1. ¹Experimental Molecular Pathology, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy
2. ²Molecular Simulation Engineering Laboratory, DICAMP University of Trieste, Trieste, Italy
3. ³Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy
4. ⁴Department of Surgery, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy
5. ⁵Department of Clinical Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy
6. ⁶IFOM, FIRC Institute of Molecular Oncology, Milan, Italy

Correspondence: Dr S Pilotti, Unit of Experimental Molecular Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Via G Venezian 1, Milan 20133, Italy. E-mail: silvana.pilotti@istitutotumori.mi.it

⁷These authors contributed equally to this work.

⁸Senior co-authors.

Received 9 January 2006; Revised 21 March 2006; Accepted 23 March 2006; Published online 5 June 2006.

Abstract

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 M of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.