Abstract
STI571, a selective inhibitor of Bcr-Abl, has been a successful therapeutic agent in clinical trials for chronic myelogenous leukemia. Chronic phase chronic myelogenous leukemia patients treated with STI571 have durable responses; however, most responding blast phase patients relapse despite continued therapy. Co-crystallization studies of Abl kinase and an STI571-related compound identify specific amino acid residues as critical to STI571 binding, one of which, T315, has been characterized as an acquired Thr to Ile mutation in relapsed patients. Other studies, however, suggest that mutations other than these predicted contact points are capable of conferring STI571 resistance in relapsed patients. Using a variety of models of STI571 binding to the Abl kinase, we have performed an extensive mutational analysis of sites that might alter the sensitivity of the Abl kinase to STI571. Although mutation of many of the predicted contact points between Abl and STI571 result in a kinase-inactive protein, additional mutations that render the Abl kinase less sensitive to STI571 demonstrate a broad range of possibilities for clinical resistance that are now becoming evident.
Highlights
Chronic phase chronic myelogenous leukemia patients treated with STI571 have durable responses; most responding blast phase patients relapse despite continued therapy
Mutation of many of the predicted contact points between Abl and STI571 result in a kinase-inactive protein, additional mutations that render the Abl kinase less sensitive to STI571 demonstrate a broad range of possibilities for clinical resistance that are becoming evident
Chronic myelogenous leukemia is a clonal hematopoietic stem cell disorder characterized by the presence of the Philadelphia chromosome
Summary
Mutation Construction—The Abl kinase domain consisting of c-Abl amino acids 220 – 498 was subcloned into the BamHI site of pGEX KG (Amersham Biosciences). The Abl kinase mutations were incubated with concentrations of STI571 ranging from 0 to 1 M for 10 min, after which 10 Ci of [␥-32P]ATP (111 nM total ATP) was added and the kinase reaction allowed to proceed for 30 min. Reactions were analyzed by SDS-PAGE as described above, and signal intensity was quantitated. The ATP concentrations resulting in 50% maximum signal ([ATP]50% maximum ) signal for wild type Abl and each of the mutations that showed a change in STI571 sensitivity were determined from the slope and y-intercept of double reciprocal plots of 1/[ATP] versus 1/signal. Relative Vmax values of wild type Abl and the mutants were determined by comparing the relative signal intensity of the proteins at 100 nM. Relative Vmax values were calculated using the average ATP affinities in the following equation: Vmax ϭ signal[ATP] ϭ 100 nM
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