Drug binding and resistance mechanism of KIT tyrosine kinase revealed by hydrogen/deuterium exchange FTICR mass spectrometry

Abstract

Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.

Platelet-Derived Growth Factor/Vascular Endothelial Growth Factor Receptor Inactivation by Sunitinib Results in Tsc1/Tsc2-Dependent Inhibition of TORC1

“…Treatment with sunitinib in humans results in the emergence of secondary mutations that render the oncogenic RTK resistant to the effects of sunitinib. Secondary mutations in GIST that render KIT resistant to sunitinib typically map to the A-loop (68)(69)(70) and are thought to shift the kinase toward the active state (67,75,76). Residues that are mutated in KIT include D816, D820, N822, Y823, and A829 (68)(69)(70).…”

Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity

“…Therefore, we consider a disturbing effect on the JM-Switch folding as indicative of the JMR departure from its inhibitory position and general effect induced by KIT hotspot mutations. Such departure of JMR from the kinase domain favors the stabilization of the inactive non-autoinhibited state [107], [108], where the JMR displays an increased solvent accessibility, as was revealed by hydrogen/deuterium exchange mass spectrometry analysis of KIT D816H and KIT V560D mutants [107]. Nevertheless, the contribution of this change to KIT activation is different in the two kinds of mutation – in the A-loop and in the JMR.…”

“…It was shown that imatinib binds to the inactive non-autoinhibited state in which the JMR is detached from the kinase domain [107], [108]. The inactive non-autoinhibited conformations of KIT V560G/D mutants, which is induced by JMR detachment from the kinase domain, would be the most appropriate targets for imatinib binding.…”

Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell– and T Cell–Specific Tec Kinases

“…HDX studies of a mutant form of the tyrosine kinase c-Kit that is associated with gastrointestinal stromal tumors indicate that the D816H mutation, at the N-terminus of the activation segment, increases solvent-accessibility at the C-terminal end of the activation segment (29). Similar to the sequence changes that we introduced into Itk to produce the activated Itk_Btk loop-6 mutant, a change in activation loop dynamics induced by the D816H mutation of c-Kit correlates with a higher rate of autophosphorylation compared to that of the wild-type kinase (29). Indeed, it is likely that many disease-causing mutations fundamentally alter protein dynamics, which leads to functional deregulation.…”