Molecular and functional characterization of a new 3′ end KIT juxtamembrane deletion in a duodenal GIST treated with neoadjuvant Imatinib

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. GISTs express the receptor tyrosine kinase KIT, and the majority of GISTs present KIT gain-of-function mutations that cluster in the 5′ end of the receptor juxtamembrane domain. On the other hand, little information is known about GISTs carrying mutations in the 3′ end of the KIT juxtamembrane domain. Here we report and discuss a clinical case of localized duodenal GIST whose molecular characterization revealed the presence of a new 21 nucleotide/7 amino acid deletion in the 3′ end of KIT juxtamembrane domain (Δ574–580). The patient was treated with Imatinib at standard regimen dose (400 mg/day), and responded well as the original tumor mass reduced, ultimately allowing conservative surgery. In line with these clinical evidences computer simulations, biophysical techniques and in vitro experiments demonstrated that the receptor tyrosine kinase KIT carrying the Δ574–580 mutation displays constitutive phosphorylation, which can be switched-off upon Imatinib treatment. In addition, results from this study showed that a clinical useful procedure, neoadjuvant treatment, can occasionally be of value for the understanding of the molecular pathogenesis of GIST.


Computational details
The optimized structure of KIT was taken from our previous work (Tamborini et al., 2006a(Tamborini et al., , 2006b . Each protein/ligand system was relaxed in a box of TIP3P water molecules (Jorgensen et al., 1983). To achieve electroneutrality, a suitable number of neutralizing ions were added; further, the solution ionic strength was adjusted to the physiological value of 0.15 M by adding the required amounts of Na + and Cl − ions. Each hydrated complex system was then gradually heated to 25°C and then equilibrated for 5 ns via molecular dynamics (MD) simulations under isobaric/ isothermal conditions (i.e., NPT ensemble). Finally, each MD equilibrated system was subjected to 50 ns of NPT MD data collection runs to estimate the corresponding drug/protein free energy of binding (Pierotti et al., 2010;Pierotti et al., 2011).
All simulations were carried out using the Pmemd modules of Amber 16 (Case et al., 2016).
All energetic analysis was carried out for only a single MD trajectory of each KIT/imatinib complex considered, with unbound protein and substrate snapshots taken from the snapshots of that trajectory.
The affinity of the mutant KIT isoforms for imatinib and ATP, expressed as the value of the corresponding free energy of binding ∆G bind , was estimated using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method (Kollman et al., 2000, Puerotti et al., 2011. Accordingly, the binding free energy between two biological entities (e.g., a drug and its protein target) in a solvent was obtained as the sum of the interaction energy between the receptor and the ligand (∆E MM ), the solvation free energy (∆G so l), and the conformational entropy contribution (−T∆S), averaged over a series of snapshots from the corresponding MD trajectories: The ΔE MM term in Eq. (1) can be obtained directly from the molecular mechanics interaction energies as: where ΔE int , ΔE vdW , and ΔE el e are the internal, van der Waals and electrostatic components of the nonbonded interaction energy, respectively. Since in this work we adopted the "single trajectory protocol", then ΔE int = 0 in Eq. (S2).
The second term Eq. (S1), the solvation energy ∆G sol , can also be partitioned into two different contributions: The polar term of ΔG solv , ΔG PB , was estimated by solving the Poisson-Boltzmann equations numerically and calculating the electrostatic energy according to the electrostatic potential. In these calculations, the interior and exterior dielectric constant values were set equal to 1 and 80, respectively. A grid spacing of 0.5 per Å, extending 20% beyond the dimensions of the solute, was employed. The value of nonpolar component of ∆G solv , ∆G NP , was calculated using the following relationship (Sitkoff et al., 1994): in which γ = 0.00542 kcal (mol Å2) −1 , β = 0.92 kcal/mol, and SA is the molecular surface area estimated by means of the MSMS software (Sanner et al., 1993).
The change in solute entropy upon association (−T∆S in Eq. (S1)) was evaluated using the Nmode module of Amber 16. The normal-node analysis was performed for the minimized structures of the complexes, KIT, and ligands using a distance-dependent dielectric constant ε = 4r ij . In the first step of this calculation, an 8-Å sphere around the ligand was cut out from an MD snapshot for each ligand-protein complex. This value was shown to be large enough to yield converged mean changes in solute entropy. On the basis of the size-reduced snapshots of the complex, we generated structures of the uncomplexed reactants by removing the atoms of the protein and ligand, respectively. Each of those structures was minimized, using a distance-dependent dielectric constant ε = 4r, to account for solvent screening, and its entropy was calculated using classical statistical formulas and normal mode-analysis. To minimize the effects due to different conformations adopted by individual snapshots we averaged the estimation of entropy over 50 snapshots.
A per-residue binding free energy decomposition (PRBFED) was performed exploiting the MD trajectory of each complex. This analysis was carried out using the MM/GBSA approach (Tsui and Case, 2000), and was based on the same snapshots used in the binding free energy calculation.
Cloning and KIT mutants purification for experimental Imatinib binding studies ∆559, T670I, and ∆574-580 mutant KIT constructs were produced according to the established methodology described by Gajiwala et al. (2009). Accordingly, a construct encoding an N-terminal 6X-His tag + r3c protease site + KIT residues 544-976 was cloned into the baculovirus expression vector pVL1393 (Invitrogen). Proteins were expressed in Sf9 cells for 48 h; cells were then harvested and frozen (−80°C). Cellular lysates were obtained by treatment with 25 mM Tris (pH 7.4), 250 mM NaCl, 0.25 mM TCEP, and 20 mM imidazole. After binding to a Ni-NTA column, each protein was step-eluted using the same buffer employed for pellet lysing with 250 mM imidazole. Each peak eluted fraction was subjected to dialysis at 4°C for 12 h. Next, the His tag was cleaved with the r3C protease and removed by reloading the proteins on the Ni-NTA column. Each Flowthrough was collected from the column and concentrated. Next, each protein was feeded to a Superdex-200 column equilibrated in 25 mM Tris (pH 7.4), 250 mM NaCl, 0.5 mM TCEP, and 1mM EDTA. The peak fractions were pooled, concentrated (up to 6 mg/ml) and quickly frozen.

Isothermal titration calorimetry (ITC) experiments
A solution of each purified KIT mutant protein (10 µM, sample cell) was titrated with 36 step-by-step injections (spacing 150 s) of 2 µL volume of Imatinib (250 µM, syringe). Solutions and buffer were degassed for 30 min at room temperature under stirring at 750 rpm prior to each experiment.