Mechanisms of Autoinhibition and STI-571/Imatinib Resistance Revealed by Mutagenesis of BCR-ABL

Abstract

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.

Combined effects of novel heat shock protein 90 inhibitor NVP-AUY922 and nilotinib in a random mutagenesis screen

“…The observed data from the isobologram indicated the synergistic effect on simultaneous exposure to AUY922 and nilotinib in WT BCR-ABL-expressing BaF3 cells (Figure 2a). M351T is a sensitive mutation to nilotinib, on the other hand, E255K is an insensitive mutation to The approach uses a DNA repair-deficient Escherichia coli strain to produce random mutagenesis of a BCR-ABL retroviral plasmid, infection of BaF3 cells, and selection for BaF3 clones conferring varying degree of drug resistance using methods previously described (Azam et al, 2003). BaF3 cells expressing the random mutagenesis of BCR-ABL were kindly provided by Dr James D Griffin (Dana-Farber Cancer Institute) (Ray et al, 2007).…”

Quantitative monitoring by polymerase colony assay of known mutations resistant to ABL kinase inhibitors

“…Template preparation and direct sequencing Genomic DNA and/or RNA from patient samples or from the murine BaF3 cell lines engineered to express either wild-type BCR-ABL or imatinib-resistant mutated BCR-ABL (Azam et al, 2003) were purified using DNeasy (no. 69506, Qiagen, Valencia, CA, USA) and RNeasy (no.…”

“…Sensitivity of the polony assay To assess the mutation detection sensitivity of the polony assay by controlled dilution experiments, we performed the polony assay on mixtures of native BCR-ABL plasmid and plasmids carrying the BCR-ABL-G250E mutant (Azam et al, 2003). We serially diluted the wild-type plasmid from 10 7 molecules to 10 3 molecules, leaving the mutant plasmid at 10 3 molecules.…”

“…In most cases, resistance to imatinib is caused by amino-acid substitutions in the BCR-ABL protein that impede drug binding (Azam et al, 2003). Consequently, new kinase inhibitors are being developed that are active against imatinib-resistant mutants.…”

Origines de la résistance au traitement par imatinib mésylate : un exemple riche d’enseignements

“…Ainsi, une étude récemment publiée démontre que la fréquence des mutations détectées par séquençage augmente considérablement chez des patients en phase accélérée, dont le diagnostic de LMC a été posé quatre ans au moins avant la date de la recherche de mutations (27 patients porteurs de mutations sur 104 étudiés, quatre ans après le diagnostic, contre 0 sur 40 patients étudiés dont le diagnostic date de moins de quatre ans) [24]. In vitro, une approche de mutagenèse aléatoire a permis de constituer une banque de clones cellulaires BaF3 porteurs de mutations ponctuelles du gène bcr-abl [27]. Les clones analysés ont été sélectionnés pour leur résistance à l'IM.…”