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

Abstract

ROS1 fusion proteins resulting from chromosomal rearrangements of the ROS1 gene are targetable oncogenic drivers in diverse cancers. Acquired resistance to targeted inhibitors curtails clinical benefit and response durability. Entrectinib, a NTRK/ROS1/ALK targeted tyrosine kinase inhibitor (TKI), was approved for the treatment of ROS1 fusion-positive non-small cell lung cancer (NSCLC) in 2019. In addition, lorlatinib and repotrectinib are actively being explored in the setting of treatment-naïve or crizotinib-resistant ROS1 fusion driven NSCLC. Here, we employed an unbiased forward mutagenesis screen in Ba/F3 CD74-ROS1 and EZR-ROS1 cells to identify resistance liabilities to entrectinib, lorlatinib, and repotrectinib. ROS1^F2004C emerged as a recurrent entrectinib resistant mutation and ROS1^G2032R was discovered in entrectinib and lorlatinib-resistant clones. Cell-based and modeling data show that entrectinib is a dual type I/II mode inhibitor, and thus liable to both types of resistant mutations. Comprehensive profiling of all clinically relevant kinase domain mutations showed that ROS1^L2086F is broadly resistant to all type I inhibitors, but remains sensitive to type II inhibitors. ROS1^F2004C/I/V are resistant to type I inhibitors, entrectinib and crizotinib, and type II inhibitor, cabozantinib, but retain sensitivity to the type I macrocyclic inhibitors. Development of new, more selective type II ROS1 inhibitor(s) or potentially cycling type I and type II inhibitors may be one way to expand durability of ROS1-targeted agents.

Figure 1.. Characterization of subcellular localization, activity, and inhibitor sensitivity of ROS1 fusions.

A, Domain organization of ROS1 fusions. TKD - ROS1 tyrosine kinase domain; TM – Transmembrane domain; FERM – Protein 4.1 Ezrin Radixin, Moesin domain; CC – coiled-coiled domain; YWTD Propeller - β Propeller domain; FN III – fibronectin type III repeat. B, Live cell imaging (spinning disk confocal microscopy) of monomeric yellow fluorescent protein (mYFP)-tagged ROS1 fusion proteins. White arrowheads indicate endoplasmic reticulum (ER), juxta-plasma membrane (juxta-PM), cytoplasm (cyto), and membraneless cytoplasmic granules (granules). C, Immunoblot analysis of the phosphorylated and total proteins shown in transfected HEK293T cell lysates. Anti-Flag antibody was used to detect Flag-tagged ROS1 fusion. D, IC₅₀ values indicate relative potency (nanomolar) of the indicated ROS1i for Ba/F3 CD74-ROS1 or EZR-ROS1; six replicate data points are shown. E & F, Immunoblot analysis of the phosphorylated and total proteins shown DMSO or ROS1i-treated (25 nmol/L, 6 hours) in cell lysates generated from Ba/F3 CD74-ROS1 and EZR-ROS1, respectively. Representative immunoblots from two independent experiments are shown.

Figure 2.. ROS1^F2004C induces resistance to entrectinib.

A & B, Immunoblot analysis of the phosphorylated and total proteins shown from Ba/F3 CD74-ROS1^F2004C (A) and Ba/F3 EZR-ROS1^F2004C (B) lysates treated with the indicated inhibitors for 4 hours. Vehicle indicates DMSO treatment.

Figure 3.. Structural assessment of ROS1^F2004C with molecular dynamic simulation and inhibitor docking studies reveal entrectinib binds both DFG-in and DFG-out ROS1 kinase domain.

A & B, Superimposed wildtype ROS1 (A) and ROS1^F2004C mutant crystal structure models in DFG-in and DFG-out conformations are shown to highlight distinctions in structural features: Activation loop (A-Loop), αC Helix, P-loop, ATP-binding pocket, and the positioning of the aspartic acid (D), phenyalanine (F) and glycine (G), i.e., DFG motif in the two conformations. C & D, Superimposed crystal structures show conformational differences between wildtype ROS1 and ROS1^F2004C in their DFG-in (C) and DFG-out (D) states. E & F, Histograms show binned probability distribution of binding energy (kcal/mol) for ROS1^WT and ROS1^F2004C in the DFG-in (E) and DFG-out (F) kinase conformation. Binding energies were determined with Yasara; in this case the higher binding energies reflect more favorable binding. G, Percentage of docks whose binding energy was greater than the median binding energy of indicated inhibitor docking to ROS1^WT DFG-in and ROS1^WT DFG-out is plotted. The median binding energy ± standard deviation and the total number of docks for the ROS1^WT DFG-in & ROS1^WT DFG-out conformations is indicated inset within graph and panel below graph. H & I, IC_50s of crizotinib, entrectinib, lorlatinib, repotrectinib and cabozantinib as derived from dose-response cell viability assay with Ba/F3 CD74-ROS1 (H) and Ba/F3 EZR-ROS1 (I), wildtype or F2004C mutant cells.

Figure 4.. Activity of first and next-generation ROS1 inhibitors against spectrum of ROS1 intracellular mutations in CD74-ROS1 and EZR-ROS1.

A, Dose response cell viability assays of clinically relevant (S1986F, F2004C, F2004V, L2026M, G2032R, D2033N and L2086F compared to wildtype) ROS1 kinase domain mutations in Ba/F3 CD74-ROS1 cells. Average ± SEM shown for all data. B & C, Immunoblotting of Ba/F3 CD74-ROS1^L2086F and EZR-ROS1^L2086F cells treated for 6 h with 100 nM of indicated inhibitors. Phosphorylated and total ROS1 and ERK1/2 are shown. D & E, Heat map of IC₅₀ values (nanomolar) of crizotinib, entrectinib, lorlatinib, repotrectinib, cabozantinib and foretinib for Ba/F3 CD74-ROS1 (D) and EZR-ROS1 (E) mutant cell lines. IC₅₀ are average of two to four replicates for each mutant and each inhibitor. Color scale for heatmap in indicated in the figure.