Polycomb Target Genes Are Silenced in Multiple Myeloma Antonia Kalushkova1., Ma˚ rten Frykna¨ s1., Miguel Lemaire2, Charlotte Fristedt1, Prasoon Agarwal1, Maria Eriksson1, Sarah Deleu 2 , Peter Atadja3, Anders O¨ sterborg4, Kenneth Nilsson 1 , Karin Vanderkerken 2 , Fredrik O¨ berg1, Helena Jernberg-Wiklund 1 * 1 Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala Sweden, 2 Department Hematology and Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium, 3 Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States of America, 4 Department of Hematology, Karolinska University Hospital Solna, Stockholm, Sweden Abstract Multiple myeloma (MM) is a genetically heterogeneous disease, which to date remains fatal. Finding a common mechanism for initiation and progression of MM continues to be challenging. By means of integrative genomics, we identified an underexpressed gene signature in MM patient cells compared to normal counterpart plasma cells. This profile was enriched for previously defined H3K27-tri-methylated genes, targets of the Polycomb group (PcG) proteins in human embryonic fibroblasts. Additionally, the silenced gene signature was more pronounced in ISS stage III MM compared to stage I and II. Using chromatin immunoprecipitation (ChIP) assay on purified CD138+ cells from four MM patients and on two MM cell lines, we found enrichment of H3K27me3 at genes selected from the profile. As the data implied that the Polycomb- targeted gene profile would be highly relevant for pharmacological treatment of MM, we used two compounds to chemically revert the H3K27-tri-methylation mediated gene silencing. The S-adenosylhomocysteine hydrolase inhibitor 3- Deazaneplanocin (DZNep) and the histone deacetylase inhibitor LBH589 (Panobinostat), reactivated the expression of genes repressed by H3K27me3, depleted cells from the PRC2 component EZH2 and induced apoptosis in human MM cell lines. In the immunocompetent 5T33MM in vivo model for MM, treatment with LBH589 resulted in gene upregulation, reduced tumor load and increased overall survival. Taken together, our results reveal a common gene signature in MM, mediated by gene silencing via the Polycomb repressor complex. The importance of the underexpressed gene profile in MM tumor initiation and progression should be subjected to further studies. Citation: Kalushkova A, Frykna¨s M, Lemaire M, Fristedt C, Agarwal P, et al. (2010) Polycomb Target Genes Are Silenced in Multiple Myeloma. PLoS ONE 5(7): e11483. doi:10.1371/journal.pone.0011483 Editor: Axel Imhof, Ludwig-Maximilians-Universita¨t M u¨nchen, Germany Received February 24, 2010; Accepted June 2, 2010; Published July 9, 2010 Copyright: 2010 Kalushkova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from The Swedish Cancer Society, The Swedish Research Council, The Multiple Myeloma Research Foundation, VonKantzows stiftelse, Stichting tegen Kanker, FWO-Vlaanderen and GOA-Vrije Universiteit Brussel. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Helena.Jernberg_Wiklund@genpat.uu.se . These authors contributed equally to this work. Introduction Multiple myeloma (MM) remains a fatal hematopoietic malignancy. MM is characterized by the clonal expansion of tumor cells with plasma cell features in the bone marrow and is considered a genetically heterogeneous disease [1]. Although the predominant translocations in MM do not fully explain the pathogenesis of the malignant plasma cell, the identification of genetic entities has facilitated the development of targeted therapy, such as FGFR3-kinase inhibitors in t(4;14) MM [2]. However, such treatment does not apply to all MM patients. New therapeutic strategies targeting common pathogenetic events in MM are therefore imperative. An emerging strategy to fight cancer complexity is to establish gene expression profiles and connect them to specific signaling pathways contributing to the disease [3]. In MM, gene expression profiles have, in some cases, been linked to an underlying genetic event such as a translocation to the immunoglobulin heavy-chain locus or hyperdiploidy [4,5]. In further attempts to dissect the disease phenotype, gene expression profiles have been used to refine the underlying mechanisms in molecular subsets, to discover predictors of drug response and identify novel drug targets [6,7,8,9]. However, it still remains unclear how MM, although phenotypically representing mature plasmablasts/plasma cells, preserves the capacity of self-renewal and whether this capacity may be identified as a common gene expression signature. Recently, components of the Polycomb group (PcG) proteins have gained a wide interest as prominent players in carcinogenesis [10,11]. The PcG proteins function in large multimeric complexes, of which the Polycomb repressive complexes PRC1 and PRC2 are most well characterized. The PRC2 core complex consists of EED, SUZ12, RBAP48 and the catalytic subunit EZH2 [12]. PcG mediated gene repression requires a complex series of events, initiated by the recruitment of PRC2 to target genes resulting in the tri-methylation of histone H3K27, which then preserves silencing of the transcriptional program through consecutive cell divisions [13]. PcG mediated silencing is also suggested to predispose target genes to DNA-methylation in various cancers PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11483