Co-Targeting Jak Signaling Pathway and Histone Deacetylases Produces Synergistic Activity in Haematologic Malignancies Cell Lines

Background and Objectives. Jak pathway is abnormally activated in multiple hematologic malignancies. Ruxolitinib, a JAK1/2 inhibitor, is currently prescribed in the clinical practice for the treatment of myelofibrosis; it is orally available and has a very manageable toxicity profile. Aberrant expression and activity of HDACs are also implicated in several types of tumors and HDAC inhibitors are under investigation in vitro and in clinical trials for the treatment of cancer. Vorinostat is a pan HDAC inhibitor with recognized efficacy in hematological disease, and ongoing studies are evaluating the best combination. Based on these considerations we screened a series of hematologic malignancies cell lines, in order to explore whether low doses of Ruxolitinib and Vorinostat might identify a new possible drug combination, with low toxicity profile. Methods. We tested Ruxolitinib and Vorinostat alone and in combination in 12 cell lines: Hodgkin Lymphoma (L1236, L540), B cell lymphomas (RPMI, WSU-NHL, Karpas422, RL) mantle cell lymphoma (Granta519, Jeko1), multiple myeloma (U266, RPMI8266), chronic limphocytic leukemia (MEK1), anaplastic lymphoma (Karpas299) and cutaneous T cell lymphoma (HUT78). The synergism was assessed by Chou-Talalay method. Effect of the treatment on cell cycle, apoptosis, caspase activation and ROS generation was evaluated by flow cytometry. Jak expression, apoptosis-regulating proteins and the phosphorylation status of protein kinases were studied by western blot analysis. Co-coltures with bone marrow stromal cells were also performed. Results. At 24h of treatment with ruxolitinib all the cell lines tested showed an homogeneous pattern of response, with IC50 around 20 nM; at 48h IC50 ranged from 0.1 to 2nM, with 4 cell lines more sensitive to the treatment (WSU-NHL, Karpas422, RL, HUT78) compared with the rest. The treatment with vorinostat induced an homogeneous response, both at 24h (IC50=40 uM) and 48h (IC50=5-10 uM). The combination treatment with a ratio R:V=0.1 nM:1 uM and 0.5nM:5uM showed a various range of results: a clear synergistic interaction of Ruxolitinib and Vorinostat was observed at 24h using low concentrations of two compounds in L1236, RL, RPMI, MEK1 and Karpas 299. There was no synergistic effects in mantle cell lymphoma cell lines, and a minor synergism in Karpas 422, a cell line that is known for carrying both t:(14;18) and t:(4;11) chromosomal translocation. The effect of the combination was additive on the remaining cell lines. The results were similar at 48 hours of treatment. The combination induced a marked increase in G2-M arrest and enhanced cell apoptosis in all cell lines. Accordingly the expression of apoptosis-regulating proteins, in particular BIM and Bad (SET-2), were up-regulated with down-regulation of Bcl-2 and Mcl-1 (HEL). Particularly evident was the effect of the combination on autophagy with decreased expression of p62 and a dramatic reactive oxygen species (ROS) accumulation in the cell lines in which the combination of the two drugs resulted highly synergistic. In addition, cell lines more sensitive to the combination of the two drugs showed a greater effect of the treatment on phosphorylation status of Jak, STAT3, STAT5, Akt and mTOR proteins. By co-culturing cells lines and primary BMSCs, we observed that Ruxolitinib and Vorinostat alone did not exert their anti-tumour activity in some cell lines. Conclusions. This study showed that the combination of Ruxolitinib and Vorinostat provokes significant changes in cell viability, apoptosis, cell cycle arrest, autophagy and ROS generation in several cell lines of hematologic malignancies alone and co-cultured. Because the bone microenvironment plays such an important role in the resistance to conventional therapies, the ability of Ruxolitinib combined with Vorinostat to overcome these factors is encouraging. Collectively, these findings create a compelling rationale to determine the in vivo activity of Ruxolitinib/Vorinostat combination.