Systematic Analysis of Tyrosine Kinase Inhibitor Response to RET Gatekeeper Mutations in Thyroid Cancer

The proto-oncogene protein RET is a receptor tyrosine kinase that plays an important role in the development and progress of various human cancers. Currently, targeting RET with small-molecule tyrosine kinase inhibitors (TKIs) has been established as promising therapeutic strategy for thyroid carcinoma (TC). However, two gatekeeper mutations V804M and V804L in RET kinase domain have been frequently observed to cause drug resistance during the targeted therapy, largely limiting the application of reversible TKIs in TC. Here, we described an integrative protocol that combined literature curation, computational analysis, and in vitro kinase assay to systematically investigate the response profile of 9 approved RET TKIs to the two clinical RET gatekeeper mutations. It was revealed that the two mutations exhibit a similar energetic behavior to influence TKI binding, which can moderately decrease competitive inhibitor affinity and modestly increase substrate ATP affinity simultaneously. However, the binding potency of few second-generation kinase inhibitors such as Ponatinib and Alectinib can be improved to overcome the increased ATP affinity, thus restoring their inhibitory activity against the kinase mutants. Subsequently, the established protocol was employed to investigate the response profile of 4 commercially available RET TKIs that are under preclinical or clinical development. Three out of the four TKIs were found to become resistant upon the mutations due to steric hindrance effect introduced by the mutated residues, while the remaining one was moderately sensitized by the mutations since the mutated residues can form additional hydrophobic and van der Waals interactions with the inhibitor.