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IC50-values are based on studies performed in cell lines, which express BCR-ABL mutations that were identified in patients with CML or Ph+ ALL and resistance to imatinib (Source: From Refs. 4,10,11,59,75). Imatinib resistance mutations in up to 50 per cent of the cases are located within the nucleotide-binding loop (P-loop), with Y253 and E255 being most frequently affected. The most abundant exchanges affecting single positions are T315I (20% of cases) and M351T (15% of cases) (Source: From Refs. 21,84).

IC50-values are based on studies performed in cell lines, which express BCR-ABL mutations that were identified in patients with CML or Ph+ ALL and resistance to imatinib (Source: From Refs. 4,10,11,59,75). Imatinib resistance mutations in up to 50 per cent of the cases are located within the nucleotide-binding loop (P-loop), with Y253 and E255 being most frequently affected. The most abundant exchanges affecting single positions are T315I (20% of cases) and M351T (15% of cases) (Source: From Refs. 21,84).

This classification does not allow in estimating the degree of resistance. As an example, the class of P-loop mutations includes moderately (G250A, Q252H, E255D) as well as highly (G250E, Y253H, E255V) imatinib-resistance mutations (10,21).

Meanwhile, mutations conferring clinical resistance to therapeutically used kinase inhibitors were also identified in several other target kinases in various malignant diseases. Imatinib resistance mutations were identified in FIP1L1-PDGFR alpha in patients with hypereosinophilic syndrome (23,24), and in cKit in patients with gastrointestinal stromal tumors (GIST) (25,26). In addition, a resistance mutation in the kinase domain of FLT3 in a patient with acute myeloid leukemia (AML) treated with the kinase inhibitor PKC412 has been described (27). Similarly, in patients with non small cell lung cancer (NSCL), treated with the kinase inhibitor gefitinib (Iressa), an exchange of threonine at position 790 to methionine in the epidermal growth factor receptor (EGFR) was described (28,29). This mutation, together with the imatinib-resistant mutations KIT/T670I and FIP1L1-PDFGRalpha/T674I, is homologous to position T315 in the ABL-kinase domain,

FIGURE 2 Functional domains of the Abl kinase domain and position of imatinib resistance mutations. Ribbon representation of the c-Abl kinase domain in complex with imatinib, with C-helix in light green, P-loop in pink, and A-loop in magenta, with the A-loop in an open conformation. Labels indicate the residue number of human c-Abl kinase type Ia. The colors of the spheres represent the degree of cellular resistance to imatinib expressed as fold cellular IC50 of wild-type Bcr-Abl in Ba/F3 cells. Source: Adapted from Ref. 21.

FIGURE 2 Functional domains of the Abl kinase domain and position of imatinib resistance mutations. Ribbon representation of the c-Abl kinase domain in complex with imatinib, with C-helix in light green, P-loop in pink, and A-loop in magenta, with the A-loop in an open conformation. Labels indicate the residue number of human c-Abl kinase type Ia. The colors of the spheres represent the degree of cellular resistance to imatinib expressed as fold cellular IC50 of wild-type Bcr-Abl in Ba/F3 cells. Source: Adapted from Ref. 21.

confirming the critical role of this residue for the binding of ATP-competitive kinase inhibitors. Thus, mutations in kinase domains appear to be a general mechanism of resistance against tyrosine kinase inhibitors.

BCR-ABL Gene Amplification and Protein Overexpression

BCR-ABL gene amplification and protein overexpression as causes of imatinib resistance were both identified in vitro (1-3) as well as in CML patient samples

(4,29). Amplification of a kinase inhibitor target could also be demonstrated for KIT in imatinib-resistant GIST patients (30). Amplification of the target gene results in a shift of the inhibitor/target ratio toward a surplus of the target protein. Consequently, the amount of inhibitor available within the cell is not sufficient to effectively block all target protein molecules. In the case of CML, BCR-ABL overexpression allows for residual kinase activity even in the presence of imatinib, which enables the leukemic clone to survive.

Phamacokinetic- and Drug Transport-Issues

Alterations in the distribution of imatinib and its delivery to target cells can result in suboptimal drug concentrations within leukemic cells and thereby may give rise to resistant disease. The intracellular concentration of imatinib is determined by membrane-bound, active import and export pumps, and by its binding to plasma proteins. It was shown that imatinib is bound to plasma proteins, such as a-1-acid-glycoprotein (a-1-GP) (31). It has been proposed that increased plasma a-1-GP levels might reduce the plasma concentration of free unbound imatinib that is available for inhibition of BCR-ABL (31). Indeed, the tumor burden in a mouse model (32) and the CML disease stage in patients (33) correlated with plasma a-1-GP levels and elevated a-1-GP levels prior to treatment led to a less rapid response to imatinib in CML patients (33). However, elevated a-1-GP plasma levels in patients were reversible in the course of treatment (33), and a-1-GP did not alter the efficacy of the drug in vitro (33,34). Therefore, it is currently unclear whether plasma proteins such as a-1-GP contribute to imatinib resistance or not.

Imatinib is a substrate of the multi drug resistance-associated membrane transporter MDR-1 and thus can be actively pumped out of the cell (35,36). Overexpression of MDR-1 was found in imatinib-resistant cell lines, and by inhibition of MDR-1 imatinib resistance was partially reverted (2). Increased expression of MDR-1 was also demonstrated on progenitor cells of patients with CML in myeloid blast crisis when compared to healthy controls, although the level of expression did not predict response to imatinib. In contrast, low levels of MRP-1, another drug transporter, correlated with response to treatment (37). Recently, a membrane transporter was identified (human organic cation transporter-1; hOCT-1), which may be involved in the active transport of imatinib into the cell (import) (38). A small study demonstrated a correlation of hOCT-1 expression and response to imatinib (39). Larger studies are required to substantiate the impact of hOCT-1 expression for imatinib activity.

On the whole, drug transport mechanisms may be of potential importance for the survival of CML cells in the presence of imatinib. However, clinical evidence that pharmacokinetic mechanisms play an important role for imatinib resistance is still inconclusive, since no single critical mechanism has been identified. However, it is conceivable that pharmacokinetic mechanisms may be important for a cell in the course of acquiring secondary resistance due to mutations or secondary genetic alterations in the presence of imatinib.

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