Ricolinostat (ACY-1215), a Selective HDAC6 Inhibitor, Alone and in Combination with Bendamustine Is Effective in Preclinical Studies in Lymphoma Cell Lines

Background. Histone deacetylase inhibitors (HDACi) have emerged as a new class of anticancer agents, targeting the biological processes including cell cycle and apoptosis. The discovery of isoform selective compounds may offer a therapeutic advantage by minimizing toxicity. Ricolinostat (ACY-1215) inhibits HDAC6, resulting in tubulin hyperacetylation. Previous studies have investigated the synergistic effects of the combination of HDACis and alkylating agents offering new therapeutic strategies. The aim of this in vitro study was to investigate the activity of ricolinostat alone and in combination with bendamustine in a panel of non-Hodgkin's lymphoma (NHL) cell lines.

Methods. Ricolinostat was kindly provided by Acetylon Pharmaceuticals (Boston). NHL cell lines (WSU-NHL, RL, Granta-519, Jeko-1, Hut-78 and Karpas-299) were treated with ricolinostat and bendamustine alone and in combination. The interaction between the drugs was evaluated by the Chou-Talalay method and cell viability and clonogenicity were also evaluated. Apoptosis, ROS generation, Bcl-2 protein expression, cell cycle progression and tubulin expression were determined by flow cytometry. The effects of ricolinostat alone and in combination on caspases, PI3K/Akt, ER stress and UPR pathways were assessed by immunoblotting.

Results. Ricolinostat was able to affect the cell viability in a dose dependent manner, with IC₅₀ values ranging from 1.51 to 8.65 μM. Ricolinostat with bendamustine synergistically induced anti-proliferative and pro-apoptotic effects in lymphoma cells, even in the presence of the bone marrow microenvironment. Combination treatment decreased the percentage of viable peripheral blood mononuclear cells (PBMCs) from patients with lymphoma but had minimal or no cytotoxic effect on PBMCs from healthy donors. Clonogenic assay revealed that the drug combination significantly inhibited the colony formation compared with the drugs alone. Drug combination reduced the proportion of cells in the G0/G1 and S phases and caused an increase of "sub-G0/G1" peak with a decrease of cyclin D1 and cyclin E and an increase of p21 and p27 proteins. The synergistic effect was accompanied by increased reactive oxygen species (ROS) generation, which is linked to a decrease of thioredoxin-1 (Trx1) expression, activation of caspase -8, -9, and -3, the cleavage of PARP and modulation of the Bcl-2 protein family. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT. In addition, the exposure of ricolinostat induced the acetylation level of α-tubulin, the extent of which was not further modified by bendamustine. The direct cytotoxicity of ricolinostat/bendamustine may be mediated by an effect on microtubule stabilization. Finally, ricolinostat alone induced a significant down-regulation of IL-10 that was especially evident in WSU-NHL with a fold decrease of 6.6 compared to control. The drug combination affected IL-10 production in all three cell lines with a fold decrease of 5.77 in WSU-NL; 11.5 in Hut-78; and 10.9 in Jeko-1 cells compared with ricolinostat alone.

Conclusion. These preclinical studies suggest that bendamustine in combination with epigenetic therapy, such as ricolinostat, may be a promising treatment regime for managing lymphomas.