Dissecting the molecular landscape of resistance to ROS1 tyrosine kinase inhibitors with improved NSCLC pre-clinical models

ROS1+ NSCLC can be targeted with tyrosine kinase inhibitors (TKIs). However, point mutations affecting kinase domain that impair drug binding are a therapeutical challenge to address. Thus, unraveling the impact of mutations like ROS1 G2032R, L2026M, S1986Y in representative pre-clinical models is needed. The ROS1+ cell line HCC78 was edited using CRISPR/Cas9 to knock-in the mutations G2032R, L2026M and S1986Y. Mutant lines were treated with crizotinib, lorlatinib, ceritinib, crizotinib and repotrectinib in a monolayer (2D) and spheroids (3D) using the OrBITS platform to track cell viability. Western-blotting was performed to assess MAP kinase pathway. In parallel, ROS1 kinase models for WT and mutants were investigated for conformational landscape using molecular dynamics suite GROMACS. Protein-drug interactions via molecular docking were studied with SMINA. G2032R line showed in 2D the strongest resistance towards crizotinib, entrectinib and ceritinib, having all a weak activity (IC50 s around 1,1 µM), opposed to repotrectinib (93,25 nM) and lorlatinib (317,17 nM). L2026M clone is more sensitive to TKIs, being repotrectinib (5,9 nM) and lorlatinib (6,85 nM) more effective. S1986Y clone showed a similar response as the WT line. 3D spheroids confirmed these results. p-ROS1 levels were maintained across mutants. The kinase active state, selected as the starting conformation for computational simulations did not shift from active to inactive mode. DFG and HRD motifinteractions are conserved while some change is observed in loop regions that define ATP binding pocket. Mutations distant from the active have less impact on drug binding. Surface area and volumetric changes observed are less informative for such mutants. We conclude that G2032R is the most aggressive mutation, being partially inhibited by lorlatinib. This TKI showed remarkable activity against L2026M and S1986Y likewise, however both mutants were refractory to entrectinib, crizotinib, repotrectinib and ceritinib. These results were confirmed by western blot. Our approach allows the generation of patient-derived mutant cell lines and screened for TKI sensitivity guided by the in silico modelling of ROS1 variants.