Combined Effects of a Pan-Aurora Kinase Inhibitor MK-0457 and Dasatinib Against T315I Mutant Form of BCR-ABL: In Vitro and In Vivo Studies.

Abstract

MK-0457 is a pan-Aurora kinase inhibitor that has firstly shown clinical activity in BCR-ABL positive leukemia patients harboring the T315I mutation. Combining MK-0457 with ABL kinase inhibitors may provide several advantages, including enhanced efficacy and the potential to reduce the emergence of new resistant mutations. In the present study, we investigated the combined effects of MK-0457 and dasatinib in T315I mutant-expressing cells. Co-treatment with MK-0457and dasatinib caused significantly more inhibition of colony growth of primary leukemia cells and BaF3 cells expressing T315I BCR-ABL than either drug alone. In contrast, we did not observe the enhanced effects of MK-0457 and imatinib in T315I BCR-ABL-expressing cells. Treatment with 5 μM of MK-0457 for 48 hrs induced apoptosis in T315I BCR-ABL BaF3 cells, whereas, exposure to combination with 1.0 μM of MK-0457 and 50 nM of dasatinib exerted enhanced apoptotic effect. Combined treatment with MK-0457 and dasatinib also associated with more PARP cleavage, which is due to increased activation of caspase-3 and caspase-9 during apoptosis. Following co-treatment with MK-0457 and dasatinib caused more attenuation of the level of phospho-Stat5 and the downstream signal transducer, including Bcl-XL, Mcl-1, and cyclin D1. We also observed that combined treatment with MK-0457 and dasatinib enhanced the activation of p38MAP kinase and MAPKAP Kinase-2 (MK2). Pretreatment with p38MAP kinase or MP2 siRNA in T315I BCR-ABL BaF3 cells reduced the induction of apoptosis after MK-0457 and dasatinib exposure. These results suggest that p38MAP kinase and MK2 have a critical role for the induction of apoptosis after MK-0457 and dasatinib exposure. To assess the mechanism of combination effect between MK-0457 and dasatinib on T315I BCR-ABL-expressing cells, we used RNA interference to determine whether reduction of SRC-family kinases affected the growth inhibition. BaF3 cells expressing T315I BCR-ABL pretreated with Lyn or Hck siRNA showed enhanced growth inhibition with MK-0457. These results demonstrate that the enhanced growth inhibition by MK-0457 and dasatinib in T315I-expressing cells may be mediated by Lyn and Hck. To assess the in vivo efficacy of MK-0457 and dasatinib, athymic nude mice were injected i.v. with BaF3 cells expressing T315I mutant form of BCR-ABL. 24 hrs after injection, the mice were divided four groups (5 mice per group), with each group receiving either vehicle, MK-0457 (30mg/kg b.i.d.; ip for 5 days), dasatinib (10mg/kg q.d.; po for five days), MK-0457 (30mg/kg b.i.d.; ip for 5 days) + dasatinib (10mg/kg q.d.; po for five days). The treatment was repeated in every 3 weeks. The vehicle or dasatinib-treated mice died of a condition resembling acute leukemia by 28 days; the MK-0457 only-treated mice survived more than 56 days, and the combination of MK-0457 + dasatinib -treated mice survived more than 70 days. Histopathologic analysis of vehicle or dasatinib-treated mice revealed infiltration of the spleen and bone marrow with leukemic blasts. In contrast, histopathologic analysis of organs from MK-0457 plus dasatinib-treated mice demonstrated normal tissue architecture and no evidence of residual leukemia. Taken together, the present study shows that the combination of MK-0457 and dasatinib exhibits a desirable therapeutic index that can reduce the in vivo growth of T315I mutant form of BCR-ABL-expressing cells in an efficacious manner.