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HDAC + MEK or BCL-2 inhibition in multiple myeloma
family proteins. In pursuit of demonstration of this, we immunoprecipitated BIM and examined its binding pat- tern with BCL-2, BCL-XL and MCL-1 before and after drug treatment. AZD6244 increased the relative amount of BIM bound to all three anti-apoptotic proteins, which may partly explain why even though the drug markedly increases BIM levels, it has limited cytotoxic effects in MM as a single agent (Figure 3C). Interestingly, LBH589 dissociated BIM from MCL-1 and BCL-XL, but not BCL-2 (Figure 3C). This effect was particularly evident when the anti-apoptotic proteins were “primed” with more BIM by AZD6244. We also noted this dissociation in the recipro- cal experiment when pulling down with BCL-2 and MCL- 1, and probing for BIM (Figure 3C). Furthermore, we observed increased BIM bound to both BAX and BAK after AZD6244 and LBH589 treatment individually, which was markedly increased after the combination treatment (Figure 3D). We also noted that LBH589 dissociated BAK from MCL-1 in a similar manner to BIM, which theoreti- cally would facilitate increased BIM:BAK complexes in the
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presence of LBH589 to further activate the intrinsic apop- totic cascade (Figure 3D).
MCL-1/BCL-2 expression and BIM binding profile cor- relate with sensitivity to MEK + HDAC inhibition
In light of LBH589 dissociating BIM from MCL-1 and BCL-XL, but not from BCL-2, we wondered if the cell lines that were resistant to the MEK+HDAC inhibitor combi- nation expressed higher baseline levels of BCL-2. Western blots confirmed that this was partly true, but many of the AZD6244/LBH589-sensitive cell lines, including MM1R, MM1S, RPMI8226 and U266, had high expression of both BCL-2 and MCL-1 (Figure 4A). To interrogate the possibil- ity that expression is a poor indication of functional dependence on either anti-apoptotic protein, we immuno- precipitated BIM and examined baseline levels of BIM:BCL-2, BIM:MCL-1 and BIM:BCL-XL complexes in cell lines that were either sensitive or resistant to the MEK + HDAC inhibitor combination. Notably, BIM was mostly bound to MCL-1 in all of the cell lines which were sensi-
Figure 2. MEK + histone deacetylase inhibition drives synergistic apoptosis in multiple myeloma cell lines and inhibits target proteins. (A) Flow cytometric cell via- bility of the RAS mutant human multiple myeloma (MM) cell lines H929 and MM1S measured as the proportion of annexin–/propidium iodide (PI)– cells, after 24, 48 and 72 h of treatment with AZD6244 and LBH589 at the indicated doses. Viability is shown as percent of control on the Y-axis. (B,C) H929 and MM1S were treated with AZD6244/LBH589 for 24 h, then whole-cell lysates were separated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blotting for the indicated proteins. (D) MM1S was electroporated with scrambled siRNA, ERK1, ERK2 siRNA or the combination, then left untreated or treated with 5 nM LBH589. 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. (E) Whole-cell lysates from these cells were separated using SDS-PAGE and subjected to western blotting to confirm ERK1 and ERK2 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. All experiments were performed in triplicate.
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