Negri, Tiziana and Pavan, Giovanni Maria and Virdis, Emanuela and Greco, Angela and Fermeglia, Maurizio and Sandri, Marco and Pricl, Sabrina and Pierotti, Marco A and Pilotti, Silvana and Tamborini, Elena (2009) T670X KIT mutations in gastrointestinal stromal tumors: making sense of missense. Journal of the National Cancer Institute, 101 (3). pp. 194-204. ISSN 1460-2105
Full text not available from this repository.Abstract
BACKGROUND: Chronic myeloid leukemia, gastrointestinal stromal tumors (GISTs), and idiopathic hypereosinophilic syndrome are associated with pathological deregulation of the tyrosine kinases BCR-ABL, KIT, and PDGFRA, respectively. Patients who become resistant to imatinib treatment often develop secondary mutations, the most common of which results in a substitution of isoleucine for threonine at the same location in the ATP-binding domain in all three kinases (in KIT this occurs at amino acid 670). We sought to determine why Thr is always replaced by Ile. METHODS: All possible point mutations in the DNA triplet codon that could result in amino acid substitutions at Thr670 (Thr670Arg, Thr670Ile, Thr670Lys, Thr670Ala, Thr670Ser, Thr670Pro) were introduced by site-specific mutagenesis of the complementary DNA for a constitutively active, imatinib-sensitive form of the KIT receptor, Delta559/KIT. The resulting mutant KIT proteins were transiently expressed in COS1 African green monkey kidney cells grown with and without imatinib, and cell extracts were analyzed for KIT activation by immunoprecipitation and immunoblotting to determine autophosphorylation levels. We also performed molecular modeling to estimate the relative affinities of wild-type (Thr670) KIT and the KIT mutants for ATP and imatinib. RESULTS: Like the parental strain, Thr670Ala, Thr670Ser, and Thr670Lys mutants were inhibited by 5 microM imatinib, but in comparison, they were only weakly active and Thr670Pro and Thr670Arg were not active at all. Only the Thr670Ile mutant was fully active (autophosphorylated) and resistant to imatinib. These findings were consistent with computer modeling predictions that ranked these mutants Thr - Ile > Ala, Ser > Lys >> Pro according to their affinity for ATP but Thr > Ala, Ser > Lys >Pro - Arg - Ile according to their affinity for imatinib. CONCLUSIONS: This combination of in vitro and molecular modeling analyses shows why, among all possible amino acid substitutions at position 670 of KIT, only Ile is naturally selected as a resistance mutant in imatinib-treated GIST patients.
| Item Type: | Scientific journal article, Newspaper article or Magazine article |
|---|---|
| Subjects: | Physical sciences > Chemistry > Medicinal chemistry > Pharmaceutical chemistry |
| Depositing User: | Giovanni Maria Pavan |
| Date Deposited: | 23 Aug 2016 07:30 |
| Last Modified: | 23 Aug 2016 07:30 |
| URI: | http://repository.supsi.ch/id/eprint/7614 |
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