Synergistic Activity of the Novel Src/Abl Inhibitor Bosutinib in Combination with Imatinib.
Authors: Redaelli Sara , Boschelli Frank , Viltadi Michela , Meneghetti Iris , Gambacorti Carlo 
Published: October 2019

Abstract
The oncogenic fusion protein Bcr-Abl is the underlying cause of Chronic Myeloid Leukemia (CML), and it is present in up to 35% of Acute Lymphoblastic Leukemia (ALL). The discovery of a selective Abl inhibitor, Imatinib mesylate, has revolutionized the treatment of CML. Recently, several new inhibitors have been developed with the aim of increasing both potency and selectivity against Abl. Bosutinib (SKI-606, Wyeth) is a dual Src/Abl inhibitor that showed an in vitro activity in the low nanomolar range on several BCR-ABL positive cells and it is, at present, in phase II clinical trials. Bosutinib is devoid of activity against some known “off-target” kinases blocked by imatinib, such as PDGFR and c-Kit. In addition structural and modelling data attribute to Bosutinib the ability to bind Bcr-Abl in the intermediate/active conformation, while Imatinib is able to bind only the inactive conformation of Bcr-Abl. In this study we analyzed in vitro the combination of Imatinib and Bosutinib in Bcr-Abl expressing cell lines to evaluate the possibility to decrease dosage of both drugs, increasing or maintaining the same efficacy but avoiding toxic effects. Combination effects were evaluated according to the method of Chou and Talalay, in which the combination index (CI) value is calculated for a combination of two drugs and allows the quantification of synergism: CI <1, =1 or >1 indicate synergistic, additive or antagonistic interactions, respectively. Proliferation assays on a panel of Imatinib-sensitive and Imatinib-resistant BCR-ABL positive cell lines were performed. Cells were treated with Imatinib and Bosutinib as single agents or in three ratio combinations (1:3, 1:10, 1:33 in favour to Imatinib) across a wide range of concentrations. Combination indexes (CI) calculated at IC50, IC75 and IC90 for K562 cells (Imatinib-sensitive), suggest a synergistic to very strong synergistic effect (CI= 0.01-0.53). Similarly, in KCl22, KU812 and Lama84 cells (Imatinib-sensitive) moderate to strong synergistic effects were observed. A slight to moderate synergism was also obtained in three Imatinib-resistant cell lines tested: Lama84R (CI=0.63-0.88), K562R (CI=0.63-0.82) and KCL22R (CI=0.62-0.92). Western blot analysis of the tyrosine phosphorylation status of K562S cells treated with a mixture of 100nM Imatinib and 10nM Bosutinib revealed a substantially more pronounced inhibition compared with either 100nM Imatinib or 10nM Bosutinib alone. The effect of the combination was also assessed in murine Ba/F3 cells transfected with either wild type (WT) or mutated forms of BCR-ABL. Parental Ba/F3 cells were not affected by the presence of both drugs, while in Ba/F3 BCR-ABL WT the CI ranged from 0.49 to 0.85, indicating moderate synergism. The combination of Imatinib and Bosutinib inhibited the growth of Ba/F3 BCR-ABL Y253F with a slight synergism (CI 0.77-0.87). No synergistic effect was observed on Ba/F3 BCR-ABL E255K and on the highly resistant T315I mutant. Fresh leukemic cells obtained from three CML patients were also studied. In these samples synergistic effects between Bosutinib and Imatinib were confirmed (CI=0.52, 0.73, 0.62). The different binding modes of Imatinib and Bosutinib may justify the synergistic effect observed in the CML lines. This results support a possible therapeutic advantage for the combination of Bosutinib and Imatinib against Philadelphia positive leukemias.