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V.G. Ramakrishnan et al.
death (Online Supplementary Figure S3A). These results were confirmed by knocking down HDAC6 using siRNA (Online Supplementary Figure S3B). Considering all the find- ings, we concluded that HDAC6 inhibition does not seem to be important for the synergistic cell death induced by the AZD6244/LBH589 combination.
Simultaneous inhibition of HDAC1 and HDAC2 is suffi- cient to enhance cell death induced by MEK or BCL-2 inhibition
We next examined whether inhibiting class I HDAC augmented cell death in combination with MEK or BCL-
AB
2 inhibitors. For this, we first used MS275 (entinostat), which inhibits HDAC1, 2 and 3. We observed potent syn- ergy when MS275 was used with AZD6244 or ABT-199 in MM cell lines (Online Supplementary Figure S4A, B) and patients’ cells (Online Supplementary Table S2) similar to the synergy observed with LBH589. This synergy occurred even at very low doses of MS275 (i.e. 150 nM), which would be unlikely to inhibit HDAC3 (the IC50 for HDAC3 is 1.7 μM).32 To determine whether inhibiting HDAC1 and 2 would be sufficient to synergize with AZD6244 or ABT-199, we used the HDAC1 and 2 inhibitor FK228 (romidepsin) in combination with
CD
E
Figure 6. Concomitant inhibition of histone deacetylases 1 and 2 replicates synergy with MEK or BCL-2 inhibition in multiple myeloma cell lines. (A) The histone deacetylase (HDAC)-1 and -2 inhibitor FK228 (romidepsin) was combined with AZD6244 in increasing doses in the MCL-1 primed, RAS/RAF mutant human multiple myeloma (MM) cell lines H929, MM1R, RPMI8226 and U266. Cellular viability was assessed using MTT at 72 h. Viability is shown as percent of control on the Y- axis. Combination index (CI) values <1.0, indicating synergy, are shown for each cell line. (B) FK228 was combined with ABT-199 in increasing doses in the BCL-2- primed human MM cell lines KMS18, OPM1, OPM2 (RAS/RAF wild-type) and KMS28 (RAS mutant). Cellular viability was assessed using flow cytometry by analyzing the proportion of annexin–/propidium iodide (PI)– cells, shown as percent of control on the Y-axis. (C) KMS28 was treated with 100 nM ABT-199 and 0.5 nM FK228 for 24 h, then immunoprecipitates for BCL-2, MCL-1 and BCL-XL, or whole cell lysates (input) were separated using sodium dodecylsulfate polyacrylamide gel elec- trophoresis (SDS-PAGE) and probed for the indicated proteins. Light and dark film exposures are shown for the BCL-2, MCL-1 and BCL-XL immunoprecipitates so the BIM:MCL-1 and BIM:BCL-XL dissociations are apparent. (D) MM1S was electroporated with scrambled siRNA, HDAC1 siRNA, HDAC2 siRNA or HDAC1 and 2 siRNA, then left untreated or treated with 250 nM AZD6244. At 72 h, cell viability was assessed using flow cytometry by analyzing the proportion of annexin–/PI– cells, shown as percent of control on the Y-axis. In addition, whole-cell lysates were separated using SDS-PAGE and probed for the indicated proteins to confirm silencing. (E) KMS18 was electroporated with scrambled siRNA or HDAC1 and HDAC2 siRNA, then left untreated or treated with 50 nM ABT-199. At 72 h, cell viability was assessed using flow cytometry by analyzing the proportion of annexin–/PI– cells, shown as percent of control on the Y-axis. Also whole-cell lysates were separated using SDS- PAGE and probed for the indicated proteins to confirm silencing. Error bars represent the standard error of mean of triplicate experiments. Differences between groups were calculated with the Student t test. **P<0.001, ##P<0.01. All experiments were performed in triplicate.
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