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V.G. Ramakrishnan et al.
tive to the MEK + HDAC inhibitor combination, including the aforementioned MM1R, MM1S, RPMI8226 and U266, suggesting that MCL-1 that is “BIM-primed” is critical for the observed synergy to occur (Figure 4A). On the other hand, cell lines with BIM mostly bound to BCL-2 were resistant to the MEK + HDAC inhibitor combination (Figure 4A). It was noteworthy that BCL-XL was weakly expressed in most of the cell lines and there were few BIM:BCL-XL complexes at baseline (Figure 4A).
BCL-2 + HDAC inhibition induces synergistic apoptosis in MEK + HDAC inhibitor-resistant, BCL-2-primed cell lines
Since cell lines with more BIM:BCL-2 complexes rela- tive to BIM:MCL-1 complexes were resistant to the MEK + HDAC combination, we hypothesized that these BCL- 2-primed cell lines would be sensitive to BCL-2 inhibition with the BH3 mimetic ABT-199 (venetoclax) when used in
combination with LBH589. Remarkably, BCL-2 + HDAC inhibition caused synergistic proliferation arrest and cyto- toxicity in nearly all the cell lines that were resistant to the MEK + HDAC combination, including DOX40, KMS18, KMS28 and OPM2 (Figure 4B and data not shown). Pronounced induction of apoptosis was confirmed by annexin/PI staining (Online Supplementary Figure S2).
This coincided with cleavage of caspase 9/3 and PARP (Figure 4C). Immunoprecipitation experiments demon- strated that ABT-199 dissociated BIM from BCL-2, and LBH589 dissociated BIM from MCL-1 (Figure 4D). In addi- tion, we found that ABT-199 and LBH589 both increased BIM bound to both BAX and BAK, an effect that was even more pronounced with the drug combination (Figure 4E). Moreover, BAX/BAK knockdown with siRNA protected from the ABT-199/LBH589-induced cytotoxicity, confirm- ing that the observed synergistic cell death occurred via the mitochondrial intrinsic apoptotic pathway (Figure 4F).
Table 1. Changes in apoptosis of multiple myeloma patients’ plasma cells exposed to various drug combinations. Plasma cells sorted from patients with multiple myeloma were exposed to AZD6244, LBH589, and the combination, or ABT-199, LBH589, and the combination for 72 h. The proportion of cells undergoing apoptosis was measured using flow cytometry, and the relative fold change in apoptosis is indicated. The upper part of the table summarizes the results from the 14 patients treated with the AZD6244/LBH489 combination, while the lower part summarizes the results for the nine patients treated with the ABT-199/LBH589 combination.
Patient #
MC1
MC2 MC3 MC4 MC5 MC6 MC7 MC8 MC9 MC10 MC11 MC12 MC13 MC14
Control AZD6244 LBH589
1 1.35 3.15
1 1.71 3.43 1 1.07 3.47 1 0.62 2.14 1 4.21 3.07 1 1.07 7.41 1 1.67 1.54 1 4.67 4.33 1 1 1.38 1 2.75 4.75 1 2.78 1.78 1 2.85 1.23 1 2 3.75 1 2.3 2.1 1 2.15 3.11
Relative fold change in apoptosis
Drug dose (nM) AZD6244 LBH589
500 10
500 5 500 5 500 7.5 300 5 500 7.5 500 2.5 500 2.5 500 2.5 500 1 500 1 500 2.5 500 2.5 500 2.5
Relative fold change in apoptosis
AZD6244+ LBH589
Mean fold change in apoptosis
Drug dose (nM) Patient # ABT-199 LBH589
MC10 500 1
MC11 500 2.5 MC12 500 2.5 MC13 500 2.5 MC14 500 1 MC15 1000 1 MC16 500 2.5 MC17 500 2.5 MC18 500 2.5
5.96
4.07 4.47 2.62 20.71 15.19 9.44 9.56 2.85 11 6.78 4.08 7.5 5.6 7.85
Control ABT-199 LBH589
ABT-199+ LBH589
Mean fold change in apoptosis
1 2.75 4.75 13.5
1 1.78 4.89 6.22 1 2.15 1.23 3.23 1 3 3.75 12.75 1 2.2 2.1 4.6 1 12.33 3.33 21.33 1 2.33 2 7.67 1 7.71 2 10.71 1 1.75 1.38 2.16
1 4 2.82 9.13
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