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Despite promising results from clinical studies of ABL kinase inhibitors, a challenging problem that remains is the T315I mutation against which neither nilotinib nor dasatinib show significant activity. In the present study, we investigated the activity of a novel Aurora kinase inhibitor, VE-465, against leukemia cells expressing wild-type BCR-ABL or the T315I mutant form of BCR-ABL. We observed a dose-dependent reduction in the level of BCR-ABL autophosphorylation in VE-465-treated cells. Exposure to the combination of VE-465 and imatinib exerted an enhanced apoptotic effect in K562 cells. Combined treatment with VE-465 and imatinib caused more attenuation of the levels of phospho-AKT and c-Myc in K562 cells. Further, the isobologram indicated the synergistic effect of simultaneous exposure to VE-465 and imatinib in K562 cells. To assess the in vivo efficacy of VE-465, athymic nude mice were injected intravenously with BaF3 cells expressing wild-type BCR-ABL or the T315I mutant form. The vehicle-treated mice died of a condition resembling acute leukemia by 28 days; however, nearly all mice treated with VE-465 (75 mg/kg, twice daily; intraperitoneally for 14 days) survived for more than 56 days. Histopathological analysis of vehicle-treated mice revealed infiltration of the spleen. In contrast, histopathological analysis of organs from VE-465-treated mice demonstrated normal tissue architecture. Taken together, the present study shows that VE-465 exhibits a desirable therapeutic index that can reduce the in vivo growth of T315I mutant form and wild-type BCR-ABL-expressing cells in an efficacious manner.

Dasatinib is an ATP-competitive, multi-targeted SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical standpoint, dasatinib is particularly attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant chronic myeloid leukemia patients. The fact because the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on wild-type p210 BCR-ABL-expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 h, and gene expression data were obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib-responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The distinct genes that are selectively modulated by dasatinib are cyclin-dependent kinase 2 (CDK2) and CDK8, which had a maximal reduction of

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.

Imatinib and dasatinib are two clinically active ABL kinase inhibitors that serve as paradigm for the study of emergence of resistance in targeted cancer therapy. We have developed the imatinib- or dasatinib-resistant cell lines. K562 cells and TF1 BCR-ABL cells were cultured with 0.1 μM of imatinib or 0.5 nM of dasatinib for 2 weeks, and then the concentration of imatinib or dasatinib was gradually increased. Finally imatinib-resistant K562-IMR cells and TF1 BCR-ABL-IMR cells can grow in the presence of 10 μM of imatinib. Also dasatinib- resistant K562-BMSR cells and TF1 BCR-ABL-BMSR cells can grow in the presence of 10 nM of dasatinib. These resistant cell lines do not have the point mutations of BCR-ABL kinase domain determined by PCR analysis. We used DNA microarrays to identify genes whose transcription was altered in imatinib- or dasatinib-resistant K562 cells. The gene expression data was obtained from three independent microarray hybridizations. We used 1.5-fold cut off which is a reliable limit for the identification of differentially expressed genes. Imatinib- or dasatinib-resistant K562 cells up-regulate the expression of several MAP kinase genes (MAP2K7, MAP3K1, and MAP3K7), apoptosis-regulated genes (BCL2L1, BCL6, and MCL1). Immunoblotting confirmed that the imatinib-resistant cell lines and dasatinib-resistant cell lines expressed the increased amount of BCR-ABL proteins compared to parental cell lines. p42/p44 MAP kinase is more activated in both inatinib-resistant cell lines and dasatinib-resistant cell lines. Up-regulation of Lyn kinase is observed in dasatinib-resistant cell lines but not in imatinib-resistant cell lines. Up-regulation of Bcl-XL and Mcl-1 is also observed in dasatinib-resistant cell lines. We determined the sensitivity of imatinib and dasatinib in these drug resistant cell lines. Cells were treated with 10 μM of imatinib or 5 nM of dasatinib for 72 hrs. The induction of apoptosis was analyzed by APO2.7. In TF-1 BCR-ABL cells, the treatment with 10 μM of imatinib or 5 nM of dasatinib effectively induced apoptosis. In imatinib-resistant TF1 BCR-ABL cells, treatment with 5 nM of dasatinib significantly increased the APO2.7-positive cells compared with imatinib-treatment. Also treatment with 10 μM of imatinib increased the apoptotic cells in BMS-resistant TF1 BCR-ABL cells. These results suggest that relatively high dose of imatinib or dasatinib still has biological activity in each compound resistant cells. By using these drug resistant cell lines, we determined the activity of ABT-737, an inhibitor of Bcl-2 family proteins. AST-737 is a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-w with high affinity (Nature435: 677, 2005). ABT-737 did not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with imatinib or dasatinib in these resistant cell lines. Treatment of 1μM of ABT-737 exhibited more sensitive in dasatinib-resistant K562 cells as single agent mechanism-based killing compared to parental K562 cells or imatinib-resistant K562 cells. Taken together, these finding indicate that co-treatment with ABT-737 enhances cytotoxic effects of imatinib or dasatinib and may have activity against imatinib- or dasatinib-resistant BCR-ABL-positive leukemia.

Imatinib is an ABL-specific inhibitor that binds with high affinity to the inactive conformation of the BCR-ABL tyrosine kinase and has been shown to be effective in the treatment of chronic myelogenous leukemia. Dasatinib is an ATP-competitive, dual-spesific SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical stand point, dasatinib is particular attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant CML patients. In the view of the fact that the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on a wild type p210 BCR-ABL expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 hrs, and gene expression data was obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib- responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The genes whose expression was affected by imatinib and dasatinib were categorized into different functional groups based on their biological function, and genes in the cell proliferation and apoptosis categories were examined in detail. Imatinib and dasatinib affected the expression of several cyclin-dependent kinases (CDK2, CDK4, CDK6, CDK8, and CDK9), cell division cycle genes (CDC6, CDC7, CDC25C, and CDC34), and cyclones (cyclin A2, C, D2, D3, E1, E2, F, G1, G2, and H). Imatinib and dasatinib also modulated the expression of apoptosis-related genes including APAF1, BAK1, BCL2, BCL10, MCL1, CASP3, and CASP6). One of the distinct genes which are selectively modulated by dasatinib are CDK2 and CDK8, which had a maximal fold reduction of