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Research Articles: Therapeutics, Targets, and Development

Resistance to c-KIT kinase inhibitors conferred by V654A mutation

¹ School of Biomedical Sciences and ² School of Environmental and Life Sciences, University of Newcastle, and Hunter Medical Research Institute, Newcastle, New South Wales, Australia; and ³ Division of Haematology, Hanson Institute, Institute of Medical and Veterinary Sciences, Adelaide, South Australia, Australia

Requests for reprints: Leonie K. Ashman, University of Newcastle, Room 511, Medical Sciences Building, Callaghan, New South Wales, 2308 Australia. Phone: 61-2-4921-7947; Fax: 61-2-4921-6903. E-mail: leonie.ashman{at}newcastle.edu.au

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. [Mol Cancer Ther 2007;6(3):1159–66]

Grant support: National Health and Medical Research Council of Australia grant 150413 (L.K. Ashman) and the Anthony Rothe Memorial Trust. New South Wales Health via the Hunter Medical Research Institute (infrastructure support).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: K.G. Roberts and A.F. Odell contributed equally to this work.

⁴ http://www.rcsb.org

⁵ http://www.accelrys.com

Received 10/17/06; revised 12/13/06; accepted 1/24/07.

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