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V.G. Ramakrishnan et al.
cells are particularly sensitive to MEK inhibition when this is used in combination with pan-HDAC or class I HDAC inhibitors. Our results demonstrate that while sin- gle-agent MEK inhibition increased BIM, this protein remained sequestered by anti-apoptotic BCL-2 family members. Concomitant HDAC inhibition, specifically of class I HDAC, dissociated BIM:MCL-1 and BIM:BCL-XL complexes, but not BIM:BCL-2 complexes, thereby free- ing BIM to activate apoptotic cell death (Figure 7A).
Given the prevalence of RAS/RAF mutations in MM, we anticipate that this combination could be highly clin- ically effective. While we did not investigate specific dif- ferences between N-RAS and K-RAS mutations here, a prior study showed different response durations after standard therapy, i.e. bortezomib, between these two subgroups; future research should examine the signifi- cance of these isoforms in the context of MEK + HDAC inhibition.35 In addition, given the marked intra-tumoral heterogeneity of MM, the impact of the variant allele fre- quency of RAS/RAF mutations must be evaluated in future studies.
Our mechanistic studies identified that MEK + HDAC inhibitor-sensitive MM cell lines expressed high levels of p-MCL-1 (T163), a post-translational modification that enhances the stability of MCL-1 and is driven in part by ERK.36 MCL-1 is unique among the anti-apoptotic BCL-2 family because of its short half-life, but is often upregu- lated or stabilized in numerous cancers, including MM.37,38 Although p-MCL-1 (T163) does not affect the binding capacity of MCL-1, stability of the anti-apoptotic BCL-2 family proteins is part and parcel of their function.39
Interestingly, we identified for the first time that the pan-HDAC inhibitor LBH589 and class I HDAC inhibitors MS275 and FK228 act as MCL-1 and BCL-XL inhibitors at relatively low doses. Furthermore, we sug- gest that the downregulation of p-MCL-1 at the S64 residue may be responsible for the former observation.29 Unlike other studies using several-fold higher doses of HDAC inhibitors in their experiments,18,40 HDAC inhibi- tion did not increase levels of BIM, NOXA, or any other BH3-only proteins in our experiments. Thus, HDAC inhibitor-driven upregulation of BH3-only proteins does not appear to mediate synergy with MEK or BCL-2 inhi- bition, at least in MM. However, we were unable to elu- cidate specifically how HDAC inhibition downregulated p-MCL-1 (S64), or functionally prove that this was responsible for the observed BIM dissociation. In addi- tion, further studies are required to determine how LBH589 dissociates BIM from BCL-XL.
MM cell lines that were resistant to MEK+HDAC inhi- bition had markedly more BIM:BCL-2 complexes at base- line. Combining ABT-199 (venetoclax) with HDAC inhibitors synergistically killed these cell lines (Figure 7B). ABT-199 is a highly potent BCL-2 inhibitor which, in just a few years, has altered the treatment landscape of chron- ic lymphocytic leukemia.41 It has also produced promising clinical responses in many other hematologic malignan- cies and several non-Hodgkin lymphomas.42 More rele- vant to our study, single-agent ABT-199 recently pro- duced encouraging clinical responses in patients with relapsed/refractory MM, particularly those with t(11;14) who had high BCL2 expression.43 It is conceivable that HDAC inhibition could either expand the pool of patients who would be sensitive to BCL-2 inhibition, or deepen the responses of partially sensitive patients.
However, it should be noted that concomitant HDAC inhibition did not sensitize all MM cell lines to BCL-2 inhibition, as evidenced by the in vitro resistance of RAS/RAF mutant, MCL-1-primed MM cell lines to BCL-2 + HDAC inhibition. Even so, it is well documented that increased sequestration of BH3-only proteins by MCL-1 and BCL-XL is a major resistance mechanism of BH3 mimetics.44 Perhaps then, HDAC inhibition could re-sen- sitize patients, who relapse after ABT-199 treatment, to BCL-2 inhibition. It is also worth noting that several spe- cific MCL-1 inhibitors are actively being developed for use in MM.45 Speculatively, cells that acquire resistance to MCL-1 inhibition could be investigated to determine whether they become more BCL-2-dependent, and there- by sensitive to the ABT-199/LBH589 combination.
Notably, we demonstrated markedly increased apopto- sis in plasma cells sorted from MM patients with a wide variety of clinical characteristics after treatment with the MEK + HDAC and BCL-2 + HDAC inhibitor combina- tions ex vivo. However, there were too few patients’ sam- ples to discern if the type or number of prior lines of ther- apy, or particular cytogenetic abnormalities predicted for sensitivity to either combination. Even so, it is conceiv- able that readily discernible subgroups of patients, beyond the subgroups formed on the basis of RAS/RAF mutational status, may be identified and that these sub- groups could be more likely to respond to either combi- nation therapy, akin to patients with t(11;14) treated with ABT-199.43
Using both pharmacological and genetic methods, we determined that simultaneous inhibition of HDAC1 and HDAC2 was sufficient to induce apoptosis when com- bined with either MEK or BCL-2 inhibition. This is of inter- est because the clinical utility of the pan-HDAC inhibitor LBH589 in MM has been limited by toxicities, mostly related to diarrhea, fatigue, lymphopenia and thrombocy- topenia,15 which could perhaps be avoided with more spe- cific HDAC inhibition. MS275 (entinostat), an HDAC1, 2, and 3 inhibitor, is actively being investigated in clinical tri- als for numerous tumor types.46 Moreover, FK228 (romidepsin), which mostly inhibits HDAC1 and HDAC2, has been approved by the Food and Drug Administration for the treatment of several T-cell lymphomas.
Specific HDAC1+2 inhibition has previously been shown to drive apoptosis in lymphoid cell lines.47 In MM, a prior study showed that the apoptosis induced by HDAC inhibitors is mainly mediated by HDAC class I inhibition.48 Interestingly, elevated HDAC1 expression has been correlated with poor prognosis in MM.49 In our study, we observed that both HDAC1 and HDAC2 must be inhibited for the synergistic effect with AZD6244 or ABT-199. Given the genetic and functional similarity between HDAC1 and 2, we think that it will be impor- tant to target both of these proteins simultaneously to attain meaningful clinical responses. It must be noted that while our approach using HDAC inhibition holds consid- erable promise in MM and other cancers, the pharmaco- logical specificities of HDAC inhibitors have been incon- sistently described to date, and often do not include specificity for particular HDAC1/2-containing multipro- tein complexes such as CoREST.14 Speculatively, perhaps inhibition of these complexes is important for the mech- anisms described in the present study, rather than inhibi- tion of the isolated enzymatic activity of HDAC1 and 2. If so, compounds with greater specificity for CoREST or
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haematologica | 2019; 104(10)