Trametinib overcomes KRAS-G12V-induced osimertinib resistance in a leptomeningeal carcinomatosis model of EGFR-mutant lung cancer

Abstract

Leptomeningeal carcinomatosis (LMC) occurs frequently in non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third-generation EGFR-TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR-mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next-generation sequencing. We detected the Kirsten rat sarcoma (KRAS)-G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS-G12V overexpression in parental cells revealed the involvement of KRAS-G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS-G12V-harboring osimertinib-resistant LMC in EGFR-mutant NSCLC.

Keywords: epidermal growth factor receptor; leptomeningeal carcinomatosis; non-small cell lung cancer; osimertinib; trametinib.

FIGURE 1

Generation of osimertinib‐resistant PC9 cell clones in vivo. A, SHO mice were injected with PC9‐ffluc cells in the leptomeningeal space. Next, the mice were daily treated with osimertinib. After continuous treatment with osimertinib for 4 wk, the mice developed leptomeningeal carcinomatosis (LMC) with acquired osimertinib resistance. MTT assays were performed to assess the growth inhibition of PC9‐ffluc cells and the resistant clones, following treatment with osimertinib or rociletinib for 72 h (n = 3). The results are expressed as the mean ± standard error of the mean (SEM). The calculated IC₅₀ values are shown. B, The parental PC9‐ffluc cells and the resistant clones were treated with 1 µM osimertinib for 2 h. Changes in the phosphorylation levels of the indicated proteins were assessed by Western blotting. C, Control and epidermal growth factor receptor (EGFR) siRNAs were transfected into parental and resistant cells, and EGFR knockdown was confirmed by Western blotting. Cell viabilities were analyzed after 72 h by performing MTT assays (n = 3). *P < .001

FIGURE 2

Analysis of the resistance mechanism in the generated cells. A, Parental PC9‐ffluc cells and osimertinib‐resistant cells were treated with 1 µM osimertinib for 72 h. Cell lysates from each cell line were analyzed using a phospho‐RTK array kit. B, The parental PC9‐ffluc cells and the resistant clones were treated with 1 µM osimertinib for 2 h. Changes in the phosphorylation levels of the indicated proteins were analyzed by Western blotting. C, MTT assays were performed to assess the growth inhibition of PC9‐ffluc cells and the resistant clones, following osimertinib treatment in combination with 1 µM crizotinib for 72 h (n = 3). D, Genomic DNA was isolated from the parental PC9‐ffluc cells and the resistant cells, and mutations in 409 cancer‐associated genes were analyzed using the Ion AmpliSeq Comprehensive Cancer Panel. A Kirsten rat sarcoma (KRAS) codon 35G>T missense mutation (G12V) was detected in clone PC9‐OR#2

FIGURE 3

Analysis of Kirsten rat sarcoma (KRAS)‐G12V–mutated clonal cell lines. A, Copy numbers of KRAS‐G12V in parental PC9‐ffluc cells and osimertinib‐resistant PC9‐OR#2 cells were evaluated to determine the KRAS‐G12V mutation ratio by ddPCR. B, MTT assays were performed to assess the growth inhibition of PC9‐ffluc cells, PC9‐OR#2 cells, and the resistant clones, after osimertinib treatment for 72 h (n = 3). C, D, Control and KRAS siRNAs were transfected into parental PC9‐ffluc cells and osimertinib‐resistant OR#2 cells. At 24 h post transfection, the cells were treated with 1 µM osimertinib, and cell viabilities were measured after 72 h by performing MTT assays (n = 3) (C); after 1 wk, the cells were stained with crystal violet and examined visually (D). Error bars represent the SEM (D). *P < .0001. E, Control and KRAS siRNAs were transfected into the resistant clonal cell lines. At 24 h post transfection, the cells were treated with 1 µM osimertinib for 72 h, after which cell lysates were obtained and analyzed by immunoblotting with the indicated antibodies

FIGURE 4

Analysis of non–small cell lung cancer NSCLC) cells stably expressing Kirsten rat sarcoma (KRAS)‐G12V. A, MTT assays were performed to assess the growth inhibition of PC9‐ffluc, PC9‐ffluc‐KRAS‐G12V, H1975, and H1975‐KRAS‐G12V cells by osimertinib treatment for 72 h (n = 3). The calculated IC₅₀ values are shown. The results are expressed as the mean ± SEM. B, PC9‐ffluc, PC9‐ffluc‐KRAS‐G12V, H1975, and H1975‐KRAS‐G12V were treated with osimertinib for 1 wk, followed by staining with crystal violet and visual examination. C, PC9‐ffluc and PC9‐ffluc‐KRAS‐G12V cells were treated with 1 µM osimertinib for 2 or 24 h. Changes in the phosphorylation levels of the indicated proteins were assessed by Western blotting. D, MTT assays were performed to assess the growth inhibition of PC9‐KRAS‐G12V cells treated with increasing concentration of osimertinib in combination with 0.03 µM trametinib for 72 h (n = 3). E, PC9‐ffluc‐KRAS‐G12V cells were treated with 1 µM osimertinib and/or 0.03 µM trametinib for 2 or 72 h. Changes in the expression of the indicated proteins were evaluated by Western blotting

FIGURE 5

Effects of trametinib on Kirsten rat sarcoma (KRAS)‐G12V–induced osimertinib resistance. A, B, MTT assays were performed to assess the growth inhibitory effects of combined treatment for 72 h (trametinib (0.03 µM) and increasing osimertinib concentrations) on PC9‐OR#2 KRAS‐positive clonal cell lines (n = 3). The calculated IC₅₀ values are shown. The results are expressed as the mean ± SEM. C, PC9‐OR#2 KRAS mutant–positive clonal cell lines were treated with 1 µM osimertinib in combination with 0.03 µM trametinib for 1 wk, followed by staining with crystal violet and visual examination. D, PC9‐OR#2 KRAS mutant–positive clonal cell lines were treated with 1 µM osimertinib and/or 0.03 µM trametinib for 2 or 72 h. Changes in the expression of the indicated proteins were assessed by Western blotting. E, Clone #26 and clone #31 were treated with 1 µM osimertinib and/or 0.03 µM trametinib for 24 h. C‐caspase‐3 activity was measured using immunofluorescence

FIGURE 6

The effects of trametinib on osimertinib resistance in a mouse model of leptomeningeal carcinomatosis (LMC). A, PC9‐OR#2 Kirsten rat sarcoma (KRAS) mutant–positive clonal cell line clone #31 (4 × 10⁵ cells/0.1 mL) was injected in the leptomeningeal space of SHO‐SCID mice (n = 15). Beginning on day 8, the mice were orally administered osimertinib (25 mg/kg) and trametinib (0.6 mg/kg) every day depending on the group; the mice were randomized into four groups (control group, n = 4; trametinib group, n = 4; osimertinib group, n = 4; combination treatment group; n = 3). Luminescence was measured twice per week until the experiment was terminated. The error bars represent the SEM. Two‐sided Student's t‐test was used for comparisons between two groups. The threshold for significance was designated as P < .05, when compared with the combination treatment group. B, Representative luminescence images in mice and fluorescence in the brain lesions are shown C, Time course of mouse body weight.