Cancer Therapeutics

The Bcr-Abl fusion protein kinase causes chronic myeloid leukemia and is targeted by the signal transduction inhibitor STI-571/Gleevec/imatinib (STI-571). Sequencing of the BCR-ABL gene in patients who have relapsed after STI-571 chemotherapy has revealed a limited set of kinase domain mutations that mediate drug resistance. To obtain a more comprehensive survey of the amino acid substitutions that confer STI-571 resistance, we performed an in vitro screen of randomly mutagenized BCR-ABL and recovered all of the major mutations previously identified in patients and numerous others that illuminate novel mechanisms of acquired drug resistance. Structural modeling implies that a novel class of variants acts allosterically to destabilize the autoinhibited conformation of the ABL kinase to which STI-571 preferentially binds. This screening strategy is a paradigm applicable to a growing list of target-directed anti-cancer agents and provides a means of anticipating the drug-resistant amino acid substitutions that are likely to be clinically problematic. taken from: Azam M, Latek RR, Daley GQ. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.
Cell. 2003 Mar 21;112(6):831-43. (go to abstract)
BCR-ABL Mutagenesis and In Vitro Screen for STI-571 Resistance. The 10 kb plasmid pEYKBA was propagated in XL-1 red (Stratagene), which introduces random point mutations at a rate of one base pair substitution per 10 kb replicated DNA. Retroviral supernatants with titers of 106 would represent a library approximating saturation mutagenesis of BCR-ABL. Cells were selected in either 5 or 10 uM STI-571, as the numbers of colonies resulting from exposure to lower drug concentrations (1 and 2 uM) were too numerous to analyze and did not afford tight selection of resistant variants. taken from: Azam M, Latek RR, Daley GQ. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.
Cell. 2003 Mar 21;112(6):831-43. (go to abstract)
 
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Mapping of Mutations to Structural Models of ABL. (A) Two opposite views of the ABL kinase domain solvent accessible surface, encompassing amino acid residues 76-517. Labeled and highlighted in blue or red are the positions of kinase domain mutations isolated in this study. Internal residues that are not exposed on the surface of the protein are not depicted. Mutated residues that mediate contacts with either SH3 or SH2 are colored red. (B) Two opposite views of the predicted surface of a composite ABL kinase, SH3/SH2, and CD-linker model. The two perspectives of the composite ABL SH3/SH2/kinase model are colored as in (A). The SH3 domain is shown in green, the SH2 domain in cyan, and the CD linker in white. (C) Ribbon depiction of SRC structure alignment with ABL model. SRC is shown in yellow, ABL in white. Variants identified in this screen are colored as in (A). Potential complimentary mutations are labeled according to their relative positions within the model. taken from: Azam M, Latek RR, Daley GQ. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.Cell. 2003 Mar 21;112(6):831-43. (go to abstract)