Pharmacodynamic and Pharmacokinetic Properties of a Novel and Selective HDAC6 Inhibitor, ACY-1215, in Combination with Bortezomib in Multiple Myeloma
Authors: Santo Loredana , Hideshima Teru , Kung Andrew L. , Tseng Jen-Chieh , Tamang David , Yang Min , Jarpe Matthew , van Duzer John H. , Mazitschek Ralph , Ogier Walter C , Cirstea Diana D. , Rodig Scott , Eda Homare , Scullen Tyler A. , Canavese Miriam , Bradner James E. , Anderson Kenneth C , Jones Simon , Raje Noopur S. 
Published: October 2019

Abstract
Abstract 2912
HDAC enzymes are being studied as novel therapeutic targets in several cancers including multiple myeloma (MM). In particular, the combination of proteasome inhibitors (e.g. bortezomib (BZ)) with Histone Deacetylase (HDAC) inhibitors have shown very promising results in pre-clinical MM models. HDAC6, a Class II HDAC, has been linked to the activity of aggresomes that degrade unfolded and misfolded ubiquitinated proteins. 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.
Here we investigated the preclinical activity of an HDAC6 selective inhibitor ACY-1215 in MM, either alone or in combination with BZ. In vitro enzyme assays showed that ACY-1215 has potent inhibitory activity against HDAC6 (IC50 0.0054 mM) 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 μM. To investigate the specific inhibitory effect of ACY-1215 on HDAC6 activity, we evaluated its effect on acetylation of a-tubulin. ACY-1215 induces potent acetylation of a-tubulin at low doses and triggers acetylation of lysine on histone H3 and histone H4 only at much higher doses, confirming its selective inhibitory effect on HDAC6 activity. Importantly, this selective inhibition was also observed in patient MM cells, where ACY-1215 increased acetylated a-tubulin after 4 h of treatment. We next combined low doses of ACY-1215 with BZ and showed synergistic anti MM activity, resulting in apoptosis via caspase-3, -8, -9 and poly (ADP) ribosome polymerase activation. Moreover, the combination of ACY-1215 plus BZ increased the accumulation of polyubiquitinated proteins compared to either agent alone. To investigate the effect of ACY-1215 on aggresome formation, MM.1S cells treated with ACY-1215 1 μM and/or BZ 2.5 nM for 12 h were stained with immunofluorescent anti-ubiquitin antibody. BZ-treated cells showed perinuclear structures consistent with aggresome formation, which was disrupted when BZ and ACY-1215 were combined. This result supports the synergistic anti MM activity of ACY-1215 with BZ.
We also evaluated the in vivo anti-MM effect of combination therapy using two different xenograft models in SCID mice: plasmacytoma model and disseminated MM model. ACY-1215 in combination with BZ triggered more significant anti-MM activity than either agent alone in suppressing tumor growth and prolonging host survival in both models, without significant adverse effects.
To optimize the design of future clinical trials, we conducted pharmacokinetic and pharmacodynamic studies in our plasmacytoma model. ACY-1215 peak plasma levels were observed at 4 h, which were unaffected by the addition of BZ. To further characterize the activity of ACY-1215 against HDAC6 in vivo, we evaluated the acetylation of α-tubulin in mouse blood cells by flow cytometry. The maximum levels of blood cell α-tubulin acetylation were observed at 4 h, providing an important biomarker for future clinical trials. Importantly, levels of acetylated α-tubulin were also detected in tumor tissue from treated mice in a similar time frame to peak blood levels, suggesting that ACY-1215 is readily absorbed by tumor tissue. Moreover, ACY-1215 did not accumulate in tumor tissue, as shown by the decline of acetylated α-tubulin in blood cells and tumor tissue by 24 h post-dose, which parallels the elimination of ACY-1215 from blood. We further confirmed the HDAC6 selectivity of ACY-1215 in our in vivo models by investigating the effect of the drug combination on histone acetylation in tumor tissue. WB analysis and IHC did not show a significant increase in acetylated histone H3 (lys 18), while demonstrating a robust acetylation of α-tubulin, the primary marker of HDAC6 inhibition by ACY-1215 at the cellular level.
The results from our in vitro and in vivo studies therefore show significant and synergistic anti-MM activity of ACY-1215 in combination with BZ and provided the rationale for the ongoing phase I/II clinical trial in patients with relapsed or relapsed/refractory MM. Moreover, our pharmacodynamic helped inform the design of correlative studies, which will establish whether acetylated α-tubulin can be used as predictive biomarker of HDAC6 inhibition and disease response.

Disclosures:
Hideshima: Acetylon: Consultancy. Kung:Acetylon Pharmaceuticals, Inc.: Consultancy. Tamang:Acetylon Pharmaceuticals, Inc.: Employment. Yang:Acetylon Pharmaceuticals, Inc.: Employment. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment. Mazitschek:Acetylon Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees. Ogier:Acetylon Pharmaceuticals, Inc.: Employment. Bradner:Acetylon: Consultancy. Anderson:celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Acetylon Pharmaceuticals Inc: founder. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.