Ruxolitinib in Combination with Vorinostat Suppresses Tumor Growth and Alters the Metabolic Phenotype in Haematological Diseases

Background and Objectives Jak-2 dysregulation has a significative role as an oncogenic driver and as a prominent therapeutic target in hematological malignancies. Ruxolitinib is pyrrolo[2.3-d]pyrimidine derivative with inhibitory activity against JAK1 and JAK2, moderate activity against TYK2 and minor activity against JAK3. Vorinostat is an HDAC inhibitor able to reduce Jak-2 expression affecting Jak-2 mRNA expression and increasing Jak-2 proteasomal deterioration. In this study we hypothesized that Ruxolitinib and Vorinostat combination could have a synergistic effects on haematological disease. Methods. We tested Ruxolitinib and Vorinostat alone and in combination for 24 and 48 hrs in 12 lymphoma and myeloma cell lines. The synergism was assessed by Chou-Talalay method. The cell cycle, the apoptosis, the caspase activation, the autophagy, the interleukin expression, the ROS generation, the ATP levels, the lactate, the phosphorylation status of protein kinases were evaluated in all 12 cell lines after 24 hrs. Co-coltures with bone marrow stromal cells were also performed after 24 to 120 hrs of treatments. Results. Treatment of 12 cell lines with low doses of Ruxolitinib and Vorinostat as single agents for 24h was rather ineffective at blocking cell growth if compared with the efficacy of the combination of the two drugs. The combination was additive in two lines of mantle cell lymphoma, in one line of Hodgkin lymphoma, in one line of myeloma, in one line of T cell lymphoma and in on line of B cell lymphoma. In the remaining cell lines the combination of the two drugs was highly synergistic. This synergistic cytotoxic action was maintained over time up to 120 hrs also in the presence of stromal cells. Ruxolitinib and Vorinostat combination influenced the cell cycle distribution increasing G2-M phase arrest in all 12 cell lines after 24 hrs. The levels of p21 and p27 resulted up-regulated while the levels of Cyclin D and Aurora A were downregulated in all 12 cell line after combined treatment. The apoptotic AnnexinV-positive cells after 24 hrs of treatment with Ruxolitinib or Vorinostat alone was less than 10%. However, the percentage of apoptotic cells grew up to 50 % after treatment of the cells with Ruxolitinib in combination with Vorinostat. After 24 of combined treatment, we detected a cleavage of caspase 8 and caspase 3 in all 12 cell line, suggesting activation of the extrinsic apoptotic pathway. Quantification of caspase 9 in all 12 cell lines has resulted in observation of an increase in cleaved form only in the six most sensitive cell lines, suggesting a caspase activation mitochondrial events regulating the intrinsic apoptosis. Bax, Bid and BAD levels increased in all 12 cell lines treated with combined drugs, but apoptotic inhibitors BLC-2 and MCL-1 were down regulated only in the 6 more sensitive cell lines. Our experiment showed that Ruxolitinib in combination with Vorinostat reduced IL-10 and IL-6 secretion in all 12 cell lines in a comparable way. The expression of IL-17A after combined treatment was significantly decreased in only the 6 most sensitive cell lines. Ruxolitinib and Vorinostat alone and in combination induced a decrease of the amount of autophagy-related protein p62 in cancer cells, indicating the activation of autophagic system. Autophagy and apoptosis increased the oxidative stress leading to ROS production and in this work, we showed that Ruxolitinib and Vorinostat synergistically induce apoptosis in 6 more sensitive cell lines, associated with a marked increase in ROS generation. Interestingly, our data have shown that combined therapy alters tumor cell metabolism by reducing the glycolic state by causing greater sensitivity of the six cell lines to the combined treatment. Conclusions. In conclusions, the combination of Ruxolitinib and Vorinostat is able to decrease glucose metabolism and this partial reversion of Warburg effect is associated to ROS production, apoptosis and cell growth inhibition. The drugs tested under different cell culture conditions provide informations on drug efficacy and could help predict pharmacological effects that may be difficult to translate from in vitro and ex vivo conditions to the clinic. This study gives evidence of synergistic interaction between Ruxolitinib and Vorinostat in lymphoid cells and provides the rationale for clinical studies with the combination of both agents in patients.