title: Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients.
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 microM 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.
authors.affiliation: Experimental Molecular Pathology, Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Tamborini
authors.forename: S
authors.initials: S
authors.lastname: Pricl
authors.forename: T
authors.initials: T
authors.lastname: Negri
authors.forename: M S
authors.initials: MS
authors.lastname: Lagonigro
authors.forename: F
authors.initials: F
authors.lastname: Miselli
authors.forename: A
authors.initials: A
authors.lastname: Greco
authors.forename: A
authors.initials: A
authors.lastname: Gronchi
authors.forename: P G
authors.initials: PG
authors.lastname: Casali
authors.forename: M
authors.initials: M
authors.lastname: Ferrone
authors.forename: M
authors.initials: M
authors.lastname: Fermeglia
authors.forename: A
authors.initials: A
authors.lastname: Carbone
authors.forename: M A
authors.initials: MA
authors.lastname: Pierotti
authors.forename: S
authors.initials: S
authors.lastname: Pilotti
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001549:Benzamides; D019008:Drug Resistance, Neoplasm; D046152:Gastrointestinal Stromal Tumors / Q000188:drug therapy* / Q000235:genetics; D006801:Humans; D000068877:Imatinib Mesylate; D008958:Models, Molecular; D009154:Mutation; D010879:Piperazines / Q000627:therapeutic use*; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000235:genetics*; D011743:Pyrimidines / Q000627:therapeutic use*
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit