KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination

Abstract

Background: KRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions.

Methods: Cell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model.

Results: Our research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression.

Conclusions: We posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

Fig. 1

Combination of tepotinib plus omeprazole potentiates cell viability inhibition. Lung cancer cells H358 and A549 and the resistant cell lines to sotorasib (H358R) or trametinib (A549TR) were exposed to increasing concentrations of tepotinib combined with increased concentrations of omeprazole for 72 h. Cell viability was analyzed by MTT assay. The experiments were performed in triplicate. The tepotinib dose was as follows: 0, 0.75, 1.5, 3, 3.75, 4.5, 6, 9, 18 (µM) for H358 and H358R cell lines, and 0, 0.65, 2.6, 3.25, 3.9, 4.55, 5.2, and 7.9 for A549 and A549TR cell lines. The omeprazole dose range was: 0, 12.5, 25, 37.5, 50 and 150 (µM) for H358 and H358R cells and 0, 35, 52, 70, 105 and 210 (µM) for A549 and A549TR cells. Graphs show the mean +/- SD of 3 independent replicates. One-way ANOVA test was used, ⁎p < 0.05, ⁎⁎p < 0.01, ⁎⁎⁎p < 0,0001, ns = not significant

Fig. 2

Isobologram analysis and synergism determination: Dose-response matrix is depicted showing tepotinib and omeprazole dual drug assays in NSCLC cells (H358, A549) and resistant cell lines (H358R, A459TR) using Combenefit Software (Bliss & HSA Models). This matrix portrays the interaction between eight doses of tepotinib and five doses of omeprazole across four distinct cell lines: H358, A549 and their resistant derivatives, H358SR and A549TR. (A) Two-drug combination dose response surface expressed as a percentage of the control value. The plot portrays the efficacy of each of the dual-drug combinations. (B) Synergy scores, shown in matrix format, calculated according to the Bliss and HSA methods. The blue boxes indicate synergy between tepotinib and omeprazole, while the green boxes represent additivity, and the yellow, orange, or red boxes signify antagonism. The number of biological replicates (N) is indicated at the top left of the matrix. The number below the synergy score is the standard deviation

Fig. 3

Clonogenic formation assay upon treatment of KRAS-mutant cell lines grown in monolayer cultures with tepotinib, omeprazole or the double combination. The cells were grown in six-well plates (500 cells/well) for 24 h and then left untreated or treated with tepotinib, omeprazole and the double combination. After 72 h, media was replaced with fresh media without drugs. After seven more days cells were washed and stained with crystal violet and then photographed. Images are representative of at least three independent experiments

Fig. 4

Effects of single treatments or tepotinib plus omeprazole combination with or without actinomycin D on proteins involved in the downstream signaling of the Wnt, MET and KRAS pathways. Western blot analysis showed protein levels in parental H358 and H358R cells treated with the single drugs (omeprazole 100 µM, tepotinib 10 µM, actinomycin 2µM) or the combinations. Protein extracts were taken at 24 h after treatment. β-actin was used as loading control. Data were generated from a minimum of three replicates

Fig. 5

In vivo antitumor efficacy of tepotinib in combination with omeprazole and actinomycin D. A. Time schedule for the H358 cells subcutaneous implantation and drug treatment groups, mice were randomly grouped into 6 groups: vehicle group, tepotinib group, omeprazole group, actinomycin D group, tepotinib + omeprazole group, tepotinib + omeprazole + actinomycin D group. (B) Tumor volumes for subcutaneous H358 tumor. (C) Mice body weight in each group. (D) Subcutaneous tumor weight in each group. (E) Representative H358 tumor pictures. (n = 7). Data were analyzed using unpaired t-test comparisons. * P < 0.05, compared to the tepotinib + omeprazole + actinomycin D group

Fig. 6

Recurrence-free survival and overall survival curves based on the mRNA expression level of KRAS or MET. Kaplan-Meier curves were created by dividing patients with the median of mRNA expression