Resistance Profile and Structural Modeling of Next-Generation ROS1 Tyrosine Kinase Inhibitors

Area of Science:

• Oncology
• Molecular Biology
• Genetics

Background:

• ROS1 fusions are oncogenic drivers in various cancers, particularly non-small cell lung cancer (NSCLC).
• Targeted tyrosine kinase inhibitors (TKIs) show efficacy against ROS1-driven cancers, but acquired resistance limits long-term clinical benefit.
• Entrectinib, lorlatinib, and repotrectinib are key TKIs used or investigated for ROS1-positive NSCLC.

Purpose of the Study:

• To identify novel resistance mutations to entrectinib, lorlatinib, and repotrectinib in ROS1 fusion-driven cancers.
• To understand the mechanisms of resistance to different classes of ROS1 inhibitors.
• To inform the development of next-generation inhibitors or treatment strategies to overcome resistance.

Main Methods:

• Utilized an unbiased forward mutagenesis screen in Ba/F3 cells engineered with CD74-ROS1 and EZR-ROS1 fusions.
• Exposed cells to entrectinib, lorlatinib, and repotrectinib to select for resistant clones.
• Characterized identified mutations (e.g., ROS1^F2004C, ROS1^G2032R, ROS1^L2086F) through cell-based assays and modeling.

Main Results:

• Identified ROS1^F2004C as a recurrent mutation conferring resistance to entrectinib.
• Discovered ROS1^G2032R in clones resistant to both entrectinib and lorlatinib.
• Demonstrated that entrectinib acts as a dual type I/II inhibitor, susceptible to both resistance mutation types.
• Found ROS1^L2086F confers broad resistance to type I inhibitors but retains sensitivity to type II inhibitors.
• Showed ROS1^F2004C/I/V mutations confer resistance to type I (entrectinib, crizotinib) and type II (cabozantinib) inhibitors but remain sensitive to type I macrocyclic inhibitors.

Conclusions:

• Specific kinase domain mutations in ROS1 confer resistance to clinically relevant TKIs.
• Understanding inhibitor binding modes (type I vs. type II) is crucial for predicting resistance patterns.
• Developing selective type II ROS1 inhibitors or employing inhibitor cycling strategies may enhance treatment durability in ROS1-driven NSCLC.