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

Abstract

AbstractChromosomal rearrangements ofROS1generate ROS1 tyrosine kinase fusion proteins that are established oncogenes predicting effectiveness of tyrosine kinase inhibitors (TKI) treatment. The cancer genome reveals nonsynonymous missense mutations inROS1,however, their oncogenic potential remains unknown. We nominated thirty-four tumor-associated missense mutations in ROS1 kinase domain for functional interrogation. Immunoblotting revealed diverse impact of the mutations on the kinase, ranging from loss of function to significant increase in catalytic activity. Notably, Asn and Gly substitutions at the Asp-2113 position in ROS1 kinase domain were TKI- sensitive hyper-activating mutations, and transformative oncogenes in independent cell models. Molecular modeling revealed drastic alterations in the activation loop of ROS1D2113Ncompared to wildtype kinase. Proteomics studies showed that ROS1D2113Nincreases phosphorylation of known effectors akin to ROS1 fusions, and upregulates pathways not previously linked to ROS1, including mTORC2, JNK1/2, AP-1, TGFB1 and CCN1/2.In vivo, ROS1D2113Ndrove tumor formation that was sensitive to inhibition by crizotinib and lorlatinib. Taken together, these data show that select point mutations within ROS1 RTK are oncogenic, and maybe therapeutically targetable with FDA-approved TKI.