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Background: Accumulating evidence reveals effectiveness of histone deacetylase (HDAC) inhibition in various types of cancer, and resminostat is a novel and oral HDAC inhibitor. In this study, we investigated the preclinical activity of resminostat in human non-small-cell lung cancer (NSCLC) cells, especially in combination with docetaxel (DTX). Materials and Methods: Anti-proliferative activity of resminostat was evaluated in 9 human NSCLC cell lines in vitro. Monolayers of cultured cells were exposed to resminostat and cell viability was determined. The amount of p21, acetylated histone H3 and α-tublin were analyzed by Western blotting in NCI-H460 cells exposed to resminostat. In addition, caspase-3 activity was measured. In a drug combination study, cells were simultaneously exposed to resminostat and DTX, and the synergistic effects of this drug combination were analyzed using the Combination Index, calculated by the median-effect method. For in vivo studies, a suspension of NCI-H460 cells was subcutaneously inoculated into the flank of male nude mice. The antitumor efficacies of oral resminostat alone or in combination with intravenous DTX were evaluated in this NSCLC xenograft model. Results: Resminostat exhibited antiproliferative activity in all tested cell lines. Sensitivity to resminostat was not influenced by the genetic status of KRAS among the cell lines (IC50 from 9 cell lines: 0.4-8.7 μmol/L). The treatment of cells with resminostat alone increased the levels of total p21 protein and acetylated histone H3. α-tublin was also acetylated by resminostat treatment and the level of acetylation was enhanced by the combination treatment with DTX. The amount of polymerized α-tubulin was increased by DTX treatment and this effect was greater in the combination treatment with resminostat than with DTX alone. Caspase-3 was more significantly activated by the combination treatment than with each mono-treatment. The Combination Index showed synergistic or additive effects in the resminostat plus DTX combination. The antitumor activities of resminostat were demonstrated in NCI-H460 tumor-bearing mice, where an up-regulation of p21 was accompanied by acetylation of histone H3 in the tumor tissues. The tumor growth inhibition ratio (IR) in the combination treatment was 2.6 and 1.8 times higher than with DTX alone or with resminostat alone, respectively, following a tolerable drug application schedule. Conclusion: These results demonstrated the potent activity of resminostat in NSCLC. The combination of resminostat and DTX appears to be a promising regimen to be tested in clinical trials in NSCLC patients. Based on this study, we initiated a phase I/II study in NSCLC patients to examine the safety and efficacy of this combination. Citation Format: Akimitsu Takagi, Hiroaki Konishi, Momomi Tsugane, Keisuke Taniguchi, Hiroyuki Takahashi, Yu Inutake, Akinobu Watanabe, Hiroshi Kodaira, Takeshi Matsuzaki. A novel and oral histone deacetylase inhibitor, resminostat, effectively suppresses the growth of non-small cell lung cancer in a xenograft mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5534. doi:10.1158/1538-7445.AM2014-5534

Background: Perifosine is an oral anticancer compound that inhibits the PH-domain of AKT, which is frequently activated in human cancers including gastric cancer. In this study, the mode of action of perifosine, especially in association with mTOR, and the antitumor activity in combination with paclitaxel were examined in human gastric cancer cell lines. Materials and Methods: The following human gastric cancer cell lines were used in this study: MKN1, MKN45, MKN74, AGS, and NCI-N87. Monolayers of cultured cells were exposed to perifosine for 48 hours and the antiproliferative activity was measured. The phosphorylated (p-)AKT status and its downstream molecules (e.g., PRAS40, TSC2, mTOR, 4E-BP1, and p70S6K) were analyzed using immunoblotting. In perifosine-treated cells, the induction of autophagy-like cell death was detected using LC3. With or without perifosine, the p-AKT (S473) levels were examined in rapamycin-treated cells. In a drug combination study, perifosine was used in combination with paclitaxel, and the synergistic effects of these drugs were quantified using the median-effect method to estimate the Combination Index. The antitumor efficacy of daily oral perifosine alone or in combination with intravenous paclitaxel was evaluated at various doses for 4 weeks in xenografted mice. The tumors at the time of necropsy were immunohistochemically examined for TUNEL, Ki-67 and AKT signal-related molecules. Results: Perifosine exhibited antiproliferative activity in the tested cell lines (GI50: 4.7-18.0 μmol/L), reducing p-AKT. The perifosine-induced inhibition of phosphorylation was also seen in molecules downstream of AKT, such as PRAS40, mTOR, and p70S6K. Perifosine also induced autophagy-like cell death, observed as the induction of type II LC3 along with cleaved PARP. The phosphorylation of AKT (S473) was induced by rapamycin treatment. While this phosphorylation was not induced by perifosine, the rapamycin-dependent phosphorylation of AKT was inhibited by perifosine. The Combination Index showed synergistic or additive effects when perifosine was used in combination with paclitaxel. The antitumor activities of perifosine were demonstrated in a dose-dependent manner in the 3 cell lines tested in mice. When perifosine was administered with paclitaxel to NCI-N87 cells in mice, the inhibitory ratio of the tumor increased to 78.8% from 61.7% for perifosine monotherapy and from 50.0% for paclitaxel monotherapy. The population of TUNEL-positive cells was higher in the combination therapy group than in the monotherapy group in the tumor tissues. This combination decreased the number of positive cells with Ki-67 and AKT signal molecules in the tissue. Conclusion: These results demonstrated that perifosine downregulates the mTOR function. The combination of perifosine and paclitaxel appears to be a promising regimen for the treatment of gastric cancer. Citation Format: Akimitsu Takagi, Momomi Tsugane, Hiroaki Konishi, Satoru Ishii, Ken-ichi Sata, Hiroshi Kodaira, Takeshi Matsuzaki. Perifosine, an AKT inhibitor, downregulates mTOR function and suppresses the growth of human gastric cancer cells in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3994. doi:10.1158/1538-7445.AM2013-3994