T315I-mutated Bcr-Abl in chronic myeloid leukemia and imatinib: insights from a computational study Sabrina Pricl, 1 Maurizio Fermeglia, 1 Marco Ferrone, 1 and Elena Tamborini 2 1 Molecular Simulation Engineering Laboratory, Department of Chemical Engineering, University of Trieste, Trieste, Italy and 2 Experimental Molecular Pathology, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy Abstract The early stage of chronic myeloid leukemia is triggered by the tyrosine kinase Bcr-Abl. Imatinib mesylate, a selective inhibitor of Bcr-Abl, has been successful in chronic myeloid leukemia clinical trials, but short-lived remissions are usually observed in blast crisis patients. Sequencing of the BCR-ABL gene in relapsed patients revealed a set of mutants that mediate drug resistance. Previously reported work postulated that the missense T315I mutation both alters the three-dimensional struc- ture of the protein binding site, thus decreasing the protein sensitivity for the drug, and does not feature a fundamental hydrogen bond that is critical for binding with imatinib. These speculations, however, were not supported by investigations at the molecular modeling level. Here, we present the results obtained from the application of molecular dynamics simulations to the study of the interactions between T315I Bcr-Abl and imatinib. For the first time, we show that, with respect to the wild-type system, the absence of the supposedly critical H-bond is not the only cause for the failure of receptor inhibition by imatinib, but also a plethora of other protein/drug interactions are drastically and unfa- vorably changed in the mutant protein. [Mol Cancer Ther 2005;4(8):1167 – 74] Introduction Chronic myeloid leukemia (CML) is a clonal disease involving the pluripotent hematopoietic stem cell com- partment and is associated with the Philadelphia chro- mosome, a reciprocal translocation between chromosome 9 and 22 (1 – 4). This translocation links the c-Abl tyrosine kinase oncogene on chromosome 9 to the 5V half of the BCR gene on chromosome 22 and originates the fusion gene BCR-ABL (4, 5). The fusion gene produces a chimeric 8.5 kb transcript that codes for the p210 BCR-ABL protein (6), which possesses constitutive tyrosine kinase activity and is the pathogenic agent of CML. From the therapeutic perspective, as CML arises from a single genetic lesion, a research goal has been to develop kinase-specific inhibitors. The development of imatinib (also known as imatinib mesylate, Glivec, or Gleevec) is a landmark achievement in this respect as it has shown great promise in the chronic phase (7, 8), and some expectations in the accelerated and blastic phase of CML, as well as in BCR-ABL – expressing acute lymphoblastic leukemias (9). Unfortunately, however, most responding blast-phase patients relapse despite continued chemother- apy (10) and, as frequently happens with cancer chemo- therapeutics, resistance to imatinib has been reported in both Bcr-Abl – expressing cell lines and in patients with CML (11 – 14). Recently, point mutations within the DNA sequence coding for the ATP-binding pocket of the BCR-ABL gene were identified in cells from some patients with CML who had imatinib refractory disease or who had a relapse during the treatment (15 – 18). In particular, a point mutation resulting in a threonine-to-isoleucine change at amino acid position 315 (T315I) has been described in detail (14). This mutation, when engineered into wild-type (WT) p210 BCR-ABL and transiently infected into 293T cells or Ba/F3 cells, interfered with the inhibition of Bcr-Abl kinase activity in cells exposed to imatinib (14, 18). Further, Corbin et al. (15) showed that the T315I-mutated Abl kinase domain exhibited no significant inhibition at imatinib concentrations 200-fold higher than the IC 50 value of the WT kinase; it also showed a 2-fold increase in its ATP affinity relative to the WT protein. However, the extent to which this mutation contributes to imatinib resistance in vivo remains a matter of lively literature dispute (14, 17, 19 – 22). On the basis of these evidences, both Schindler et al. (23) and Corbin et al. (15) postulated that the above- mentioned point mutation T315I in the tyrosine kinase domain of Bcr-Abl alters the three-dimensional structure of the ATP pocket, thus decreasing the sensitivity of Bcr- Abl to imatinib, and does not feature a fundamental hydrogen bond that is critical for binding with the drug. These speculations, however, were not supported by further investigations at a molecular modeling level. Accordingly, we present the results obtained from a detailed molecular simulation study of both WT and T315I-mutated Abl kinase domain in complex with Received 4/4/ 05; revised 5/19/ 05; accepted 6/14/ 05. Grant support: Italian Association for Cancer Research, Italian Ministry of Health, and Italian Ministry for University and Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Sabrina Pricl, Molecular Simulation Engineering Laboratory, Department of Chemical Engineering, University of Trieste, Piazzale Europa 1, 34127 Trieste, Italy. Phone: 39-40-5583750; Fax: 39-40-569823. E-mail: sabrina.pricl@dicamp.units.it Copyright C 2005 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0101 1167 Mol Cancer Ther 2005;4(8). August 2005 Research. on December 16, 2021. © 2005 American Association for Cancer mct.aacrjournals.org Downloaded from