrolase inhibitor 3- Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 reactivate Polycomb target genes in MM cell lines We next investigated the possibility of reactivating the genes enriched for H3K27-tri-methylation. The expression of the genes selected from the integrative genomics profile was analyzed after treatment with the chemical inhibitors, DZNep and LBH589, in the RPMI 8226 and U-266-1984 cell lines. RPMI 8226 and U- 266-1984 were treated with 10 mM DZNep and 20 nM LBH589 and the expression of CIITA, CXCL12, GATA2, CDH6, ICSBP/ IRF8 and INK4A was analyzed using Q-RT-PCR after 6, 24, 48 and 72 hours. LBH589 (Figure 3A and 3B) was more potent and led to a higher increase in mRNA levels at earlier time points in both cell lines, whereas, DZNep (Figure 3C and 3D) increased mRNA levels to a lesser extent and at later time points. Changes in gene expression varied between the cell lines and had slightly different kinetic profiles. CIITA mRNA expression in RPMI 8226 increased after DZNep and LBH589 treatment. In U-266-1984, CIITA expression gradually increased only by DZNep. In RPMI 8226, CXCL12 gene expression was upregulated only after DZNep treatment. GATA2 responded strongly to both drugs in both cell lines. The CDH6 mRNA level was increased by both DZNep and LBH589 in RPMI 8226. ICSBP/IRF8 expression increased gradually by DZNep in both cell lines; LBH589 strongly increased ICSBP/IRF8 mRNA levels only in U-266- 1984. INK4A/p16 responded weakly to both drugs in both cell lines. CXCL12 and CDH6 mRNA levels were detected at very late amplification cycles in U-266-1984 and were not amenable to analysis. Both DZNep and LBH589 deplete EZH2 protein levels in MM cell lines EZH2 is the catalytically active subunit of PRC2 responsible for the establishment of tri-methylation at histone H3 lysine 27 that is associated with gene silencing [24]. The two chemical inhibitors, the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplano- cin (DZNep) and the histone deacetylase inhibitor LBH589, are reported to deplete EZH2 protein levels and reduce survival in cancer cells [25,26]. The MM cell lines, RPMI 8226 and U-266-1984 were treated with DZNep at 0.5, 2.5 and 10 mM or LBH589 at 4, 10, 20 nM PLoS ONE | www.plosone.org 2 July 2010 | Volume 5 | Issue 7 | e11483