Foretinib can overcome common on-target resistance mutations after capmatinib/tepotinib treatment in NSCLCs with MET exon 14 skipping mutation

Abstract

Background: Capmatinib and tepotinib are guideline-recommended front-line treatments for non-small-cell lung cancer (NSCLC) patients with MET exon 14 skipping mutations (METex14). However, the emergence of acquired resistance to capmatinib/tepotinib is almost inevitable partially due to D1228X or Y1230X secondary mutations of the MET. In this study, we explored agents that are active against both D1228X and Y1230X MET to propose an ideal sequential treatment after capmatinib/tepotinib treatment failure in NSCLC patients with METex14.

Methods: The inhibitory effects of 300 drugs, including 33 MET-TKIs, were screened in Ba/F3 cells carrying METex14 plus MET D1228A/Y secondary mutations. The screen revealed four-candidate type II MET-TKIs (altiratinib, CEP-40783, foretinib and sitravatinib). Therefore, we performed further growth inhibitory assays using these four MET-TKIs plus cabozantinib and merestinib in Ba/F3 cells carrying MET D1228A/E/G/H/N/V/Y or Y1230C/D/H/N/S secondary mutations. We also performed analyses using Hs746t cell models carrying METex14 (with mutant allele amplification) with/without D1228X or Y1230X in vitro and in vivo to confirm the findings. Furthermore, molecular dynamics (MD) simulations were carried out to examine differences in binding between type II MET-TKIs.

Results: All 6 type II MET-TKIs were active against Y1230X secondary mutations. However, among these 6 agents, only foretinib showed potent activity against D1228X secondary mutations of the MET in the Ba/F3 cell and Hs746t in vitro model and Hs746t in vivo model, and CEP-40783 and altiratinib demonstrated some activity. MD analysis suggested that the long tail of foretinib plays an important role in binding D1228X MET through interaction with a residue at the solvent front (G1163). Tertiary G1163X mutations, together with L1195F/I and F1200I/L, occurred as acquired resistance mechanisms to the second-line treatment foretinib in Ba/F3 cell models.

Conclusions: The type II MET-TKI foretinib may be an appropriate second-line treatment for NSCLCs carrying METex14 after campatinib/tepotinib treatment failure by secondary mutations at residue D1228 or Y1230.

Fig. 1

Summary of the acquired resistance mechanisms in MET-driven NSCLC patients in a clinical setting. A, B Summary of clinically reported acquired resistance mechanisms to MET-TKIs in METex14-positive lung cancer patients. The frequency of occurrence of on-target and off-target resistance mechanisms to MET-TKIs is based on previous data reported by Recondo G. et al. (Clin Cancer Res. 2019, reference no: 6). The details of secondary resistance mutations are summarized based on the previous literatures (reference no: 3–10). TM; Transmembrane domain, A; Alanine, C; Cysteine, D; Asparatic acid, F; Phenylalanine, G; Glycine, H; Histidine, I; Isoleucine, L; Leucine, N; Asparagine, R; Arginine, S; Serine, V; Valine, Y; Tyrosine

Fig. 2

Evaluation of eight MET-TKIs, including four candidates, against clones with MET D1228X or Y1230X. A The IC₅₀ values (nM) of each drug for Ba/F3 cells harboring METex14 plus the indicated MET secondary mutations are expressed according to the indicated color scale. * indicates Ba/F3 clones generated using the corresponding vectors. Other cell lines were derived by ENU mutagenesis. Growth inhibition curves are summarized in Additional file 2: Fig. S2A and B. B The IC₅₀ values of each drug for cells with each secondary mutation are expressed as fold increases of the IC₅₀ for parental cells with METex14 in dot plots. C Western blot analyses of Ba/F3 cells with METex14 with/without secondary mutations treated with MET-TKIs at the indicated concentrations for 3 h. D The sensitivity index (SI) was defined as the IC₅₀ value divided by the concentration max (C_max) of each drug, which was reported in a clinical study. SI is expressed in a heatmap by the indicated color scales. For cabozantinib, since no PK data at 60 mg/day were available, the values were estimated from those reported at 80 mg/day and 40 mg/day based on previous reports (Kuzrock, R. et al. J Clin Oncol. 2011)

Fig. 3

Evaluation of type II MET-TKIs in Hs746t in vitro and in vivo models. A The IC₅₀ values (nM) of each drug for Hs746t cells harboring METex14 plus the indicated MET secondary mutations are expressed according to the indicated color scale. Growth inhibition curves are summarized in Additional file 2: Fig. S3. B Western blot analyses of Hs746t cells with METex14 with/without D1228N secondary mutation treated with MET-TKIs at the indicated concentrations for 3 h. β-actin was used as a loading control. C Changes in tumor size in vivo. Clinical dose of foretinib and cabozantinib used in phase II trial or clinical practice was converted to equivalent dose in mice considering the body surface area (Nair AB, Jacob S. J Basic Clin Pharm. 2016). Tumor size was measured every 2 days. Tumor Volume (TV) was calculated as follows: TV (cm³) = length × width × width × 0.5. Statistical analysis was performed using the Kruskal–Wallis test with the Dunnett’s multiple comparison test. P < 0.05 was considered to indicate statistical significance (* means P < 0.05). D Comparison of resected tumors treated with each drug from sacrificed mice on day 10. E H&E-stained histopathological images and phosphorylated MET in resected tumors treated with each drug determined by immunohistochemistry are shown. F The change (%) in the average body weight of mice during the treatment is shown compared to body weight before treatment

Fig. 4

Comparison of the binding models of foretinib, cabozantinib and merestinib against MET with D1228V mutation. Using MolDesk Basic ver. 1.1.77, molecular docking simulations of foretinib, cabozantinib and merestinib with the c-Met kinase domain (wild type or D1228V mutant) were carried out. Using PyMOL (ver 2.5.2), the 3D docking model of each MET-TKI and wild-type MET was aligned to the structure of MET D1228V. A The closest distances (Å) between the quinoline or pyrazole group of cabozantinib, merestinib, or foretinib and MET V1228 were measured by PyMOL. B The closest distances between the quinoline group of modified foretinib and MET V1228 were measured by PyMOL

Fig. 5

Analysis of the tertiary resistance mutations against second-line foretinib. A Schematic showing ENU mutagenesis. B The number of obtained resistant clones and the percentage of clones that carried tertiary mutations are shown for each drug concentration. Those with a tertiary mutation are shown in orange, and those without are shown in blue. C The detailed tertiary mutations obtained from ENU mutagenesis screening with foretinib are summarized for each drug concentration. D The MTT assay was performed with the obtained resistant clones with a tertiary resistant mutation, and IC₅₀ values were determined. IC₅₀ values are classified based on the indicated colors. They were also evaluated for the presence of cross-resistance to capmatinib and tepotinib. Growth inhibitory curves are summarized in Additional file 2: Fig. S7