Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1

Abstract

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

Fig. 1.

Characterization of 11-18R cells. (A) Cell growth-inhibition assays show the relative sensitivity of 11-18 and 11-18R cells to erlotinib. Data are expressed as percentage of viability compared with vehicle by the cell titer blue assay. Data shown are mean ± SD of three independent experiments performed in hextuplicate. (B) (i) EGFR knockdown experiments using siRNAs show that, compared with parental cells,11-18R cells are no longer dependent upon EGFR for survival. Data shown are mean ± SD of three independent experiments performed in hextuplicate. Scr, Scramble. Two different siRNAs against EGFR were used. (ii) Immunoblotting studies using the indicated antibodies show that downstream signaling is inhibited in parental but not resistant cells after knockdown of EGFR. (C) The effect of erlotinib on EGFR pathway signaling in 11-18/11-18R cells. (D) (i) SNaPshot assay reveals that, in addition to a baseline EGFR L858R mutation, 11-18R cells have acquired an NRAS Q61K mutation but not an EGFR T790M mutation. (ii) Direct sequencing confirms presence of the NRAS 181C > A (Q61K) mutation in 11-18R cells.

Fig. 2.

Functional role of NRAS Q61K in 11-18R cells. (A) RAS GTPase-specific pulldown assay shows increased activated NRAS in 11-18R cells compared with parental cells. Erlotinib has no effect on activated NRAS activity in 11-18R cells. (B) Cell growth-inhibition assays show the relative sensitivity of 11-18/11-18R and PC-9/PC-9R cells to erlotinib, the MEK inhibitor AZD6244, or the combination of erlotinib with AZD6244. Data shown are mean ± SD of three independent experiments performed in hextuplicate. (C) Athymic nude mice with 11-18R tumors were administered vehicle, erlotinib, MEK inhibitor, GSK1120212, or erlotinib plus GSK1120212. Tumor volume was determined at the indicated times after the onset of treatment. n = 5 mice per group. Error bars indicate SE. *P < 0.05 (Student’s t test) for the combination of erlotinib plus GSK1120212 versus either erlotinib or GSK1120212 alone. (D) siRNA-mediated knockdown of NRAS combined with erlotinib or siRNA-mediated knockdown of EGFR combined with AZD6244 inhibits growth of 11-18R cells. Scr, Scramble. Two different siRNAs against NRAS were used. Data shown are mean ± SD of three independent experiments performed in hextuplicate. *, **P < 0.05 (Student’s t test) for the combination of erlotinib plus siNRAS knockdown versus erlotinib or siNRAS alone in 11-18R cells. ***P < 0.05 (Student’s t test) for the combination of siEGFR plus AZD6244 knockdown versus AZD6244 alone in 11-18R cells. (E) Immunoblotting with the indicated antibodies demonstrates that siRNA-mediated knockdown of NRAS combined with erlotinib inhibits ERK activation in 11-18R cells. These samples were run on the same gel but were noncontiguous.

Fig. 3.

Ectopic expression of NRAS Q61K or BRAF V600E in EGFR mutant cells mediates resistance to EGFR TKIs. (A and B) PC-9 cells were transiently transfected with expression plasmids encoding NRAS wild type or NRAS Q61K (A) or BRAF wild type or BRAF V600E (B) and were cultured in the absence or presence of erlotinib for 6 h. Corresponding cell lysates were subjected to immunoblotting with the indicated antibodies. Lysates from cells harboring NRAS wild type or NRAS Q61K displayed higher levels of total NRAS than seen in control-transfected cells, but only cells transfected with NRAS Q61K displayed enhanced phospho-ERK expression in the presence of erlotinib. Similarly, lysates from cells harboring BRAF wild type or BRAF V600E displayed higher levels of total BRAF than seen in control-transfected cells, but only cells transfected with BRAF V600E displayed enhanced phospho-ERK expression in the presence of erlotinib. (C and D). PC-9 cells were stably transfected with control plasmids or expression plasmids encoding NRAS Q61K (C) or BRAF V600E (D). Ectopic expression of NRAS Q61K (C) or BRAF V600E (D) mediated resistance of PC-9 cells to erlotinib. C13, clone 13; C21, clone 21; C5, clone 5; C6, clone 6. Data shown are mean ± SD of three independent experiments performed in hextuplicate.

Fig. 4.

MEK inhibition restores the sensitivity of PC-9/NRAS Q61K or PC-9/BRAF V600E stable clones to erlotinib. C13, clone 13; C21, clone 21; C5, clone 5; C6, clone 6. (A and C) The combination of erlotinib and GSK1120212 leads to greater inhibition of cell growth in PC-9 cells stably expressing NRAS Q61K (PC-9/NRAS Q61K cells) (A) or BRAF V600E (PC-9/BRAF V600E cells) (C) than seen either drug alone. Data shown are mean ± SD of three independent experiments performed in hextuplicate. (B and D) In stable transfectants the combination of erlotinib plus GSK1120212 leads to a greater reduction in phospho-ERK levels than either drug alone. PC-9/NRAS Q61K (B) or PC-9/BRAF V600E (D) cells were cultured in the absence or presence of erlotinib or/and GSK1120212 for 6 h; corresponding cell lysates were subjected to immunoblotting with the indicated antibodies.

Fig. P1.

Cell-proliferative signaling pathways in lung tumors. EGFRs signal through the PI3K/AKT and RAS/MEK/ERK cascades. (i) WT EGFRs are activated in a ligand-dependent manner. (ii) Mutant EGFRs are constitutively activated. EGFR TKIs block kinase activity from mutant EGFRs and inhibit downstream signaling. (A and D) Acquisition of NRAS or BRAF mutations (mts) leads to constitutive activation of the RAS/MEK/ERK pathway. (B and E) EGFR-TKIs inhibit EGFR, but mutant NRAS or BRAF maintain downstream ERK activation. (C) The combination of EGFR and MEK inhibitors can overcome resistance caused by the acquisition of NRAS mutations in EGFR-mutant cells. (F) The combination of EGFR and BRAF inhibitors or EGFR and MEK inhibitors can overcome resistance caused by the acquisition of BRAF mutations in EGFR-mutant cells.