TKI Type Switching Overcomes ROS1 L2086F in ROS1 Fusion-Positive Cancers
Purpose: Despite the strong efficacy of ROS1 tyrosine kinase inhibitors (TKIs) in treating ROS1-positive non-small cell lung cancer (NSCLC), resistance to TKIs limits the long-term success of these therapies. However, the resistance mechanisms of next-generation ROS1 TKIs remain poorly understood.
Design: We compared the activity of type I TKIs (crizotinib, entrectinib, taletrectinib, lorlatinib, and repotrectinib) and type II TKIs (cabozantinib and merestinib) against type I FLT3 inhibitor gilteritinib in Ba/F3 cells expressing wild-type ROS1 or various ROS1 mutants (F2004C, L2026M, G2032R, L2086F). These findings were confirmed through NIH3T3 colony formation assays and in vivo tumor growth studies. Additionally, CRISPR/Cas9 gene editing was used to generate isogenic wild-type and L2086F mutant TPM3-ROS1 expressing patient-derived cell lines. TKI activity was evaluated through cell viability assays and immunoblotting. Molecular modeling studies were conducted to understand the structural determinants of TKI sensitivity in wild-type and mutant ROS1 kinase domains. We also report clinical cases of ROS1 TKI resistance treated with cabozantinib.
Results: The ROS1 L2086F mutant kinase exhibited resistance to all type I TKIs, including crizotinib, entrectinib, lorlatinib, repotrectinib, and taletrectinib. In contrast, type II TKIs cabozantinib and merestinib maintained efficacy against this mutant. Gilteritinib, a type I FLT3 inhibitor, inhibited both wild-type and L2086F mutant ROS1; however, the G2032R solvent front mutation conferred resistance to gilteritinib. Molecular modeling revealed that cabozantinib binds in the DFG-out conformation of the kinase, while gilteritinib adopts a DFG-in binding mode, explaining their activity against ROS1 mutants. Clinical cases showed that cabozantinib was effective in tumors with TKI resistance driven by the ROS1 L2086F mutation.
Conclusion: Cabozantinib and gilteritinib effectively inhibit ROS1 L2086F, with clinical evidence confirming the activity of cabozantinib in patients with ROS1 L2086F mutant NSCLC resistant to other TKIs. Gilteritinib may provide an alternative treatment option, though its distinct off-target toxicities warrant further investigation. As both cabozantinib and gilteritinib are multi-kinase inhibitors, there remains an unmet need for more selective and better-tolerated TKIs to overcome intrinsic resistance in the ROS1 L2086F mutant kinase.
Translational Relevance: The ROS1 L2086F mutation is an emerging, recurrent resistance mechanism to type I ROS1 TKIs, including newer-generation agents. This study provides robust preclinical and clinical evidence supporting the activity of the quinolone-based type II TKI cabozantinib in the setting of ROS1 L2086F resistance. Additionally, gilteritinib, a pyrazine carboxamide-based type I TKI, shows activity in ROS1 L2086F resistance, suggesting it could be another treatment option for patients with ROS1 L2086F TKI resistance. This is the first comprehensive report of ROS1 L2086F resistance in the context of later-generation TKIs, including macrocyclic inhibitors.