Effects Of Bosutinib (Ski-606) In Cml: Kinase Target Profile, Effects On Bcr/Abl Mutants, And Synergism With Dasatinib In T315i+Cells

Abstract Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50>1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50<0.5 nM) and most of the ABL mutants tested (H396P, M351T, Q252H, and Y253F) were all completely inhibited by both drugs at 1 μM in the kinase assay, the ABL T315I mutant was inhibited by bosutinib (IC50=26 nM) almost 70 times more potently than by dasatinib. Together, these data show that bosutinib and dasatinib synergize with each other in producing antileukemic effects on CML cells including BCR/ABL T315I+ subclones. These synergistic effects may be explained by differential target kinase profiles and by the fact that bosutinib retains some activity against the BCR/ABL T315I mutant kinase.