Shang-Gin Wu

Supplementary Figure S2. Drug sensitivity of osimertinib combined with capmatinib in PE5345, PE5345 os/cp R and PE5867 cells. Cells were exposed to a fixed osimertinib concentration at 1 μM with the indicated concentration of capmatinib for 96 hr and viability was assessed by the CCK8 assay.

Mesenchymal–epithelial transition factor (MET) amplification is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mechanism that may be overcome by dual inhibition of EGFR and MET. However, acquired resistance after dual inhibition still occurs, and the mechanisms remain largely unknown. We analyzed resistance mechanisms after dual inhibition for MET amplification–mediated EGFR TKI resistance in lung cancer. Patient-derived cell lines were established before and after acquired resistance to dual inhibition, if possible. Potential resistance mechanisms were evaluated through signal pathway analysis and next-generation sequencing. Of 12 patients with resistance after EGFR/MET inhibition, 5 (42%) acquired oncogenic fusions (3 BRAF fusions, 1 ALK fusion, and 1 RET fusion), 1 (8%) developed EGFR T790M, 1 (8%) exhibited EGFR C797S, and 1 (8%) transformed to small cell. The EGFR T790M and ALK fusion responded to subsequent targeted therapies. Serial cell lines from a patient with EGFR L858R and MET amplification (PE5345), after resistance to osimertinib and capmatinib through gradual escalation of drug exposure in vitro (PE5345 os/cp R) and after clinical resistance to osimertinib and capmatinib (PE5867), were propagated. Novel acquired general transcription factor IIi–BRAF fusions emerged in PE5867. Osimertinib plus trametinib reversed resistance. PE5345 os/cp R overexpressed EGFR, which was inhibited by afatinib. Additionally, amivantamab induced significant antibody-dependent cell-mediated cytotoxicity (ADCC) against PE5345, PE5867, and PE5345 os/cp R. We found that resistance after dual inhibition is heterogeneous, but some might still be targetable. Amivantamab induced significant ADCC against both MET-amplified EGFR TKI-resistant cancer and resistant cells after dual EGFR/MET inhibition. Understanding resistance mechanisms may provide clues for subsequent therapy.

Supplementary Figure S1. Cell line authentication by short tandem repeat (STR) profiling. (A) PE5345 (B) PE5867 (C) PE5345 os/cp R These three cell lines are homologous, and comparison with CLASTR 1.5.0 revealed no match to any known cell lines.

Supplementary Figure S3. Expression of cleavage of PRAP and caspase 3 in PE5867 cells treated with osimertinib, capmatinib and either (A) trametinib or (B) AZD6244. Cells were treated for 16h with trametinib (5nM) or AZD6244 (0.5 μM) alone, osimertinib (5nM) plus capmatinib (5nM), or a combination of trametinib or AZD6244 with osimertinib and capmatinib. The expression levels of the cleavage of PRAP and caspase 3 were examined.

Supplementary Figure S4. Drug sensitivity of osimertinib combined with trametinib in PE5867 cells. Cells were exposed to a fixed osimertinib concentration at 1 μM or vehicle, with the indicated concentration of trametinib for 96 hr and viability was assessed by the CCK8 assay.

Supplementary Table S1. Primers used in this study

Supplementary Figure S5. Expression of MET, phosphorylated MET (p-MET), EGFR, phosphorylated EGFR (p-EGFR), HER2 and phosphorylated HER2 (p-HER2) in PE5345 and PE5345 os/cp R cells. The presented data are representative of three independent experiments.

Supplementary Figure S6. IGV illustration of the break points of (A) GTF2I and (B) BRAF genes in PE5867 cells (upper row) and PE5345 cells (lower row). (A) In the GTF2I region, fusion reads were detected exclusively in PE5867. GTF2I is not routinely sequenced by TSO500 and there were not any reads in PE5345. (B) In the BRAF region, fusion reads were detected exclusively in PE5867.

Supplementary Table S2. Reagents used in this study