The biological effect of small-scale ROS1 aberrations: An in silico analysis

The ROS1 proto-oncogene encodes a receptor tyrosine kinase that activates downstream signaling pathways related to cell proliferation and survival. Although ROS1 fusions are well-treatable genetic aberrations, little is known about small-scale ROS1 aberrations. Driving on our previous investigations elucidating their clinical and molecular characteristics, we aimed at analyzing the effect exerted by the detected aberrations with regard to their biological effect on the protein structure. Next-generation sequencing (NGS) was performed on tissue samples from Non-small cell lung cancer patients within the National Network Genomic Medicine. Patients with activating ROS1 fusions and solvent front mutations (SFM) were excluded. We analyzed the molecular aberrations using a mutation mapper, the protein structure prediction software AlphaFold® and the protein database models 3zbf and 4uxl. Of 8072 patients analyzed by NGS between 2018 and 2022, 129 (1.6%) patients harbored ROS1 mutations without having a ROS1 fusion or SFM. In 10.2% of patients ROS1 mutation was the only detected aberration and most ROS1 mutations were transversions (52.1%). 75.4% of all mutations are missense and 7% are truncating mutations. About 95% of all mutations are located in the cytoplasmic domain whereof 57.3% of mutations are located in the protein tyrosine kinase domain. Only 2.4% of mutations are located in the ATP binding site. We did not encounter a specific mutational hotspot. Most importantly, we determined truncating and missense mutations in the DFG-motif which controls the active, type I kinase conformation and the access to the catalytic site. These mutations either truncate the crucial phenylalanine of the motif or substitute its glycine for a valine. Structure predictions revealed considerable deformations in the motif. Furthermore we report several amino acid substitutions in the kinase domain activation loop. This evidence implies a biological impact in silico backing up the previously determined clinical impact. We warrant further research to characterize the biological impact in vitro and in vivo, and the potential to act as a drug target.