The genetic heterogeneity of multiple myeloma (MM) 
has hindered the identification of the mechanisms underlying its pathogenesis and hence, MM remains a fatal disease. Both the translocations to the immunoglobulin heavy chain (IgH) locus and the chromosomal trisomies observed in MM fail to explain a common origin of the disease and to provide a universal MM drug target. Gene expression profiles have provided clues of the importance of previously unrecognized signaling pathways in MM such as the NF-κB pathway 
, but to date have mainly focused on subdividing MM into genetic entities for prognostic or therapeutic purposes 
. Thus, the question remains if a common denominator for myeloma development and tumor maintenance exists.
By means of integrative genomics we identified a set of underexpressed genes in MGUS and MM patient cells relative to normal bone marrow plasma cells and total bone marrow cells, respectively. These genes have also been previously described as targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts 
. Importantly, the suppression of the genes found in the profile was more pronounced in the advanced stages (ISS stage III compared to stage II and I) of MM progression.
The presence of tri-methylation at histone H3 lysine 27 using chromatin immunoprecipitation (ChIP) was determined for several genes found in the profile in four MM patient samples, and two MM cell lines RPMI 8226 and U-266-1984. Among the four patients the genes selected from the integrative genomics approach were consistently enriched for H3K27me3. In addition, simultaneous enrichment for the negative H3K27me3 and lack of the H3K9-acetylation mark associated with an active transcription, suggested active Polycomb-mediated silencing also in the two MM cell lines. Notably, among the genes found in the expression profile, and associated with H3K27me3, was the major histocompatibility (MHC) class II transactivator (CIITA) gene. CIITA is the master regulator of MHC class II expression. Underexpression of CIITA and thus of MHC II genes in MM has been described as a mechanism to escape the immune response 
. Independently, Polycomb silencing of CIITA is described in uveal melanoma 
. Another gene present in the profile was ICSBP/IRF8, which frequently displays low levels of expression in patient plasma cells and MM cell lines. This silencing mechanism is attributed to DNA methylation of the ICSBP/IRF8 gene in 8 out of 10 MM cell lines, including U-266-1984, whereas the frequency is lower in CD138+ selected primary MM cells with only 1 out of 9 being methylated 
. In the present study ICSBP/IRF8 was associated with H3K27me3-mediated silencing in 3 out of 4 patients and the U-266-1984 cell line. Interestingly, DNA methyltransferases are known to be associated with the PRC2 complex 
, and thus DNA methylation following Polycomb-mediated gene silencing is suggested to contribute to permanent transcriptional repression in cancer 
. Hence, PcG mediated repression may be sufficient, and in some cases precede DNA methylation and explain the observed ICSBP/IRF8 silencing in MM patients where DNA methylation is not detected. Our results are also consistent with a previous study suggesting EZH2 as an oncogene in MM, where it is often overexpressed and contributes to cell survival 
. An alternative mechanism for the deregulation of Polycomb control of gene expression in MM was recently suggested by the finding of prevalent mutations in the H3K27-demethylase UTX 
The identification of the Polycomb repression gene profile based on differentially expressed genes in MM patient cells, strongly suggests that this profile is a common feature among the tumor cells, rather than representing a specific subpopulation. Thus, an emerging question is if targeting the common denominator for the observed gene silencing can be used in the treatment of MM. Epigenetic gene silencing is an attractive drug target due to the fact it can be reverted. However, Polycomb targets are not uniformly reactivated solely by depleting cells of PcG proteins using siRNA interference 
. In this study, two chemical inhibitors, the global histone methylation inhibitor 3-Deazaneplanocin (DZNep) 
and the histone deacetylase inhibitor LBH589, were successfully used to reactivate selected genes from the profile, which carried the H3K27me3 mark, including CIITA and INK4A/p16. Moreover, gene upregulation by LBH589 treatment in vivo
was also observed in the 5T33MM model of MM. In addition to their role on global chromatin modifications and their ability to reactivate Polycomb-silenced genes, DZNep and LBH589 have also been reported to deplete EZH2 protein from AML and breast cancer cells 
. Similarly, both DZNep and LBH589 depleted EZH2 protein also from MM cells. According to recent data LBH589 treatment of cancer cells leads to ubiquitin-dependent degradation of DNMT1 
. However, further studies are needed to clarify whether a similar mechanism may contribute to the protein downregulation of EZH2 in MM cells.
