Selective Inhibition of HDAC6 with a New Prototype Inhibitor (ACY-1215) Overcomes Bortezomib Resistance In Multiple Myeloma (MM)

Abstract 2997

HDAC enzymes, whose activity has been linked to the transcription of DNA in several cancers including multiple myeloma (MM), are being studied as novel therapeutic targets. Four classes of HDAC enzymes have been identified and several non-specific pan-HDAC and class I HDAC inhibitors have been evaluated in clinical studies. HDAC6, a class II HDAC, has recently been linked to the activity of aggresomes that degrade unfolded and misfolded ubiquitinated proteins. Importantly, targeting both proteasomal and aggresomal protein degradation systems with proteasome inhibitors and HDAC inhibitors respectively, induces accumulation of polyubiquitinated proteins, followed by activation of apoptotic cascades and synergistic cytotoxocity.

Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, either alone or in combination with bortezomib. In vitro enzyme assays showed that ACY-1215 has potent inhibitory activity against HDAC6 (IC₅₀ 0.0054 μ M) compared to the other HDACs, including Class I HDACs. Maximal cytotoxicity of ACY-1215 against MM cell lines was observed at 48h, with IC₅₀ values ranging from 2–8 μ M. ACY-1215 was also effective against patient MM cells, including bortezomib resistant MM cells, without significant cytotoxicity in normal peripheral blood mononuclear cells (PBMCs). Moreover, ACY-1215 in a dose-dependent manner inhibited DNA synthesis of MM cells at 48h triggered either by binding to bone marrow stromal cells (BMSCs) or by exogenous IL6 and IGF-1, confirming its ability to overcome the proliferative advantage conferred by BMSCs and cytokines. We next studied whether the highly selective HDAC6 inhibitor ACY-1215 could achieve the same efficacy as a pan-HDAC inhibitor such as SAHA, but with less toxicity. Compared to SAHA, ACY-1215 was not toxic to PHA stimulated PBMCs. Importantly, both ACY-1215 and SAHA induced dose-dependent acetylation of α-tubulin in MM.1S cells, but ACY-1215 induced less potent acetylation of lysine on histone H3 and histone H4 than SAHA, further confirming its specific inhibitory effect on HDAC6 activity.

We next combined low doses of ACY-1215 to inhibit aggressomal with bortezomib to inhibit proteasomal protein degradation, and showed synergistic anti MM activity (Combination Index < 0.9), resulting in apoptosis via the activation of caspase-8,-9,-3 and poly (ADP) ribosome polymerase. Annexin V⁺PI⁺ staining after 24h treatment confirmed increased cells in early and late apoptosis after combined therapy (83.6%) compared to ACY-1215 1μ M (10.2%) or bortezomib 5 nM (36.6%) treatment alone. Since NF-kB pathway plays a crucial role in MM cell survival and bortezomib triggers NF-κB activity in some MM cell lines, we next investigated the effect of combined therapy on NF-κB activity. MM.1S cells were co-cultured with BMSCs and then treated with bortezomib 5nM, ACY-1215 1μ M, or the combination. Interestingly, bortezomib treatment significantly enhanced NF-κB activity in MM.1S cells co-cultured with BMSCs, whereas ACY-1215 treatment resulted in only a modest increase in NF-κB activity. Importantly, combined therapy was associated with partial abrogation of bortezomib-induced NF-κb activity. Ongoing studies are determining whether this effect on NF-κB activity contributes to the cytotoxic effect of ACY-1215/bortezomib combination therapy.

Finally, we confirmed the synergistic anti-MM activity of ACY-1215 and bortezomib in vivo using two different xenograft mouse models: human MM injected subcutaneously (plasmacytoma model); and luciferase-expressing human MM injected intravenously (disseminated MM model). In the, plasmacytoma model, daily (5×/week) dosing of ACY-1215 (IP: 50 mg/kg) and bortezomib (IV: 0.5 mg/kg) given twice weekly significantly inhibited tumor growth (p<0.0001) and prolonged survival (34 days in the combined treatment group versus 22 days in the control group, p<0.0011). In the disseminated MM model, daily (5x/week) dosing of ACY-1215 (IP: 75 mg/kg) and bortezomib (IP: 1.5mg/kg) once weekly significantly inhibited tumor growth (p<0.0001) and prolonged survival (40 days in the combined treatment group versus 17 days in the control group, p<0.0001). Importantly, no significant adverse effects were noted in either in vivo model. These results therefore provide the preclinical rationale for clinical trials of ACY-1215 together with bortezomib in the treatment of MM.