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HDAC + MEK or BCL-2 inhibition in multiple myeloma
Isobologram analysis
The effects of combination treatments in MM cells were ana- lyzed using the CalcuSynTM software program (Biosoft, Ferguson, MO, USA), which is based on the Chou-Talalay method, as previ- ously described.19,24
Results
Single-agent MEK inhibition does not induce cell death in multiple myeloma cell lines
Prior studies have documented the lack of induction of cytotoxicity by single-agent MEK inhibitors in MM.12 We aimed to confirm these findings using a panel of MM cell lines that are wild-type or mutated for RAS/RAF. We treat- ed cell lines with increasing doses of the MEK inhibitor AZD6244 (selumetinib) and observed a lack of significant cytotoxicity, with IC50 not reached at doses up to 1500 nM (Online Supplementary Figure 1A). AZD6244 was slightly more capable at inducing proliferation arrest in the same panel of cell lines, but still largely ineffective (Online Supplementary Figure 1B). These results were also con- firmed by performing annexin/PI staining after treating two RAS mutant MM cell lines with 5000 nM of AZD6244, which is far above the concentrations at which the kinase activity of MEK is inhibited (Online Supplementary Figure 1C). Thus, it became clear that despite commonly occurring oncogenic mutations in RAS and RAF, MEK inhibitors have limited scope as single- agents in MM.
MEK + HDAC inhibition induces synergistic cell death in multiple myeloma cell lines
We examined the ability of the recently approved pan- HDAC inhibitor LBH589 (panobinostat) to enhance cell death induced by AZD6244 in MM cell lines. Low doses of the AZD6244/LBH589 drug combination induced potent synergistic cytotoxicity (Figure 1A). Interestingly, the synergy was observed in cell lines with mutations in K-RAS and N-RAS (i.e. H929, MM1R, MM1S, RPMI8226) and B-RAF (i.e. U266), but not in cell lines that are wild- type for RAS and RAF (i.e. KMS11, KMS18, OPM2). We also observed significantly more potent proliferation arrest when the drugs were used in combination (Figure 1B).
Components of the bone marrow microenvironment such as bone marrow stromal cells play an indispensable role in MM disease progression and resistance to thera- pies.25 We therefore investigated whether AZD6244/LBH589 was able to overcome the protective effects of bone marrow stromal cells. To do this, we co- cultured MM1S cells with patient-derived bone marrow stromal cells and measured the proliferation rate after treatment with either single-agent AZD6244 or LBH589, or the drug combination. We observed that the AZD6244/LBH589 combination was able to inhibit the proliferation of MM1S even when co-cultured with bone marrow stromal cells (Figure 1C). We also noted synergis- tic cell death when we used another MEK inhibitor, MEK162 (binimetinib), or the ERK inhibitor SCH772984 in combination with LBH589 (Online Supplementary Figure 1D). Furthermore, AZD6244 synergistically killed MM cells when combined with the pan-HDAC inhibitor SAHA (data not shown).
To understand whether the cytotoxicity caused by the
AZD6244/LBH589 combination occurred through the apoptotic pathway, we performed annexin/PI staining. The drug combination clearly induced apoptotic cell death by 72 h (Figure 2A). We also examined whether the com- bination induced the cleavage of caspases and PARP, both of which are markers of apoptosis. The AZD6244/LBH589 combination induced potent cleavage of caspase 9 and PARP, but not caspase 8, suggesting that the cell death occurred through the mitochondrial intrinsic apoptotic pathway (Figure 2B). Next, we examined whether the drugs, at the doses used above, were able to inhibit their target proteins. As expected, AZD6244 inhib- ited pERK and LBH589 caused an increase in acetylated histone H3 levels (Figure 2C). Finally, to confirm that MEK/ERK pathway inhibition contributed to the synergy with LBH589, and to examine whether both isoforms of ERK need to be inhibited for the synergy to occur, we nucleofected isoform-specific ERK siRNA into MM1S and treated the cells with LBH589. We observed that ERK1 or ERK2 knockdown individually enhanced the cell death induced by LBH589 (Figure 2D, E). However, simultane- ous knockdown of both isoforms led to even more pro- nounced cell death when used in combination with LBH589, supporting an important survival role for both ERK isoforms in this context. Finally, we treated plasma cells obtained from MM patients with the drug combina- tion. The characteristics of these patients are detailed in Online Supplementary Table S1. It is worth noting that sev- eral patients had high-risk features including TP53 dele- tion, t(4;14) and refractoriness to multiple lines of therapy. We observed augmented apoptosis with the AZD6244/LBH589 drug combination compared to the effects of either drug alone (Table 1).
MEK + HDAC inhibitor-induced synergistic apoptosis is mediated by BIM
Given that the MEK/ERK pathway is known to phos- phorylate the pro-apoptotic BH3-only protein BIM at ser- ine 69 (S69) to mark it for proteasomal degradation,26 we were not surprised to find that AZD6244 treatment increased BIM protein levels (Figure 3A). Although prior studies have shown that HDAC inhibition increases BIM expression in MM,18,19 we did not observe increased levels of BIM with the several-fold lower doses of LBH589 that were used in this study (Figure 3A). Even so, given that MEK inhibition increased BIM and that the drug combina- tion induced potent activation of intrinsic apoptotic mark- ers, we hypothesized that BIM might play an important role in the observed synergy. To investigate this, we nucleofected MM1S cells with BIM siRNA and observed that BIM knockdown completely protected from the syn- ergistic cell death induced by the MEK + HDAC inhibitor combination (Figure 3B).
HDAC inhibition dissociates BIM from MCL-1 and BCL-XL
From the above results, it became clear that BIM plays an essential role in the synergistic apoptosis induced by the drug combination. Since BIM was upregulated by AZD6244, but not by LBH589, we reasoned that the increased levels of BIM induced by AZD6244 were unable to activate apoptosis due to sequestration by the anti- apoptotic BCL-2 family members (i.e. BCL-2, BCL-XL and MCL-1). Thus, we hypothesized that the mechanism by which LBH589 synergizes with AZD6244 is by modulat- ing the interactions of BIM with the anti-apoptotic BCL-2
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