In addition to the reactivation of the Polycomb-target genes both DZNep and LBH589 reduced viability and induced apoptosis in two MM cell lines. As LBH589 has an advantageous efficiency at nanomolar concentrations and is currently in phase I and II clinical trials for several hematologic malignancies including MM 
; its effects on MM growth were evaluated also in vivo
using the murine 5T33MM model. Treatment with LBH589 significantly decreased tumor load and levels of paraprotein. Additionally, LBH589 prolonged survival in the murine myeloma 5T33MM model. Thus, our data strongly supports the use of LBH589 in the clinics and provides and alternative mechanism for its antitumor action. Although limited effects of LBH589 single administration have been reported, LBH589 is currently in phase I clinical trials in combinatorial regimens for relapsed MM 
due to the fact that it is able to overcome drug resistance in MM cell lines and cells from patients with refractory disease 
. Recently, a combination of both LBH589 and DZNep was reported to be an effective therapy in a mice model of AML 
; however data from clinical studies has not yet been presented.
Taken together, our results with MM cell lines and fresh MM biopsy cells are in agreement with several recent genome-wide studies implicating Polycomb-mediated gene silencing in the development of other types of malignancy 
. During embryogenesis the PcG proteins constitute an important part of the epigenetic memory by repressing lineage-specific developmental genes. During normal differentiation PcG proteins become redistributed whereby lineage-specific genes are expressed and pluripotency genes become silenced 
. The fact that an epigenetic mark maintaining self-renewal in embryonic stem cells is found in cancer supports the hypothesis that cancer cells share features with normal stem cells. This observation has previously been limited to tumors possessing a poorly differentiated phenotype 
. Using integrative genomics and experimental data, we here emphasize that Polycomb gene silencing is associated with what is considered a differentiated plasmablast/plasma cell tumor. In general, terminal differentiation is tightly linked to an irreversible arrest in G1/G0 phase of the cell cycle 
. Our finding of the underexpressed gene signature in MM suggests that Polycomb silencing enables expansion of differentiated B cells, which retain their ability to divide in conjunction with a differentiated phenotype. In acute leukemia cells, in which an early block in differentiation leads to malignancy, LBH589 induces differentiation 
. It is therefore of interest to note that the reduction of growth in MM by LBH589 is the result of cell cycle arrest 
, fulfilling at least one of the criteria of differentiation. Additional studies are required to dissect the functional role of the repressed gene signature in maintaining proliferation in MM, and the biological consequences of its reactivation.
The existence of the silenced gene expression profile in MGUS, recently proposed to precede MM in majority of the cases 
, suggests that aberrant Polycomb-mediated gene silencing might be an early event during MM development. Although a small subpopulation of clonogenic CD138 negative B cells are suggested to function as cancer stem cells in MM 
, they are unlikely to have an impact on the underexpressed gene profile, when analyzing the tumor bulk. Instead, the identification of the Polycomb-silenced gene profile in CD138 positive patient samples and cell lines implies that this is a general feature shared by a large proportion of the MM cells. The question, however, remains if MM cells have acquired the Polycomb gene repression signature de novo
in the late stages of plasma cell differentiation, or if it is a remnant from earlier stages of normal B-cell development, which remains preserved in the MM cells. Though, currently we cannot rule out any of these possibilities, our data argues that maintaining a stemness feature in the plasma cell tumor is important in order to sustain survival and self-renewal of MM.