title: Identification of tyrosine 806 as a molecular determinant of RET kinase sensitivity to ZD6474.
abstract: ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC(50): 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 (IC(50): 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 (IC(50): 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 (IC(50): 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.
authors.affiliation: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, via S. Pansini 5, 80131 Naples, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Carlomagno
authors.forename: Teresa
authors.initials: T
authors.lastname: Guida
authors.forename: Suresh
authors.initials: S
authors.lastname: Anaganti
authors.forename: Livia
authors.initials: L
authors.lastname: Provitera
authors.forename: Svend
authors.initials: S
authors.lastname: Kjaer
authors.forename: Neil Q
authors.initials: NQ
authors.lastname: McDonald
authors.forename: Anderson J
authors.initials: AJ
authors.lastname: Ryan
authors.forename: Massimo
authors.initials: M
authors.lastname: Santoro
mesh_terms: D000595:Amino Acid Sequence; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D051096:Proto-Oncogene Proteins c-ret / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D010880:Piperidines; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; C036387:tyrosinamide; D014443:Tyrosine; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; C452423:vandetanib