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This Article Accepted manuscript (PDF) All Versions of this Article: ERC-08-0213v1 16/1/233    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Carlomagno, F. Articles by Santoro, M. Search for Related Content PubMed PubMed Citation Articles by Carlomagno, F. Articles by Santoro, M.

F Carlomagno, Istituto di Endocrinologia e Oncologia Sperimentale, CNR, Napoli, Italy
T Guida, Istituto di Endocrinologia e Oncologia Sperimentale, CNR, Napoli, Italy
S Anaganti, Dept. Biol. Patol. Cell. Molec., University Federico II, Napoli, Italy
L Provitera, dept. Biol. Patol. Cell. Molec., University Federico II, napoli, Italy
S Kjaer, Structural Biology Laboratory, London Research Institute, Cancer Research UK, London, United Kingdom
N McDonald, Structural Biology Laboratory, London Research Institute, Cancer Research UK, London, United Kingdom
A Ryan, Cancer Discovery, Astra Zeneca UK, Macclesfield, United Kingdom
M Santoro, Dept. Biol. Patol. Cell. Molec., University Federico II, Napoli, Italy

Correspondence: Massimo Santoro, Email: masantor{at}unina.it

Abstract

ZD6474 (vandetanib, Zactima) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC50: 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC50: 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC50: 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC50: 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.