TKI type switching overcomes ROS1 L2086F in ROS1 fusion-positive cancers

Abstract

The grammar in this abstract is generally correct, but there's a minor issue with sentence structure in one part. Here's a slightly revised version with improved grammar and flow:ROS1 tyrosine kinase inhibitors (TKIs) are highly effective in ROS1-positive non-small cell lung cancer, but resistance remains a challenge. We investigated the activity of various TKIs against wildtype and mutant ROS1, focusing on the emerging L2086F resistance mutation. Using Ba/F3 and NIH3T3 cell models, CRISPR/Cas9-edited isogenic wildtype and mutant patient-derived cell lines, and in vivo tumor growth studies, we compared type I TKIs (crizotinib, entrectinib, taletrectinib, lorlatinib, and repotrectinib) to type II TKIs (cabozantinib and merestinib) and the type I FLT3 inhibitor gilteritinib. The ROS1 L2086F mutant kinase showed resistance to type I TKIs, while type II TKIs retained activity. Gilteritinib inhibited both wildtype and L2086F mutant ROS1 but was ineffective against the G2032R mutation. Structural analyses revealed distinct binding poses for cabozantinib and gilteritinib, explaining their efficacy against L2086F. Clinical cases demonstrated cabozantinib's effectiveness in patients with TKI-resistant, ROS1 L2086F mutant NSCLCs. This study provides the first comprehensive report of ROS1 L2086F in the context of later-generation TKIs, including macrocyclic inhibitors. While cabozantinib effectively inhibits ROS1 L2086F, its multi-kinase inhibitor nature highlights the need for more selective and better-tolerated TKIs to overcome kinase-intrinsic resistance. Gilteritinib may offer an alternative for targeting ROS1 L2086F with distinct off-target toxicities, but further studies are required to fully evaluate its potential in this setting.

Fig. 1. TKI activity in models of ROS1 kinase-intrinsic resistance.

a Heat map of IC₅₀ values shows relative potency (nM) of the indicated ROS1 inhibitors for Ba/F3 CD74-ROS1 wildtype and mutant cell lines. IC₅₀ values were calculated from three replicates from two independent experiments. b Network diagram summarizes tyrosine kinase inhibitor (TKI) activity against ROS1 G2032R, L2086F, and F2004C mutants, with type I inhibitors shown in blue, type II in green, and type I/II in purple. c Immunoblot analysis of phosphorylated and total ROS1 in cell lysates generated from Ba/F3 CD74-ROS1 wildtype, G2032R, and L2086F cell lines treated with indicated inhibitors at 50 nM for 2 h. d Scatter plot of cell-based IC50 values for Ba/F3 CD74-ROS1 wildtype, L2086F, F2004C + L2086F, and G2032R + L2086F cell lines tested with crizotinib, entrectinib, lorlatinib, taletrectinib, repotrectinib, cabozantinib and gilteritinib. e, f Colony counts anchorage-independent soft agar assays using NIH3T3 CD74-ROS1 WT (e) and L2086F (f) cell lines. Data normalized to vehicle (DMSO) treatment for indicated TKI treatment conditions. Average and standard error of means is shown in graphs (N = 4).

Fig. 2. Activity of ROS1 inhibitors in CUTO-28 patient-derived TPM3–ROS1 fusion cell line with or without L2086F.

a Heat map depicting IC₅₀ data from dose response cell viability assays conducted using CUTO-28 TPM3–ROS1 fusion parental or L2086F cell lines. Colorimetric reagent CCK-8 was added after 72-h exposure with varying concentrations of crizotinib, cabozantinib, repotrectinib, or gilteritinib. Absorbance data were normalized to vehicle-treated cells. b Fold change in IC₅₀ of indicated inhibitors is graphed relative to the Parental Cell IC₅₀. Control HDR indicates the CUTO-28 cell line that was subjected to genome editing; however, instead of L2086F editing, the HDR template retained wildtype amino acid. c Immunoblot analysis of phosphorylated and total ROS1, ERK, and downstream pathways in cell lysates generated from CUTO-28 parental and TPM3-ROS1 L2086F cells treated with indicated inhibitors at 100 nM for 4 h. d Immunoblot analysis of phosphorylated and total ROS1 from lysates generated from CUTO-28 parental and TPM3-ROS1 L2086F cells after 4 h treatment with DMSO (Vehicle) or 2.5, 25, 250, 2500 nM concentrations of entrectinib, cabozantinib and gilteritinib, as indicated. The ratio of phosphorylated ROS1 divided by total ROS1 as determined by densitometry is presented below the blots. e Immunoblot analysis of phosphorylated and total ROS1 from lysates generated from CUTO-28 parental and TPM3–ROS1 L2086F cells that were treated for 2, 4, 6, 8, and 24 h with 25 nM entrectinib, cabozantinib and gilteritinib, as indicated.

Fig. 3. Molecular docking of gilteritinib and cabozantinib to wild-type and mutant ROS1.

a Model of wild-type ROS1 kinase domain with gilteritinib in a type I conformation. L2086 is shown in blue, and gilteritinib is shown in orange. b Model of ROS1 L2086F kinase domain with gilteritinib bound in a type I conformation. F2086 is shown in blue, and gilteritinib is shown in orange. c Model of ROS1 G2032R bound to gilteritinib in the type I (DFG-in) conformation. Predicted steric clash between R2032 and gilteritinib is depicted. d Model of wild-type ROS1 kinase domain bound to with cabozantinib bound in the type II conformation. L2086 is shown in blue, and cabozantinib is shown in teal. e Model of ROS1 L2086F kinase domain bound to cabozantinib in the type II conformation. F2086 is shown in blue, and cabozantinib is shown in light teal. f Model of ROS1 G2032R bound to cabozantinib in the type II conformation.

Fig. 4. Clinical course of a patient with ROS1 fusion-positive non-small cell lung carcinoma.

a Timeline of the diagnostic and treatment history of the patient which exhibits initial response to cabozantinib. Periods of partial response (PR) are shown in light green, metabolic complete response (CR) is shown in green, and stable disease is shown in yellow. b Images of chest CT showing partial response to treatment with cabozantinib.