Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models

Abstract

ACY-241 is a novel, orally available and selective histone deacetylase (HDAC) 6 inhibitor in Phase 1b clinical development in multiple myeloma (NCT 02400242). Like the structurally related drug ACY-1215 (ricolinostat), ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drug candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. We now show that combination treatment of xenograft models with paclitaxel and either ricolinostat or ACY-241 significantly suppresses solid tumor growth. In cell lines from multiple solid tumor lineages, combination treatment with ACY-241 and paclitaxel enhanced inhibition of proliferation and increased cell death relative to either single agent alone. Combination treatment with ACY-241 and paclitaxel also resulted in more frequent occurrence of mitotic cells with abnormal multipolar spindles and aberrant mitoses, consistent with the observed increase of aneuploid cells. At the molecular level, multipolar mitotic spindle formation was observed to be NuMA-dependent and γ-tubulin independent, suggesting that treatment-induced multipolar spindle formation does not depend on centrosomal amplification. The significantly enhanced efficacy of ACY-241 plus paclitaxel observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor versus pan-HDAC inhibitors, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors.

Keywords: HDAC; mitotic spindle; paclitaxel; solid tumors.

Figure 1. ACY-241 selectively inhibits HDAC6

(A) Chemical structure of ACY-241. (B) A2780 cells were cultured with vehicle or a range of ACY-241 concentrations for 24 hours prior to immunoblotting. ACY-241 preferentially induced hyperacetylation of α-tubulin relative to H3K56.

Figure 2. Combination treatment with HDAC6 inhibitors and paclitaxel reduced tumor xenograft growth

Combination treatment of (A) TOV-21G and (B) A2780 ovarian cancer with ricolinostat plus paclitaxel resulted in significantly greater suppression of tumor growth relative to control and single agent treatments. Combination treatment of MiaPaCa-2 pancreatic cancer xenografts with ricolinostat (C) or ACY-241 (D) plus paclitaxel resulted in similar suppression of tumor growth. Shown is the mean tumor volume ± SD over the course of the treatment period. (E, F) Combination treatment of the MiaPaCa-2 model with ACY-1215 or ACY-241 did not result in body weight loss. Shown is the mean relative body weight change ± SD over the course of the treatment period.

Figure 3. Combination treatment with ACY-241 and paclitaxel reduced cancer cell proliferation

MiaPaCa-2, TOV-21G, or T47D cells were incubated with the indicated concentrations of ACY-241 and/or paclitaxel for 9 days. (A) Representative image of TOV-21G cell proliferation in response to treatment with each agent as assessed by crystal violet staining. (B) Relative proliferation was plotted as mean total crystal violet absorption of three independent experiments ± SD over time. *p < 0.05, **p < 0.005, and ***p < 0.0001

Figure 4. Reduction of S-phase population, induction of cell death and impact on DNA content after combination treatment with ACY-241 and paclitaxel

(A) Dual parameter Click-iT Plus Edu (y axis, DNA synthesis) and FxCycle Far Red (x axis, DNA content) staining in A2780 ovarian cancer cell cells indicate the percentage of cells in S-phase and SubG1 after 3 days of treatment with ACY-241, paclitaxel, or the combination. In addition to reduced S-phase and increased subG1 frequency, combination treatment results in an aneuploid cell population (DNA content distributed between 2 N and 4 N; red arrowhead). Percentage of cells in S-phase (B) or in the SubG1 population (C) was determined after treatment of A2780 or TOV-21G ovarian cancer (3 days) or MiaPaCa-2 pancreatic cancer cells (5 days) with the indicated concentrations of each agent. Shown is the mean ± SD of three independent experiments. *p < 0.05, **p < 0.005, and ***p < 0.0001

Figure 5. Combination treatment caused enhanced hyperacetylation of α-tubulin

Immunoblotting demonstrated enhanced hyperacetylation of α-tubulin after combination treatment with ACY-241 and paclitaxel in A2780 ovarian cancer and MDA-MB-231 breast cancer cells. A2780 and MDA-MB-231 cells were treated for 48 hours with the indicated concentrations of each drug and whole cell protein lysates were subjected to immunoblotting using antibodies for acetylated-α-tubulin and β-actin.

Figure 6. Combination treatment increased the frequency of multipolar mitotic spindle formation and abnormal nuclei

TOV-21G cells were treated with vehicle or ACY-241 +/− paclitaxel for 24 hours prior to fixation. (A) Staining for α-tubulin (green), NuMA (red), and DNA (blue) demonstrated that all multipolar spindles formed with NuMA-containing spindle poles. (B) The frequency of mitotic cells with multipolar spindles was scored in at least 50 cells from each treatment condition. Shown is the mean ± SD of three independent experiments. (C) Staining for α-tubulin (green), γ-tubulin (red), and DNA (blue) demonstrated that additional spindle poles formed in the absence of centrosomal γ-tubulin (red arrowheads). (D) TOV-21G cells were treated with vehicle, ACY-241, paclitaxel, or the combination for 48 hours prior to fixation. Microtubules and DNA were visualized by staining with anti-α-tubulin antibody (green) and DAPI (blue), respectively. The frequency of interphase cells with visually abnormal nuclei, including satellite nuclei and multinucleation, was scored in at least 100 cells per treatment condition. Shown is the mean ± SD of three independent experiments.