ROS1 S1986Y

In this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.

Results 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). 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.