Histone deacetylase (HDAC) 6, a class-IIb HDAC, is the only HDAC with two functional deacetylase domains and a zinc-finger motif.HDAC6 was initially described as tubulin deacetylase; however, it also deacetylates other non-histone proteins, including heat-shock protein (Hsp90).Importantly, HDAC6 has an essential role in the aggresomal protein degradation pathway, an alternative system to the proteasome for degradation of polyubiquitinated misfolded/unfolded proteins.Specifically, HDAC6 binds to both polyubiquitinated proteins and dynein motors, thereby acting to recruit protein cargo to dynein motors for transport to aggresomes for lysosomal degradation1 We have previously shown that inhibition of both proteasomal and aggresomal protein degradation using bortezomib (BTZ) to inhibit the proteasome and tubacin to inhibit HDAC6, resp., induces accumulation of polyubiquitinated proteins and significant multiple myeloma (MM) cell stress, followed by apoptosis.2 More recently, a hydroxamic acid HDAC6-selective inhibitor ACY-1215 combined with BTZ shows significant anti-MM activities in preclin. studies,3 which have been rapidly translated to phase I/II clin. studies to treat relapsed/refractory MM.4 Previous studies have also shown that the non-selective HDAC inhibitor vorinostat in combination with BTZ also triggers synergistic cytotoxicity in MM,5 which has provided the framework for combination clin. trials.6 Carfilzomib (CFZ) is an epoxyketone proteasome inhibitor, which recently received accelerated Food and Drug Administration approval to treat relapsed refractory MM.7, 8 In this study, we asked whether HDAC6 inhibitors enhance anti-MM toxicity induced by CFZ.2 More recently, a hydroxamic acid HDAC6-selective inhibitor ACY-1215 combined with BTZ shows significant anti-MM activities in preclin. studies,3 which have been rapidly translated to phase I/II clin. studies to treat relapsed/refractory MM.4.Previous studies have also shown that the non-selective HDAC inhibitor vorinostat in combination with BTZ also triggers synergistic cytotoxicity in MM,5 which has provided the framework for combination clin. trials.6 Carfilzomib (CFZ) is an epoxyketone proteasome inhibitor, which recently received accelerated Food and Drug Administration approval to treat relapsed refractory MM.7, 8.In this study, we asked whether HDAC6 inhibitors enhance anti-MM toxicity induced by CFZ.We also delineated differential mol. mechanisms that trigger synergistic MM toxicity triggered by CFZ in combination with either HDAC6-selective or class-I HDAC-selective inhibitors ACY-1215 and MS275 (entinostat), resp.9 We first compared the combination effect of the HDAC6-selective inhibitor ACY-1215 with either BTZ or CFZ in MM cell lines.Synergistic cytotoxicity induced by CFZ with ACY-1215 was greater than that by BTZ with ACY-1215 in both RPMI8226 (Figure 1a, upper panel) and MM.1S cells (Figure 1a, lower panel).To further confirm the role of HDAC6 inhibition, we carried out similar combination cytotoxicity experiments using the chem. unrelated HDAC6-selective inhibitor tubastatin-A.Consistent with the ACY-1215 results, tubastatin-A synergistically enhanced both BTZ- and CFZ-induced cytotoxicity.Interestingly, synergistic cytotoxicity, on the basis of combination index (CI) <1, was more significant in RPMI8226 cells than in MM.1S cells (Supplementary Figure 1).Although BTZ has a remarkable clin. activity in MM, acquired resistance limits its long-term utility.We, therefore, next examined whether HDAC6 inhibition can overcome BTZ resistance in vitro using the BTZ-resistant ANBL (ANBL-R) human MM cell line.Importantly, the synergistic cytotoxicity of ACY-1215 with BTZ was observed even in ANBL-R cells.Similar to Figure 1a, this synergistic cytotoxicity was even more significant with CFZ compared with BTZ (Figure 1b).These results suggest that CFZ may have a greater clin. activity in MM than in BTZ when combined with HDAC6 inhibitors.We and others have shown that non-selective HDAC inhibitors in combination with BTZ trigger synergistic cytotoxicity in MM.10, 11.These non-selective HDAC inhibitors block not only HDAC6 but also the class-I HDAC activity,9 and whether synergistic cytotoxicity induced by non-selective HDAC inhibitors with BTZ is due solely to HDAC6 blockade remains unclear.Indeed, we have recently shown that both HDAC3 knockdown and a small-mol. HDAC3-selective inhibitor BG45 trigger synergistic MM cytotoxicity in combination with BTZ.12.To address this question, we first examined the cytotoxicity of class-I HDAC-selective inhibitor MS275 in combination