Abstract C148: Type II ROS1 inhibitors and their liability on ROS1 rearranged non-small cell lung cancer with L2086F mutation
Authors: Thawani Rajat , Keddy Clare , Repetto Matteo , Jones Kristen , Nicholson Katelyn , Beach Zoe , Drilon Alexander , Davare Monika 
Published: December 2023

Introduction: Fusion oncoproteins involving the ROS1 receptor tyrosine kinase have been established as validated therapeutic targets in non-small cell lung cancer. FDA-approved ROS1 tyrosine kinase inhibitors (TKIs) such as crizotinib and entrectinib have shown substantial response rates in these patients, leading to improved outcomes. Historically, the solvent-front ROS1 kinase mutation, G2032R, was a recurrent resistance liability to first generation ROS1 TKI. Next-generation TKIs, including NVL-520, repotrectinib, and taletrectinib are active against the ROS1 G2032R and shown activity in both treatment-naïve and ROS1 G2032R acquired resistance setting. We and others showed that ROS1 L2086F a mutation in catalytic-spine 6 within the kinase domain confers resistance to all first and next-generation type I binding mode TKIs. Cabozantinib, a type II ROS1 inhibitor, is the only FDA-approved TKI that has showed clinical activity against ROS1 L2086F. Recent reports indicated that gilteritinib, an FLT3 TKI, exhibits activity against ALK L1256 (homologous to ROS1 L2086F). In this study, we investigated the efficacy of gilteritinib compared to cabozantinib in ROS1 L2086F resistance model systems.

Methods: We tested the effectiveness of ROS1 TKI compared to gilteritinib in Ba/F3 cell lines expressing CD74-ROS1 and EZR-ROS1 resistant mutations. Additionally, we quantified the efficacy of cabozantinib and gilteritinib in ROS1 L2086F using soft agar assays. We engineered CRISPR-Cas9 genome-edited CUTO-28 cells expressing endogenous L2086F mutation, and to validate the activity of gilteritinib and cabozantinib against ROS1 L2086F.

Results: The ROS1 kinase mutations F2004C, G2032R and L2086F are resistant to entrectinib inhibition. Cabozantinib, consistent with our previous published data, retains inhibitory activity against ROS1 G2032R and ROS1 L2086F, however the ROS1 F2004C mutation imposes cabozantinib-resistance. Gilteritinib, intriguingly, is active with IC50 ≤ 25 nM against ROS1 F2004C and ROS1 L2086F but not ROS1 G2032R. Immunoblotting studies showed on-target inhibition of ROS1 auto-phosphorylation and effector ERK1/2 activation in the case of ROS1 L2086F mutant cells treated with cabozantinib and gilteritinib.

Conclusion: Gilteritinib demonstrates inhibitory activity against ROS1 L2086F mutation. Currently, patients with ROS1 L2086F resistance can be treated with cabozantinib as a second-line treatment option. The preclinical data presented herein suggest that gilteritinib could be an additional, potentially viable alternative to target the highly resistant ROS1 L2086F mutation. Additional studies are needed to explore the use of gilteritinib for ROS1 rearranged NSCLC.

Citation Format: Rajat Thawani, Clare Keddy, Matteo Repetto, Kristen Jones, Katelyn Nicholson, Zoe Beach, Alexander Drilon, Monika Davare. Type II ROS1 inhibitors and their liability on ROS1 rearranged non-small cell lung cancer with L2086F mutation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C148.