Both DZNep and LBH589 independently inhibit growth of MM cell lines To elucidate the effects of Polycomb inhibitors on the growth and survival of MM cells, RPMI 8226 and U-266-1984 were treated with DZNep or LBH589 for 6, 24, 48 and 72 hours and cell growth was analyzed by the resazurin assay. Both treatments decreased the relative number of viable MM cells in a dose- dependent manner (Figure S5A and S5B). On the contrary, treatment with the same concentrations of both drugs of normal peripheral blood mononuclear cells (PBMC) and normal human fibroblast SK 1064 cells showed no decrease in cell viability (Figure S6A and S6B). Treatment with 10 mM DZNep exhibited over 60% growth inhibition in RPMI 8226 and over 40% in U- 266-1984 at 72 hours of treatment, compared to the untreated cells (Figure 5A). Treatment with 20 nM LBH589 was more potent in reducing the relative amount of viable cells and led to a 70% and 60% growth inhibition at 72 hours in RPMI 8226 and U-266-1984, respectively (Figure 5B). To further characterize the effect of DZNep and LBH589 in reducing the number of viable cells, induction of apoptosis was quantified by AnnexinV/PI staining using FACS analysis. At 72 hours DZNep treatment increased the fraction of late Polycomb Silencing in MM apoptotic/necrotic cells in RPMI 8226 and U-266-1984 more than two fold (Figure 5C). Treatment with LBH589 was more potent in inducing apoptosis and led to approximately 3 fold increase of AnnexinV/PI positive apoptotic cells in RPMI 8226 and greater than 3 fold increase in U-266-1984 cells at 72 hours (Figure 5D). LBH589 treatment upregulates Polycomb target gene expression, decreases tumor load and promotes survival in the murine 5T33MM model Similar to human MM cell lines, treatment with LBH589 induces decreased DNA synthesis, cell cycle arrest and apoptosis in 5T33MM cells in vitro (data not shown). Here, the murine syngeneic immunocompetent 5T33MM model [27,28] was used to evaluate the effect of LBH589 treatment in vivo. The C57BL/ KalwRij mice were injected with the myeloma 5T33MM cells and after 14 days subjected to treatment with LBH589 (10 mg/kg) as described in materials and methods. After 5 days of treatment mice were sacrificed and their BM harvested. To confirm the reactivation of CIITA and CXCL12 gene expression in the in vivo 5T33MM model, RNA was extracted from the total BM fraction and from CD11b-depleted fraction of BM cells. We found that Figure 5. DZNep and LBH589 reduce growth and induce apoptosis in MM cell lines. (A) and (B): U-266-1984 and RPMI 8226 cells were treated for 6, 24, 48 and 72 hours with DZNep (10 mM) or LBH589 (20 nM) followed by a resazurin assay. At least 2 independent experiments were performed in triplicates, data is presented as mean percentage of control 6SD. (C) and (D): U-266-1984 and RPMI 8226 were treated with DZNep (10 mM) and LBH589 (20 nM) for 72 hours followed by AV/PI staining and flow cytometry analysis. At least 2 independent experiments per cell line were performed; data is presented as mean percentage apoptotic cells 6SD. doi:10.1371/journal.pone.0011483.g005 PLoS ONE | www.plosone.org 6 July 2010 | Volume 5 | Issue 7 | e11483