(393a) Changes and Challenges: Gist, Mutated C-Kit and Imatinib Resistance

Pricl, S., University of Trieste
Tamborini, E., Istituto per lo Studio e la Cura dei Tumori
Pilotti, S., Istituto per lo Studio e la Cura dei Tumori
Pierotti, M. A., Istituto per lo Studio e la Cura dei Tumori
Ferrone, M., University of Trieste
Paneni, M. S., University of Trieste
Fermeglia, M., University of Trieste

A new era of targeted cancer therapy was inaugurated with the approval of imatinib mesylate (or STI 571/Gleevec) for the treatment of chronic myeloid leukemia (CML). Imatinib is a phenyl-aminopyrimidine compound, initially identified from a high-throughput screening for inhibitors of protein kinase C and subsequently found to be a potent and selective inhibitor of the Abl, platelet-derived growth factor beta receptor, and kit tyrosine kinases. Imatinib binds in a pocket close to the ATP-binding site of the Abl catalytic domain, and effectively inhibits Abl kinase activity in vitro and in vivo at concentrations of 0.1-1.0 microM. One year after its approval by FDA and EMEA for CML treatment, in 2001 imatinib was also approved for advanced gastrointestinal stromal tumors (GISTs) chemotherapy. GISTs are the most common mesenchymal tumors of the gastrointestinal tract; they represent a spectrum of tumors, ranging from benign to highly malignant. In CML, Imatinib is highly effective both in early and late stages of the disease. Nonetheless, several relapses do occur after initial response, despite continued treatment. In patients who developed resistance to imatinib, reactivation of the bcr-abl kinase signaling was observed, due to either a secondary mutation, resulting in a missense substitution of a residue belonging to the drug binding site and critical for binding, or to a progressive BCR-ABL gene amplification. In GISTs, primary resistance seems to involve at least 15% of patients with advanced disease, and its occurrence could be correlated with different c-kit mutations. The molecular mechanisms at the bases of imatinib resistance are poorly understood and scarcely investigated. Among a series of patients surgically treated in our Institute all showing clinical/radiological evidence of progressing disease despite of Imatinib treatment, 8 patients were molecularly and biochemically investigated for KIT and PDGFRA gene alterations. None of them showed mutations in PDGFRA gene, and FISH analysis reveal neither KIT or PDGFRA gene amplification. The sequencing of the whole coding sequence of KIT gene was performed in the tumoral specimens, revealing activating mutations in exon 11 in all patients. In two patients, two different adjunctive point mutations in KIT gene, one in exon 14 responsible for T670I substitution in the kit protein, and one in exon 13 causing the V654A substitution in the receptor, were detected. Biochemical analyses showed c-kit phosphorylation in cells transfected with vectors carrying the specific mutant genes and treated with different doses of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib binding-site, but with different mechanism. By the application of molecular simulations we were able to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance at a molecular level.


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