BIM was not increased above the levels induced by I-BET151 alone in the Me1007 cell line and may indicate that single drug treatment of I-BET151 induced maximal levels in this cell line. in the AKT and Hippo/YAP signaling pathways. Xenograft studies showed that the combination of inhibitors was more effective than single drug treatment and confirmed upregulation of BIM and downregulation of XIAP as seen [17, 18]. Additionally I-BET151 has strong inhibitory effects on activation of NF-kB . In the present study we have examined whether combining the HDAC inhibitor LBH589 (panobinostat) and the BET protein inhibitor I-BET151 can potentiate the changes seen when the inhibitors are used as single agents. We report that combination of these two inhibitors has strong synergistic effects in induction of apoptosis, cell cycle arrest and against growth of melanoma xenografts. Moreover apoptosis was mediated by the mitochondrial, caspase-dependent pathway and involved downregulation of the AKT and Hippo/YAP signaling pathway. RESULTS Combined treatment with I-BET151 and LBH589 synergistically induces apoptosis in melanoma cells To determine whether combined treatment Bz 423 of I-BET151 and LBH589 can potentiate sensitivity of melanoma cells to apoptosis we examined the cytotoxic capacity of both inhibitors in a panel of melanoma cell lines. Dose response curves in a number of cell lines revealed dose-dependent cytotoxicity of the drugs individually or in combination (Supplementary Figure 1A). For subsequent experiments, 2 M I-BET151 and 30 nM LBH589 were chosen as these concentrations were only slightly toxic individually, but highly cytotoxic in combination. Melanoma cells were treated with these concentrations for 48 h before apoptosis was measured by Annexin-V/PI staining. As shown in Figure ?Figure1A1A single drug treatment of Me1007 cells with I-BET151 or LBH589 showed slight induction of Annexin-V/PI positive cells when compared to DMSO treated cells. Treatment with a combination of both inhibitors markedly increased cell death. The same effect could be shown in other tested cell lines including melanoma cell lines from patients resistant to treatment with the BRAFi vemurafenib (Patient-1-post and Patient-3-post) which were relatively resistant to both drugs alone (Figure ?(Figure1B).1B). To test if the induction of apoptosis was synergistic rather than merely additive, we performed a combination index (CI) study and calculated synergy using CalcuSyn software. A CI less than 1.0 was obtained in all tested cell lines, indicating a synergistic interaction of both inhibitors with Patient-1-post cells showing the strongest synergistic effect (Figure 1C, 1D). Open in a separate window Figure 1 Combination of I-BET151 and LBH589 synergistically induces apoptosis in melanoma cellsA. Me1007 melanoma cells were treated with 2 M I-BET151, 30 nM LBH589, combination or control for 48 h. Induction of apoptosis was determined by staining with Annexin-V/PI and flow IL3RA cytometry analysis. B. Histogram represents mean ( SEM) of = 3 experiments of different melanoma cell lines and melanocytes (HEM) drug-treated as described above. Combination treatment significantly induced apoptosis ( 0.05) compared to single drug treatment in all tested melanoma cell lines. C. Combination index (CI) of the I-BET151 and LBH589 co-treatment are plotted at increasing drug concentration and fractional effect. CI 1.0 indicates synergistic interaction. A representative Fa-CI plot (Chou-Talalay plot) for Patient-1-post cells is shown. D. CI values for different melanoma cell lines at a fractional effect (Fa) of 0.5 (dose required to kill 50% of cells). CI experiments were performed twice. Studies on the melanoma cell growth Bz 423 showed that the combination of I-BET151 and LBH589 inhibited cell growth and resulted in changes in cell morphology characterized by enlarged and flattened cell bodies (Supplementary Figure 2A). Cell cycle analysis showed the expected sub-G1 population associated with apoptosis and an increase in cells with either 2N DNA content or 4N DNA content, suggestive of arrest in G0C1 or G2-M respectively (Supplementary Figure 2BC2C). Alone, I-BET151 treatment predominantly increased the percentage of melanoma cells with 2N DNA content (G0C1 phase) while reducing the percentage of S-phase cells. LBH589-treated cells increased the proportion of cells with 4N DNA content. This increase in cells with 4N DNA content may indicate cells arrested in G2-M or cells which have failed to undergo cytokinesis and then arrested in G1 but with a 4N DNA content. A similar increase in cells with 4N DNA content was observed in combination-treated cells (except Patient-1-post) suggesting that this growth inhibitory effect is mostly Bz 423 a result Bz 423 of LBH589 inhibitor treatment. Treatment with I-BET151 increased the 4N population in melanocytes. Cell cycle arrest was associated with increases in the cell cycle inhibitor p21 (Supplementary Figure 2D) which was shown previously to be responsible for cell cycle arrest by I-BET151 . Taken together, these results indicate that the combination of I-BET151 and LBH589 synergistically induces apoptosis and cell cycle arrest in melanoma, even in cells with acquired resistance to BRAF inhibitors. Apoptosis induced by co-treatment with I-BET151 and LBH589 is caspase dependent and associated.