MET or NRAS amplification is an acquired resistance mechanism to the third-generation EGFR inhibitor naquotinib

As a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), osimeritnib is the standard treatment for patients with non-small cell lung cancer harboring the EGFR T790M mutation; however, acquired resistance inevitably develops. Therefore, a next-generation treatment strategy is warranted in the osimertinib era. We investigated the mechanism of resistance to a novel EGFR-TKI, naquotinib, with the goal of developing a novel treatment strategy. We established multiple naquotinib-resistant cell lines or osimertinib-resistant cells, two of which were derived from EGFR-TKI-naïve cells; the others were derived from gefitinib- or afatinib-resistant cells harboring EGFR T790M. We comprehensively analyzed the RNA kinome sequence, but no universal gene alterations were detected in naquotinib-resistant cells. Neuroblastoma RAS viral oncogene homolog (NRAS) amplification was detected in naquotinib-resistant cells derived from gefitinib-resistant cells. The combination therapy of MEK inhibitors and naquotinib exhibited a highly beneficial effect in resistant cells with NRAS amplification, but the combination of MEK inhibitors and osimertinib had limited effects on naquotinib-resistant cells. Moreover, the combination of MEK inhibitors and naquotinib inhibited the growth of osimertinib-resistant cells, while the combination of MEK inhibitors and osimertinib had little effect on osimertinib-resistant cells. Clinical assessment of this novel combination (MEK inhibitors and naquotinib) is worth considering in osimertinib-resistant lung tumors.


Short Tandem Repeat Polymerase Chain Reaction Analysis
The genetic identity between each resistant cell line and parental cell line was confirmed by short tandem repeat polymerase chain reaction analysis using the AuthentiFiler PCR Amplification Kit Inhibitory effect of naquotinib on cell proliferation and the EGFR signaling pathway in PC-9 and RPC-9 cells.
Error bars, SE. All experiments were performed in triplicate. B. Effects of naquotinib or gefitinib on EGFR pathway signaling in PC-9, RPC-9, and PC-9/BRc1 cells. Each cell line was incubated with naquotinib or gefitinib (0 to 1.0 µmol/L) for 4 h.
A. Schematic. Parental cells were cultured with increasing concentrations of naquotinib (beginning at half of the IC50) to establish naquotinib-resistant cell lines. The dose was increased to 1.0 µmol/L in a stepwise pattern. Fresh drug was added every 96 h. Clones were derived from PC-9/NaqR and RPC-9/NaqR cells using a single-cell cloning method. B. Time course of the development of naquotinib-resistant cell lines. C. Identical test by short tandem repeats analysis between parental and resistant cells. D. Feature of the epithelial-mesenchymal transition in each naquotinib-resistant cell line. All cells were treated with 1.0 µmol/L naquotinib for 4 h. 293T cell lysate was used as an indicator of the epithelial-mesenchymal transition.

Supplementary Figure S3.
A to C. Direct sequencing of EGFR exons 19 and 20 in naquotinib-resistant cell lines. A. PC-9 and PC-9/NaqR cells. B. RPC-9 and RPC-9/NaqR cells. C. PC-9/BRc1 and PC-9/BRc1/NaqR cells. D and E. Targeted RNA-sequencing analysis of 612 kinases and kinase-related genes from parental and naquotinib-resistant cell lines. D. Changes in the RPKM levels of PC-9/NaqR cells against PC-9 cells. The top 10 upregulated genes relative to parental cells are listed. E. Changes in the RPKM levels of PC-9/BRc1/NaqR cells against PC-9/BRc1 cells. The top 10 upregulated genes relative to parental cells are listed.
A. Co-inhibitory effect of MET inhibitors and EGFR-TKIs in PC-9/NaqR cells. Inhibitory effect of EGFR-TKIs and MET inhibitors on cell proliferation in PC-9/NaqR cells. All drugs were exposed at 1.0 µmol/L for 96 h.
B. Effect of naquotinib on the EGFR signaling pathway in PC-9 and PC-9/NaqR cells. Each cell line was incubated with naquotinib (0 to 1.0 µmol/L) for 4 h. C. Effect of naquotinib on the EGFR signaling pathway in RPC-9 and RPC-9/NaqR cells. Each cell line was incubated with naquotinib (0 to 1.0 µmol/L) for 4 h.
A. Phospho-RTK arrays in HCC827 and HCC827/NaqR cells. Resistant cells were cultured in the absence of naquotinib for 5 days. B. FISH analysis of the MET gene in HCC827 and HCC827/NaqR cells. Red, MET gene; green, CEP7 gene. C. MET copy number. DNA derived from HCC827 and HCC827/NaqR cells. Each copy number relative to GAPDH was determined by qPCR. Error bars, SE. ***, p < 0.001. All experiments were performed in triplicate. D. Inhibitory effect of EGFR-TKIs and MET inhibitors on cell proliferation in HCC827/NaqR cells. All drugs were exposed at 1.0 µmol/L for 96 h. Data are presented as the mean ± SE from three independent experiments. **, p < 0.01; ***, p < 0.001. E. Effects of EGFR-TKIs and MET inhibitors on the EGFR pathway in HCC827/NaqR cells. All drugs were exposed at 1.0 µmol/L for 4 h. F. Immunohistochemistry of MET in HCC827 and HCC827/NaqR cells. Scale bars, 50 µm.
A. Phospho-RTK arrays in RPC-9 and RPC-9/NaqR cells. Resistant cells were cultured in normal medium in the absence of naquotinib for 4 days. B. Changes in the RPKM levels of RPC-9/NaqR cells relative to RPC-9 cells by RNA-sequencing transcriptome analysis for 612 kinases and kinaserelated genes. Key genes related to the PI3K/AKT signaling are shown. C. Direct sequencing of NRAS exons 2 and 3 in RPC-9 and RPC-9/NaqR cells. D and E. Effects of the combination of EGFR-TKIs and selumetinib in RPC-9/NaqR cells using the PathScan AKT Pathway Array Kit. All drugs were exposed at 1.0 µmol/L for 4 h. Expression levels shown in the bar graph were derived from the average of two dots from the PathScan AKT Pathway Array Kit.
A. Cellular proliferation of RPC-9/OsiR cells treated with the indicated concentrations of EGFR-TKIs. Error bars, SE. All experiments were performed in triplicate. B. Direct sequencing analysis of EGFR exon 20 in RPC-9/OsiR cells. C. Phospho-RTK arrays in RPC-9 and RPC-9/OsiR cells.
Resistant cells were cultured in normal medium for 4 days. D. Effects of osimertinib on NRAS expression and activation in RPC-9 and RPC-9/OsiR cells. Each cell line was incubated with osimertinib (0 or 1.0 µmol/L) for 4 h. Cell lysates were analyzed by Western blotting. E.

Supplementary Figure S8.
Uncropped immunoblot images for Figs

A C G C A G C T C A T G C C C T T C G G C T G C T
Thr 790

Cys 797 Gln Gly Ile
Pro Phe

Leu Gly Ile
Pro Phe Gln EGFR Exon20 B

RPC-9 A C G C A G C T C A T G C C C T T C G G C T G C T
Thr 790

Cys 797 Gln Gly Ile
Pro Phe

Leu Gly Ile
Pro Phe Gln EGFR Exon20 PC-9 PC-9/NaqR G G C T C C C T G Gly 796 Cys 797 Leu 798 EGFR Exon20

Thr
Glu 63 Gly Gln 61 Glu Ala 59 Asp 57 A T