The EGFR-mutant NSCLC cell lines, HCC827, PC-9 and gefitinib-resistant PC-9/GR (T790M), were provided by Dr. P. A. Jänne (Dana Faber Cancer Institute, Boston, MA) in 2009. The EGFR-wild type NSCLC cell lines, H226 and H596, were originally obtained from Dr. R. Lotan (M. D. Anderson Cancer Center, Houston, TX) in 2003. Erlotinib-resistant HCC827/ER and AZD9291-resistant HCC827/AR cell lines were established in our laboratory and described previously (14 (
link),17 (
link)). The AZD9291-resistant cell lines, PC-9/AR and PC-9/GR/AR, were newly established in our laboratory by exposing PC-9 or PC-9/GR cells to gradually increasing concentrations of AZD9291 (starting at 10 nM and ending with 500 nM) for approximately 6 months (
Fig. S1). These cell lines were routinely cultured in the presence of 500 nM AZD9291 and maintained resistance to AZD9291 even after withdrawal of AZD9291 from the culture medium for over 6 months, indicating an irreversible phenotype.
MET gene amplification and protein hyperactivation were detected in HCC827/ER and HCC827/AR cells (14 (
link)). Using ICE COLD-PCR developed by Transgenomic, Inc (Omaha, NE), EGFR exon 20 T790M mutation was detected in PC-9/GR/AR cells. However, C797S mutation in EGFR exon 20 and other mutations around codon C797S region and in EGFR exon 21 were not detected in HCC827/AR, PC-9/AR and PC-9/GR/AR cells (
Fig. S1). Moreover, no K-RAS exon 2 mutations were detected in these cell lines. HCC827/Mcl-1 and PC-9/Mcl-1 stable cell lines (pooled populations) were generated by infecting cells with lentiviruses carrying ectopic Mcl-1 or vector for 48 h followed with selection with zeocin (500 ng/ml) for another 7 days as described previously (18 (
link)). PC-9 cell line with EGFR 19del+T790M+C797S triple mutation used in a previous study (12 (
link))(we named it PC-9/3M) was kindly provided by Dr. A. N. Hata (Harvard Medical School, Boston, MA) on January of 2017. This cell line was resistant to AZD9291 as well as CO1686 and erlotinib (
Fig. S2), thus confirming its resistance to these EGFR inhibitors. These cell lines were not genetically authenticated. Mycoplasma was detected annually or upon receiving using MycoAlert@ Mycoplasma Detection Kit (Lonza; Rockland, ME) to ensure mycoplasma negative. All cell lines were cultured in RPMI 1640 containing 5% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO
2 and 95% air.
Shi P., Oh Y.T., Deng L., Zhang G., Qian G., Zhang S., Ren H., Wu G., Legendre B J.r., Anderson E., Ramalingam S.S., Owonikoko T.K., Chen M, & Sun S.Y. (2017). Overcoming acquired resistance to AZD9291, a third generation EGFR inhibitor, through modulation of MEK/ERK-dependent Bim and Mcl-1 degradation. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(21), 6567-6579.
