T670X KIT mutations in gastrointestinal stromal tumors: making sense of missense.
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.
Negri
Tiziana
T
Experimental Molecular Pathology, Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Pavan
Giovanni Maria
GM
Virdis
Emanuela
E
Greco
Angela
A
Fermeglia
Maurizio
M
Sandri
Marco
M
Pricl
Sabrina
S
Pierotti
Marco A
MA
Pilotti
Silvana
S
Tamborini
Elena
E
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001549:Benzamides; D003198:Computer Simulation; D019008:Drug Resistance, Neoplasm; D046152:Gastrointestinal Stromal Tumors / Q000235:genetics*; D006801:Humans; D017681:Hypereosinophilic Syndrome / Q000235:genetics; D000068877:Imatinib Mesylate; D015151:Immunoblotting; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D008958:Models, Molecular; D020125:Mutation, Missense; D010879:Piperazines / Q000494:pharmacology*; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D011743:Pyrimidines / Q000494:pharmacology*; D015203:Reproducibility of Results; D012107:Research Design; D013912:Threonine
D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013912:Threonine; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit