Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors

ROS1 encodes the largest receptor tyrosine kinase (RTK) in the human genome. Chromosomal rearrangements of ROS1 generate oncogenic kinase fusion proteins sensitive to inhibition by tyrosine kinase inhibitors (TKIs). To date, ROS1 fusions are the only validated biomarkers for therapeutic implementation of ROS1 TKIs. While the cancer genome reveals nonsynonymous missense mutations in ROS1, the impact of these mutations on catalytic activity or pathogenic potential is unknown. We queried the AACR Genie database for ROS1 aberrations and nominated thirty-four non-synonymous missense mutations residing in the ROS1 tyrosine kinase domain for functional interrogation. Immunoblotting revealed diverse impact of the mutations on kinase function, ranging from loss of function to significant increase in catalytic activity. Notably, Asp and Gly substitutions at the D2113 position within the ROS1 kinase domain dramatically increased autophosphorylation and phosphorylation of downstream effectors. ROS1 D2113N/G were ROS1 TKI-sensitive, transformative oncogenes in independent cell models. Molecular dynamic simulations revealed drastic local alterations in the activation loop of of ROS1 D2113N as compared to the wildtype kinase. Global proteomics and phospho-proteomics showed that ROS1 D2113N upregulates phosphorylation of known effectors (e.g., SHP2 and STAT3) akin to ROS1 fusions, but also increases phosphorylation of pathways not previously linked to ROS1, including mTORC2, JNK1/2, AP-1, TGFB1 and CCN1. In vivo, ROS1 D2113N drove tumor formation that was sensitive to inhibition by crizotinib and lorlatinib. Taken together, these data establish that select point mutations within ROS1 RTK drive tumorigenesis and maybe therapeutically targetable with existing FDA-approved ROS1-TKIs. ### Competing Interest Statement The authors have declared no competing interest.