with CFZ.MS275 with CFZ induced synergistic cytotoxicity in RPMI8226 cells and additive cytotoxicity in MM.1S cells (Supplementary Figure 2).Consistent with our studies, MS275 with BTZ also shows significant anti-tumor activities in other cancer cell types.13 We next determined whether different mol. mechanisms mediate the synergistic MM cell cytotoxicity of HDAC6 vs. class-I HDAC inhibition in combination with CFZ.We first defined the doses of either ACY-1215 (2 μM) or MS275 (2 μM) with CFZ (10 nM) that induced equivalent cytotoxicity (35%) (Supplementary Figure 3) and used these culture conditions for subsequent experimentsWe have shown that ACY-1215 with BTZ triggers the accumulation of polyubiquitinated proteins associated with cell stress.3 Although not all polyubiquitinated proteins are degraded by the proteasome, recent studies have shown that proteins specifically linked with lysine (K)48 polyubiquitin are degraded by proteasomes.14 Our studies showed that CFZ induced an accumulation of K48-linked polyubiquitinated proteins and that ACY-1215, but not MS275, markedly enhanced this effect (Figure 2a).These results further confirm our previous observations showing involvement of HDAC6 in mediating protein degradation via aggresomes and conversely demonstrating that blocking HDAC6 increases proteasomal protein breakdown.RPMI8226 was the most sensitive MM cell line in response to the combined treatment of HDAC6 inhibitors with proteasome inhibitors.Moreover, our previous studies have shown that RPMI8226 cells constitutively express an XBP-1 spliced form, a marker of unfolded protein response upon endoplasmic reticulum (ER) stress.15 RPMI8226 cells are therefore under constitutive ER stress at physiol. conditions.We consequently hypothesized that combining ACY-1215, but not MS275, with CFZ would further enhance ER stress due to accumulating polyubiquitinated proteins, thereby resulting in an enhanced sensitivity to the combination treatment.Indeed, among MM cell lines, RPMI8226 cells are the most sensitive to ER stressor tunicamycin (Supplementary Figure 4).Importantly, we observed that downstream mols. of ER stress (p-IRE1α, p-eIF2α, p-JNK and CHOP) were significantly enhanced by the treatment with combination ACY-1215 with CFZ (Figure 2a).We further confirmed ER stress triggered by this combination treatment using quant. PCR to demonstrate enhanced XBP-1 splicing (Figure 2b).These results indicate that proteasome inhibition with HDAC6 inhibition, but not class-I HDAC inhibition, triggers accumulation of polyubiquitinated proteins followed by ER stress.We further delineated downstream apoptotic signaling in cells treated with these combinations.Caspases-3 and -7, hallmarks of caspase activation, were cleaved by treatment with ACY-1215 or MS275 in combination with CFZ; however, caspase-9 was more significantly cleaved by ACY-1215 compared with MS275 in RPMI8226 cells.In contrast, caspase-10 was cleaved by CFZ with MS275 but not with ACY-1215.An apoptosis-inducing factor associated with caspase-independent apoptosis was not induced (Figure 2c).Consistent with RPMI8226 cells, we observed enhanced CHOP and capase-9 cleavage triggered by ACY-1215 with CFZ, as well caspase-8 cleavage triggered by MS275 with CFZ in patient MM cells (Supplementary Figure 5A).To confirm the significance of caspase cleavage (activation), we used selective caspase inhibitors in the combination treatments.As expected, caspase-9 inhibitor has a more prominent inhibitory effect on cytotoxicity induced by ACY-1215 with CFZ than that by MS275 with CFZ in RPMI8226 cells (Supplementary Figure 5B) and patient MM cells (Supplementary Figures 5C and 5D).In summary, we show that BTZ and CFZ trigger significant cytotoxicity in MM cells when combined with selective HDAC6 inhibitors (ACY-1215, tubastatin-A), and that more potent synergistic cytotoxicity of HDAC6 inhibitors was observed with CFZ than with BTZ.We also showed synergistic cytotoxicity triggered by combination CFZ with class-I HDAC inhibitor MS275.Importantly, HDAC6 inhibitor ACY-1215, but not MS275, triggered a marked accumulation of K48-linked polyubiquitinated proteins and ER stress, followed by unfolded protein response.Moreover, CFZ combined with ACY-1215 predominantly triggered intrinsic apoptotic signaling, whereas CFZ with MS275 triggered the extrinsic apoptotic pathway.Our results therefore delineate distinct apoptotic pathways induced by CFZ combined with HDAC6 vs. class-I HDAC inhibition, providing the preclin. framework for clin. evaluation of combination therapy to improve patient outcome in MM.
