9P Small-scale ROS1 aberrations: Functional impact and therapeutic potential

ROS1-directed Tyrosine-Kinase-Inhibitors (TKIs) target activating fusions in the ROS1 proto-oncogene efficiently in non-small cell lung cancer (NSCLC) patients. Besides solvent-front mutations (SFMs) in resistance to targeted therapy, ROS1 aberrations other than fusions remain biologically unexplored. Driving on our thus far clinical investigations, we aimed at determining the functional impact and the potential to act as a drug target. Tumor samples from NSCLC patients were screened with two next-generation sequencing (NGS) panels. Patients with activating ROS1 fusions, SFMs and benign Single-Nucleotide Polymorphisms (SNPs) were excluded using the gnomAD database. The Provean and PolyPhen-2 tools predicted the functional impact of the detected aberrations. ChimeraX was used for drug binding analysis. Of 8072 patients analyzed by NGS between 2018 and 2022, 110 (1.4%) patients harbored small-scale ROS1 aberrations. Our cohort consists of 113 aberrations leading to 95 (84.1%) missense, 12 (10.6%) truncating and 1 (0.1%) in-frame aberrations. In 10% of patients ROS1 aberration was mutually exclusive and most ROS1 aberrations were transitions (55%). Polyphen predicts 75.5% of aberrations to be ‘possibly’ (6.4%), respectively ‘probably damaging’ (68.1%, mean score 0.75, median score 0.999). Provean predicts 50% of aberrations to be ‘deleterious’. Polyphen-2 and Provean coherently predict 46.8% of aberrations to be ‘deleterious’ respectively ‘possibly/probably damaging’. Amid the tyrosine kinase domain almost all aberrations are predicted to be ‘probably damaging’ respectively ‘deleterious’ by both tools coherently and feature a higher-than-average score. Besides, no distinct molecular pattern occurs. The M2029T and R2083T missense mutations interfere with single hydrogen-bonds and Van-Der-Waals forces in the ROS1 drug-binding pocket between the respective residues and the TKIs crizotinib and lorlatinib. However, drug binding analysis revealed that the overall binding of the TKIs is not affected. This evidence proves a functional and deleterious impact of specific aberrations and indicates a high potential to be targetable. We warrant further studies to elaborate these findings in vitro and in vivo.