Inhibition of Related JAK/STAT Pathways with Molecular Targeted Drugs Shows Strong Synergy with Ruxolitinib in Chronic Myeloproliferative Neoplasms

Abstract

AbstractAbstract 5054 Objectives:In the present study we have determined the antitumor effects, molecular mechanisms of action and potential synergies between Ruxolitinib and the molecular targeted drugs Sorafenib, KNK437, Dasatinib and Perifosine, in Philadelphia negative chronic myeloproliferative neoplasms (MPN). Materials and methods:The cytotoxic and cytostatic effects of the different compounds were determined in the JAK2V617F positive cell lines HEL and Ba/F3 JAK2V617F EPOR, and in mononuclear and bone marrow CD34 positive cells from 19 MPN patients. The effects of different drugs at the molecular level were analyzed by flow cytometry and western blot. The IC50 and the synergy combination index (CI) were determined with GrafPath Prism software and Calcusyn software respectively. Results:Ruxolitinib (IC50PV=15nM), as well as Sorafenib (IC50PV=8uM), KNK437 (IC50PV=100uM) and Perifosine (IC50PV=15uM), was able to inhibit proliferation in cell line models and in cells from MPN patients. Dasatinib (IC50PV=12nM), however, was only effective in patient samples, with similar effects in both MPN patients and control donors. Moreover, Dasatinib, KNK437 and Sorafenib showed a strong synergistic effect in combination with Ruxolitinib (CIPV<0. 3, table 1). The Western blot analysis confirmed that Sorafenib inhibited the activation of ERK and P38, and, as a consequence, of STAT5. Ruxolitinib blocked the ERK and STAT5 activation, Dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the protein JAK2, reducing its expression. Conclusions:The blockage of JAK2 related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of Ruxolitinib with inhibitors targeted against these pathways has a strong synergistic effect. This may be related to a decrease in the activation of the final common effector STAT5.Table 1:IC50 and Synergic effect for the different compounds.RuxolitinibSorafenibKNK437DasatinibIC50(nM)IC50synCIIC50(uM)IC50synCIIC50(uM)IC50synCIIC50(nM)HEL274.4249.91.07b3.876.670.41b17.1111.10.30c-BAF3mut157.558.910.60b8.4943.110.22c313.259.130.31b-BAF3wt137.421.110.43b2.3729.430.39c79.842.080.33b-PV15.50.530.052a8.111.030.251a1000.350.14a11.68(CI>1. 1 Antagonists. 1. 1<CI<0. 9 Additive effect. 0. 9<CI<0. 3, Synergic effect. CI<0. 3, strong synergism). [Ruxolitinib]:a10nM,b50nM,c100nM. The IC50synergy was determinate to Ruxolitinib in the presence of constant concentrations of Sorafenib (1uM), KNK437 (1uM) y Dasatinib (1nM). Disclosures:No relevant conflicts of interest to declare.