Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated ␣-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated ␣-tubulin use as a pharmacodynamic biomarker in future clinical trials.

Purpose Histone deacetylase (HDAC) inhibition improves the efficacy of proteasome inhibition for multiple myeloma but adds substantial toxicity. Preclinical models suggest that the observed synergy is due to the role of HDAC6 in mediating resistance to proteasome inhibition via the aggresome/autophagy pathway of protein degradation. Experimental Design We conducted a phase 1/2 trial of the HDAC6-selective inhibitor ricolinostat to define the safety, preliminary efficacy, and recommended phase 2 dose in combination with standard proteasome inhibitor therapy. Patients with relapsed or refractory multiple myeloma received oral ricolinostat on days 1–5 and 8–12 of each 21-day cycle. Results Single agent ricolinostat therapy resulted in neither significant toxicity nor clinical responses. Combination therapy with bortezomib and dexamethasone was well tolerated during dose escalation but led to dose-limiting diarrhea in an expansion cohort at a ricolinostat dose of 160 mg twice daily. Combination therapy at a ricolinostat dose of 160 mg daily in a second expansion cohort was well tolerated, with less severe hematologic, gastrointestinal, and constitutional toxicities compared with published data on non-selective HDAC inhibitors. The overall response rate in combination with daily ricolinostat at ≥160 mg was 37%. The response rate to combination therapy among bortezomib-refractory patients was 14%. Samples taken during therapy showed dose-dependent increases of acetylated tubulin in peripheral blood lymphocytes. Conclusions At the recommended phase 2 dose of ricolinostat of 160 mg daily, the combination with bortezomib and dexamethasone is safe, well tolerated, and active, suggesting that selective inhibition of HDAC6 is a promising approach to multiple myeloma therapy.

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Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A3 is a neutrophil chemo-attractant produced by pathogen infected epithelial cells hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with P. aeruginosa, we explored the role of hepoxilin A3 in neutrophil trans-epithelial migration. Pharmacological inhibitors of enzymatic pathways necessary to generate hepoxilin A3, including phospholipase A2 and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil trans-epithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A3 precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A3 are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A3 and lipid derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A3 serving a chemotactic role. Stable inhibitory structural analogues of hepoxilin A3 are capable of impeding P. aeruginosa-induced neutrophil trans-epithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A3 and 12-HETE. Data generated from multiple models herein provide further evidence that hepoxilin A3 is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.