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. 2012 Mar 15;119(11):2579-89.
doi: 10.1182/blood-2011-10-387365. Epub 2012 Jan 19.

Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma

Loredana Santo  1 , Teru Hideshima, Andrew L Kung, Jen-Chieh Tseng, David Tamang, Min Yang, Matthew Jarpe, John H van Duzer, Ralph Mazitschek, Walter C Ogier, Diana Cirstea, Scott Rodig, Homare Eda, Tyler Scullen, Miriam Canavese, James Bradner, Kenneth C Anderson, Simon S Jones, Noopur Raje
Affiliations

Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma

Loredana Santo et al. Blood. .

Abstract

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 α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.

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Figures

Figure 1
Figure 1
ACY-1215 selectively inhibits HDAC6. (A) Chemical structure of ACY-1215. (B) MM.1S cells were cultured with control medium or ACY-1215 (0-5μM) or SAHA (0-5μM) for 6 hours. MM.1S, MM.1R, and RPMI8226 cells were cultured with control medium or ACY-1215 (0.25 and 1μM) for 18 hours. (C) CD138+ patient cells were treated with control medium or ACY-1215 (2μM) for 4 hours. Whole-cell lysates were subjected to Western blotting using the indicated Abs. Increased acetylated α-tubulin was observed. CD138+ patient cells were fixed and double-stained for anti–human CD138+ and acetylated α-tubulin (left panel) and for anti–human CD138+ and acetyl-histone H3 (right panel). A significant increase in acetylation of α-tubulin was observed, whereas no significant difference was observed in the acetyl-histone H3 in the treated sample compared with control. (D) PBMCs from 4 healthy donors were stimulated with PHA and cultured with increasing doses (0-4μM) of ACY-1215 and SAHA for 48 hours. Cell growth was assessed by MTT assay (left panel). CD4+ T cells purified from human blood were stimulated by CD3/CD28 Dynabeads for 7 days in the presence of compounds. Cell viability was assessed using alamarBlue (right panel)
Figure 2
Figure 2
ACY-1215 induces dose-dependent cytotoxicity in MM cells. (A) ACY-1215 decreases MM-cell viability in a dose-dependent manner. Cells were treated with increasing doses of ACY-1215 (0-8μM) for 48 hours, and cell viability was measured by MTT assay (left panel). ANBL-6.BR cells were treated with increasing doses of ACY-1215 (0.4μM) and bortezomib (0-5nM) to show their bortezomib resistance for 48 hours; cell viability was measured by MTT assay (right panel). (B) CD138+ patient MM cells (patients 1-4) were similarly tested in cytotoxicity assays (MTT) at 48 hours. (C) MM.1S cells were cultured for 48 hours with ACY-1215 (0-4μM) in the presence or absence of BMSCs (left) and in the presence or absence of IL-6 (10 ng/mL) and IGF-1 (50 ng/mL). 3H-thymidine incorporation was measured during the last 8 hours of incubation to measure DNA synthesis.
Figure 3
Figure 3
ACY-1215 in combination with bortezomib induces synergistic anti-MM activity. (A) MM.1S and RPMI8226 cells were cultured with bortezomib in the presence or absence of ACY-1215 (0-4μM) for 24 and 48 hours. Cytotoxicity was assayed by MTT. The isobologram analysis confirms the synergistic effect. (B) MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 24 hours, followed by annexin/PI staining and flow cytometry analysis (left). MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 16 hours. Whole-cell lysates were immunoblotted with the indicated Abs. (C) MM.1S cells were treated with ACY-1215 (3μM), bortezomib (2.5nM), or combined therapy for 24 hours. Whole-cell lysates were immunoblotted with the indicated Abs. (D) MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 12 hours. Cells were fixed and stained with 4′,6-diamidino-2-phenylindole (blue) and antiubiquitin Ab. The arrow indicates the ubiquitin-conjugated proteins. (E) MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 4 hours. Whole-cell lysates were immunoblotted with the indicated Abs. XBP-1 splicing was determined by WB and RT-PCR.
Figure 4
Figure 4
ACY-1215 in combination with bortezomib induces significant anti-MM activity in vivo. (A) CB17 SCID mice were treated with saline (n = 7), ACY-1215 (50 mg/kg n = 7), bortezomib (0.5 mg/kg, n = 6), or the combination of ACY-1215 plus bortezomib (n = 7) for 3 weeks. Tumor growth was significantly inhibited in the combination-treated group compared with controls (P < .0001). (B) Using Kaplan-Meier and log-rank analysis, the median OS of animals treated with combination therapy was significantly prolonged (22 days in the control group vs 34 days in the treated group, P < .0011). WB analysis of tumors taken from mice after 3 days of treatment showed a significant accumulation of polyubiquitinated proteins in the group treated with the combination of ACY-1215 plus bortezomib compared with either agent alone (bottom panel). (C) Treatment with ACY-1215 or ACY-1215 plus bortezomib did not significantly affect the body weight of the animals. (D) SCID-beige mice were inoculated intravenously with MM.1S-LucNeo cells and then treated with saline (n = 10), ACY-1215 (n = 10), bortezomib (n = 10), or the combination of ACY-1215 plus bortezomib (n = 10) for 2 weeks. Combined treatment with ACY-1215 and bortezomib induced significant suppression of tumor growth, as demonstrated by bioluminescence imaging (log scale). (E) Treatment with ACY-1215 and ACY-1215 plus bortezomib induces between 4% and 12% of body weight loss. (F) Combined treatment with ACY-1215 and bortezomib significantly prolonged survival (17 days in the control group vs 40 days in the combination-treated group, P < .0001).
Figure 5
Figure 5
ACY-1215 PK/PD profile. (A) CB17 SCID mice were treated with saline, ACY-1215, bortezomib, or the combination of ACY-1215 plus bortezomib for 3 days. ACY-1215 levels were detected in plasma collected from each animal at 1, 4, and 24 hours after the last dose. (B) The acetylation status of α-tubulin was assessed in mouse serum at 1, 4, and 24 hours after the last dose. The fold change in acetylation of α-tubulin relative to baseline was calculated. (C) Tumor was excised from each mouse at 1, 4, and 24 hours after the last dose. IHC analysis showed a significant increase in acetylation of α-tubulin staining at 1 and 4 hours after the last dose of ACY-1215 alone or in combination. (D) WB analysis confirmed a significant increase in acetylated α-tubulin at 1 and 4 hours after treatment with ACY-1215 alone or in combination.
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