title: Resistance to c-KIT kinase inhibitors conferred by V654A mutation.
abstract: Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution, has been documented in imatinib-resistant GIST patients. We expressed c-KIT cDNA constructs encoding the V654A substitution alone and in combination with a typical activating exon 11 mutation characteristic of GIST, V560G, in factor-dependent FDC-P1 cells. The V654A substitution alone resulted in enhanced proliferation in c-KIT ligand (stem cell factor) but not factor independence. Cells expressing the double mutant were, like those expressing single V560G mutant c-KIT, factor independent. Analysis of cellular proliferation in the presence of imatinib showed that the V654A substitution alone conferred resistance. The difference in sensitivity was especially pronounced for cells expressing single mutant V560G c-KIT compared with double mutant V560G/V654A c-KIT. The findings were supported by studies of c-KIT phosphorylation. Analysis of the crystal structure of imatinib in complex with the kinase domain of c-KIT predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT inhibitors, nilotinib (AMN107) and PKC412, were also less active on V560G/V654A c-KIT than on the V560G single mutant; however, nilotinib, like imatinib, potently inhibited the V560G mutant. PKC412 strongly inhibited imatinib-resistant D816V c-KIT.
authors.affiliation: University of Newcastle, Room 511, Medical Sciences Building, Callaghan, New South Wales 2308, Australia.
authors.forename: Kathryn G
authors.initials: KG
authors.lastname: Roberts
authors.forename: Adam F
authors.initials: AF
authors.lastname: Odell
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Byrnes
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Baleato
authors.forename: Renate
authors.initials: R
authors.lastname: Griffith
authors.forename: Alan Bruce
authors.initials: AB
authors.lastname: Lyons
authors.forename: Leonie K
authors.initials: LK
authors.lastname: Ashman
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D005091:Exons / Q000235:genetics; D005455:Fluorescent Antibody Technique; D046152:Gastrointestinal Stromal Tumors / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D047468:Immunoprecipitation; D051379:Mice; D009154:Mutation / Q000235:genetics*; D023461:Myeloid Progenitor Cells / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit; C498826:nilotinib; D019311:Staurosporine; C059539:midostaurin