Polycomb Silencing in MM CIITA was upregulated after LBH589 treatment only in the CD11b-depleted bone marrow cells (Figure 6A), whereas CXCL12 was upregulated both in the crude BM cell fraction and in the CD11b-depleted cells (Figure 6B). Secondly, the effect of LBH589 as single drug treatment (continuous treatment) was determined on the in vivo tumor burden. The C57BL/KalwRij mice were injected with 5T33MM cells and the effect of LBH589 treatment compared to a vehicle treatment was analyzed. A significant decrease (p,0.0001) in the tumor load after LBH589 treatment was determined by the amount of detected paraprotein (M spike, Figure 6C) and the percentage of MM positive cells in isolated bone marrow cells (Figure 6D). Finally, LBH589 Figure 6. LBH589 upregulates gene expression, reduces tumor load and increases survival in the 5T33MM mice model. (A) and (B): At day 0 C57BL/KalwRij mice were injected with 0.56106 5T33MM cells. After 14 days mice were either assigned to a treatment group receiving 10 mg/ kg LBH589 (n = 4; daily i.p. injection) or to a vehicle group receiving 0.9% NaCl solution (n = 4; daily i.p. injection). After 5 days of treatment mice were sacrificed, BM harvested and a negative selection for CD11b positive cells performed. CIITA and CXCL12 fold difference in gene expression was calculated relative to the vehicle by Q-RT-PCR. GAPDH was used as a reference gene. The error bars represent SEM from two independent Q-RT-PCR runs. Each group contained one mouse, excluding the vehicle non-depleted, which contained two mice. (C) and (D): Mice were treated as described above, with the difference that mice were either assigned to a treatment group receiving 10 mg/kg LBH589 (n = 10; daily i.p. injection), to a vehicle group receiving 0.9% NaCl solution (n = 10; daily i.p injection) or to an untreated, disease-free group (n = 10) as a control group at day 0. The animals were sacrificed when the vehicle group showed signs of morbidity. Tumor load as determined by serum electrophoresis (C) and tumor load as determined by May Gr u¨nwald-Giemsa staining of BM cytospin samples (D) Mean values 6 SD for groups of 10 mice are shown (*p,0.0001). (E) Survival was studied using the Kaplan-Meier analysis method. Mice were treated as described above with the exception that treatment continued until each animal showed signs of morbidity. Each group contained 12 mice (p,0.0003). doi:10.1371/journal.pone.0011483.g006 PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11483