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HDAC + MEK or BCL-2 inhibition in multiple myeloma
AZD6244 or ABT-199 and observed marked synergy in MM cell lines (Figure 6A, B) and patients’ cells (Online Supplementary Table S2). Using western blotting and immunoprecipitation, we also replicated the mechanistic finding that FK228 downregulates p-MCL-1 (S64) and causes BIM dissociation from MCL-1 and BCL-XL in KMS28 (Figure 6C) and H929 (Online Supplementary Figure S4C). This strongly suggested that HDAC1 and 2 are involved in the mechanism. To confirm these results, we performed knockdown studies with HDAC1- and HDAC2-specific siRNA. Knocking down HDAC1 and HDAC2 individually in combination with AZD6244 or ABT-199 caused minor increases in apoptosis. However, simultaneous knockdown of both HDAC1 and HDAC2, when combined with AZD6244 or ABT-199, caused sig- nificantly more apoptosis (Figure 6D, E). Inhibiting HDAC3 in addition to HDAC1 and HDAC2 did not aug- ment the synergy (data not shown). Dovetailing with our results with pharmacological HDAC inhibitors, we observed p-MCL-1 (S64) downregulation when HDAC1 and HDAC2 were silenced in tandem (Figure 6D). We also noted that knocking down either HDAC1 or HDAC2 individually caused a reciprocal upregulation of HDAC2 and HDAC1, respectively. We speculate that this could be the reason why inhibition of HDAC1 or HDAC2 individ- ually was not sufficient to synergize with either MEK or BCL-2 inhibition (Figure 6D, E). Taken together, these
results showed that simultaneous inhibition of HDAC1 and HDAC2 is sufficient to markedly enhance the apop- tosis induced by AZD6244 or ABT-199 in MM.
Discussion
The sequestration of pro-apoptotic BCL-2 family pro- teins, such as BIM, by their anti-apoptotic counterparts, i.e. BCL-2 and MCL-1, is a pervasive survival strategy in cancer.20 Hence, treatments that alter the pro/anti-apop- totic BCL-2 family member ratio or modulate their bind- ing dynamics hold considerable promise, particularly in hematologic malignancies. In the present study, we iden- tified two drug combinations, i.e. MEK + HDAC and BCL-2 + HDAC inhibition, which target two distinct sub- groups of MM: MCL-1 or BCL-2 primed, respectively (summarized in Figure 7). In support of these being dis- crete phenotypes, none of the MM cell lines that we test- ed was sensitive to both drug combinations. Furthermore, sensitivity aligned mostly based on RAS/RAF mutational status.
Mutations in the RAS/RAF pathway are present in near- ly half of all malignant tumor types.33 In lieu of a direct way to inhibit RAS, MEK inhibitors have shown signifi- cant clinical benefit in several RAS/RAF-mutated can- cers.34 Herein, we identified that RAS/RAF-mutated MM
AB
Figure 7. Proposed mechanism of MEK or BCL-2 inhibition in combination with histone deacetylase inhibition in multiple myeloma. (A) MCL-1-primed lines, which all had mutated RAS/RAF, were sensitive to the MEK + histone deacetylase (HDAC) inhibitor combination. MEK inhibition increased BIM levels, and HDAC1+2 inhi- bition dissociated BIM:MCL-1 and BIM:BCL-XL complexes, the former perhaps by means of downregulation of p-MCL-1 (S64). (B) BCL-2-primed cell lines tended to be wild-type (WT) for RAS/RAF, and were sensitive to the BCL-2+HDAC inhibitor combination. Likewise, HDAC1+2 inhibition dissociated BIM:MCL-1 complexes. Both drug combinations in effect increased free BIM levels, which we found were able to engage BAX and BAK, ultimately leading to synergistic apoptotic cell death.
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