Abstract 3926: Activating missense mutations in ROS1 receptor harbor oncogenic potential and are sensitive to tyrosine kinase inhibition

Abstract ROS1 encodes the largest receptor tyrosine kinase (RTK) in the human genome. Chromosomal rearrangements of the ROS1 gene generate oncogenic ROS1 fusion proteins that have been successfully targeted with tyrosine kinase inhibitors (TKIs), most notably in lung cancer. To date, ROS1 fusions are the only known biomarkers for therapeutic implementation of ROS1 TKIs. However, unlike known oncogenic mutations in EGFR and ALK RTKs, the functional impact of point mutations within the full-length ROS1 RTK is unknown. Here we queried the AACR Genie database, which consists of pan-cancer clinical genomic sequencing data from thousands of patient samples, for ROS1 aberrations and nominated thirty-four missense mutations residing in the ROS1 tyrosine kinase domain for functional interrogation. Immunoblotting revealed distinct impact of the mutations on kinase function, ranging from complete loss to significant increase in catalytic activity. Notably, Asp and Gly substitutions at the D2113 position within the ROS1 kinase domain dramatically increased autophosphorylation and activation of downstream signaling pathways. ROS1D2113N/G were found to be ROS1 TKI-sensitive transformative oncogenes in independent cell-based models. Molecular dynamic simulations of ROS1D2113N kinase revealed dramatic alterations in the activation loop of the mutant kinase as compared to wildtype. Global proteomics and phosphoproteomics showed that ROS1D2113N upregulates signaling via phosphorylation of known effectors (SHP2 and STAT3) similar to ROS1 fusions, but also upregulates other pathways, including mTORC2, JNK1/2, AP-1 and TGFB1. In vivo, ROS1D2113N drove tumor formation that was inhibited with ROS1 TKIs. Taken together, these data demonstrate for the first time that select point mutations within ROS1 drive tumorigenesis and can be therapeutically targetable with existing FDA-approved ROS1-TKIs. Citation Format: Sudarshan R. Iyer, Kevin Nusser, Kristen Jones, Pushkar Shinde, Catherine Z. Beach, Clare Keddy, Erin Aguero, Jeremy Force, Ujwal Shinde, Monika A. Davare. Activating missense mutations in ROS1 receptor harbor oncogenic potential and are sensitive to tyrosine kinase inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3926.