Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients

Abstract

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 lM of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.

Imatinib resistance and microcytic erythrocytosis in a Kit V558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor

“…It is of interest that the KIT T670I mutation is found only in imatinib-resistant GIST patients in combination with a primary KIT mutation. There are no reports to indicate that the KIT T670I mutation may be found alone as a single-site mutation and on its own may have a role in GIST tumorigenesis, although in vitro experiments in COS cells with single-mutant KIT T670I in the absence of KitL indicated KIT autophosphorylation (27). Although GISTs of Kit V558Δ/+ and Kit V558Δ;T669I/+ mice were similar in histology and oncogenic signaling, the Kit V558Δ;T669I/+ mice were resistant to imatinib and dasatinib therapy.…”

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

“…However, because acquired resistance is often observed in GIST and other cancers treated with TKIs or other targeted therapies, further rationally developed therapies against oncogenic KIT are necessary (12)(13)(14)(15)(16)(17). Moreover, it is not clear why anti-KIT mAbs that are ∼1,000-fold more potent than the TKI imatinib and 100-fold more potent than the TKI sunitinib in blocking the tyrosine kinase activity of WT KIT fail to block the activity of the oncogenic V560D KIT mutant.…”

Imatinib response in two GIST patients carrying two hitherto functionally uncharacterized PDGFRA mutations: An imaging, biochemical and molecular modeling study

“…Eventual missing force field parameters for the inhibitor were taken from our previous work. 10,14 The involved mutations were introduced into the wild-type structure of the corresponding PDGFRA/imatinib complex following a well-validated procedure. 10,14,15 Each mutant complex was then solvated and energy minimized using a combination of molecular dynamics (MD) techniques.…”

“…10,14 The involved mutations were introduced into the wild-type structure of the corresponding PDGFRA/imatinib complex following a well-validated procedure. 10,14,15 Each mutant complex was then solvated and energy minimized using a combination of molecular dynamics (MD) techniques. 10,14 The 2-ns MD simulations at 37 C were then employed for system equilibration, and further, 4-ns MD were run for data production.…”

“…10,14,15 Each mutant complex was then solvated and energy minimized using a combination of molecular dynamics (MD) techniques. 10,14 The 2-ns MD simulations at 37 C were then employed for system equilibration, and further, 4-ns MD were run for data production.…”