The EGFR C797S Mutation Confers Resistance to a Novel EGFR Inhibitor CLN-081 to EGFR Exon 20 Insertion Mutations

Abstract

Introduction: EGFR exon 20 insertion mutations account for 5% to 10% of EGFR-mutated NSCLC. CLN-081 (formerly known as TAS6417), a novel covalent EGFR tyrosine kinase inhibitor, exhibits pan-mutation selective efficacy, including exon 20 insertions, in the clinical setting. Nevertheless, some patients may not respond to CLN-081 and resistance to CLN-081 may emerge over time in others.

Methods: We exposed Ba/F3 cells transduced with EGFR exon 20 insertions (Y764_V765 insHH or A767_S768insSVD) to increasing concentrations of CLN-081 to generate resistant cells and then subjected their complementary DNA to sequencing to identify acquired mutations. We then evaluated effects of small molecules on engineered Ba/F3 cells on the basis of proliferation assays, Western blotting, and xenograft models.

Results: All CLN-081 resistant clones harbored the EGFR C797S mutation. Ba/F3 cells with C797S (Ba/F3-C797S) were resistant to EGFR tyrosine kinase inhibitors targeting EGFR exon 20 insertion mutations, including CLN-081. Pimitespib, a selective heat shock protein 90 inhibitor, induced apoptosis in Ba/F3-C797S cells in vitro and inhibited growth of Ba/F3-C797S tumors in vivo. Ba/F3 cells with A763_Y764insFQEA-C797S remained sensitive to erlotinib.

Conclusions: We conclude that the EGFR C797S mutation confers resistance to CLN-081. Our preclinical data suggest a potential small molecule to overcome CLN-081 resistance, which may benefit patients with lung cancer with EGFR exon 20 insertions.

Keywords: C797S; CLN-081; EGFR; Non–small cell lung cancer; Resistant mechanism.

Figure 1

Chronic exposure to CLN-081 induces the C797S mutation in Ba/F3 cells harboring EGFR exon 20 insertions. (A) Parental and CLN-081–resistant Ba/F3 clones (Ba/F3-CR) expressing Y764_V765insHH or A767_S768insSVD were seeded on 96-well plates and treated with various concentrations of CLN-081 for 48 hours. Cell viability was measured using the Cell Counting Kit-8. Results are illustrated as means ± SD from three independent experiments. CLN-081 sensitivity of Ba/F3-insHH-CR or Ba/F3-insSVD-CR cells was higher than that of the parental cells. (B) Sequencing chromatogram of RT-PCR products from parental and CLN-081–resistant Ba/F3 clones expressing insHH or insSVD. Note that all resistant clones exhibit the C797S (c.2390G>C) point mutation in both insertions. #, number; RT-PCR, reverse-transcriptase polymerase chain reaction.

Figure 2

The C797S mutation confers resistance to EGFR TKIs targeting EGFR exon 20 insertions. (A) Cell viability assay of Ba/F3-insHH cells, with or without C797S (left), or Ba/F3-insSVD cells, with or without C797S (right), treated with or without varying CLN-081 concentrations for 48 hours. (B) Western blotting revealing phospho- and total EGFR levels after CLN-081 treatment of Ba/F3-insHH, Ba/F3-insHH-C797S, Ba/F3-insSVD, or Ba/F3-insSVD-C797S cells. All cells were incubated for 3 hours with CLN-081 at indicated concentrations. (C) Cell viability assay using Ba/F3-insHH, Ba/F3-insHH-C797S, Ba/F3-insSVD, or Ba/F3-insSVD-C797S cells treated with various concentrations of EGFR TKIs for 48 hours. pEGFR, phosphorylated EGFR; TKI, tyrosine kinase inhibitor.

Figure 3

HSP90 inhibition overcomes CLN-081 resistance conferred by C797S mutation. (A) Western blotting revealing phosphor- and total EGFR levels after pimitespib treatment of Ba/F3-insSVD or Ba/F3-insSVD-C797S cells. All cells were incubated 3 hours with pimitespib at indicated concentrations. (B) Western blotting of factors downstream of EGFR after pimitespib treatment of Ba/F3-insSVD-C797S cells. All cells were incubated 24 hours with pimitespib at indicated concentrations. (C) Cell viability assay of Ba/F3-insHH, Ba/F3-insHH-C797S, Ba/F3-insSVD, or Ba/F3-insSVD-C797S cells treated 48 hours with pimitespib. (D) Effects of pimitespib, CLN-081, or vehicle in mouse xenograft tumor models harboring Ba/F3-insSVD-C797S cells. Nude mice bearing Ba/F3-insSVD-C797S cells were treated with pimitespib (n = 10: 14 mg/kg orally once daily), CLN-081 (n = 10: 100 mg/kg orally once daily), or vehicle (n = 4) for 14 days and monitored for changes in tumor volume. Data are presented as the mean % change in tumor volume ± SD. Asterisks indicate p value: ∗ < 0.05. NS, not significant. (E) Survival of mice (%) described in (D). pAKT, phosphorylated AKT; pEGFR, phosphorylated EGFR; pERK, phosphorylated ERK.

Figure 4

Cells harboring EGFR A763_Y764insFQEA remain erlotinib sensitive. (A) Cell viability assay of Ba/F3-insFQEA or Ba/F3-insFQEA-C797S cells treated with various concentrations of EGFR TKIs for 48 hours. (B) IC₅₀ ratios (Ba/F3-insFQEA-C797S relative to Ba/F3-insFQEA) of indicated drugs based on cell viability assays described in (A). (C) Western blotting revealing EGFR and downstream signaling factors after erlotinib treatment of Ba/F3-insFQEA-C797S cells. All cells were incubated 24 hours with pimitespib at indicated concentrations. (D) Apoptosis assay using annexin V staining of Ba/F3-insFQEA-C797S cells treated with CLN-081, erlotinib, or vehicle control for 24 hours. Data are illustrated as means ± SD. Asterisks indicate the p value: ∗∗∗ < 0.005. (E) Effects of erlotinib or vehicle in mouse xenograft tumor models harboring Ba/F3-insFQEA-C797S cells. Nude mice bearing Ba/F3-insFQEA-C797S cells were treated with erlotinib (n = 6: 50 mg/kg orally once daily) or vehicle (n = 6) for 12 days and monitored for changes in tumor volume. Tumor volume is illustrated as the mean ± SD. (F) Weight of tumors in mice analyzed in (E). Values are presented as means ± SD. IC₅₀, Concentration that inhibits 50%; pAKT, phosphorylated AKT; pEGFR, phosphorylated EGFR; pERK, phosphorylated ERK; TKI, tyrosine kinase inhibitor.