Concurrent Alterations in EGFR-Mutant Lung Cancers Associated with Resistance to EGFR Kinase Inhibitors and Characterization of MTOR as a Mediator of Resistance

Abstract

Purpose: To identify molecular factors that determine duration of response to EGFR tyrosine kinase inhibitors and to identify novel mechanisms of drug resistance, we molecularly profiled EGFR-mutant tumors prior to treatment and after progression on EGFR TKI using targeted next-generation sequencing.Experimental Design: Targeted next-generation sequencing was performed on 374 consecutive patients with metastatic EGFR-mutant lung cancer. Clinical data were collected and correlated with somatic mutation data. Erlotinib resistance due to acquired MTOR mutation was functionally evaluated by in vivo and in vitro studies.Results: In 200 EGFR-mutant pretreatment samples, the most frequent concurrent alterations were mutations in TP53, PIK3CA, CTNNB1, and RB1 and focal amplifications in EGFR, TTF1, MDM2, CDK4, and FOXA1 Shorter time to progression on EGFR TKI was associated with amplification of ERBB2 (HR = 2.4, P = 0.015) or MET (HR = 3.7, P = 0.019), or mutation in TP53 (HR = 1.7, P = 0.006). In the 136 posttreatment samples, we identified known mechanisms of acquired resistance: EGFR T790M (51%), MET (7%), and ERBB2 amplifications (5%). In the 38 paired samples, novel acquired alterations representing putative resistance mechanisms included BRAF fusion, FGFR3 fusion, YES1 amplification, KEAP1 loss, and an MTOR E2419K mutation. Functional studies confirmed the contribution of the latter to reduced sensitivity to EGFR TKI in vitro and in vivoConclusions:EGFR-mutant lung cancers harbor a spectrum of concurrent alterations that have prognostic and predictive significance. By utilizing paired samples, we identified several novel acquired alterations that may be relevant in mediating resistance, including an activating mutation in MTOR further validated functionally. Clin Cancer Res; 24(13); 3108-18. ©2018 AACR.

Conflict of interest statement

Conflict of Interests: Dr. Helena Yu has consulted for AstraZeneca and Lilly. Dr. Mark Kris has consulted for AstraZeneca. Dr. Bob Li has consulted for Genentech, ThermoFisher Scientific, and Guardant Health. All other authors do not report any relevant conflicts of interest.

©2018 American Association for Cancer Research.

Figures

Figure 1.

Oncoprint of alterations identified in tumor samples from patients with EGFR-mutant lung cancer pre-treatment (green) and after treatment (pink) with an EGFR TKI. The frequency is noted on the right. The type of genetic alteration (missense, inframe, truncated, amplification, deletion, fusion) is described in the legend, and the comutations present in ≥5% of cases were included in the figure. HER2 and MET amplification were included due to their relevance in acquired resistance.

Figure 2:

Progression-free survival on EGFR TKI and overall survival from start of EGFR TKI stratified by concurrent alteration present in pre-treatment tumor sample in patients with EGFR-mutant lung cancers. (A) Time on EGFR TKI stratified by presence/absence of HER2 amplification (B) Time on EGFR TKI stratified by presence/absence of MET amplification (C) Time on EGFR TKI stratified by presence/absence of p53 mutation (D) Overall survival stratified by presence/absence of p53 mutation.

Figure 3:

(A) Enrichment of genomic alterations in tumors from patients with EGFR-mutant lung cancers prior to EGFR TKI versus after progression on EGFR TKI. The level of enrichment is represented as different in frequency between the two states (X axis) and its significance (P value, Y axis). The type of alterations is represented by color. (B, C) Cancer cell fractions of mutations based on FACETS analysis comparing pre-treatment (X axis) and post-treatment tumor samples (Y axis) in two representative patients.

Figure 4:

Mechanisms of resistance in the paired samples. These mutations are acquired alterations when comparing pre-treatment samples to samples obtained after clinical progression on an EGFR TKI.

Figure 5

A. TMB (tumor mutation burden) normalized per MB in pre-TKI cohort compared to post-TKI cohort. B: Number of concurrent mutations in pre-TKI sample and paired post-TKI sample for 38 patients with paired samples.

Figure 6:. Functional analysis of acquired mTOR E2419K mutation in elrotinib-resistant EGFR mutant non-small cell lung cancer

A. MSK-IMPACT analysis of paired samples before and after EGFR-TKI resistance revealed an acquired mTOR E2419K mutation. B. 293T cells were transiently transfected with pcDNA3 Flag-mTOR (WT, E2419K, S1483F, S2215F), vector control, or HA-S6K1. Thirty-six hours after the transfection, cells were serum starved overnight and subsequently nutrition starved in PBS for 1 hour. Lysates were subjected to immunoblotting. Band intensities were quantified using ImageJ software, and data are representative of two independent experiments (mean ± SE). ***p<0.001, compared to the respective WT+S6K group. C. 293T cells were transiently transfected with pcDNA3 mTOR (wild-type, E2419K), vector control, or HA-S6K1. Forty-eight hours after the transfection, cells were treated with everolimus (100nM), AZD8055 (500nM), BEZ235 (500nM), or erlotinib (1μM) for 3 hours and then subjected to immunoblotting. D. Isogenic stable PC9-mTOR lines were treated with increasing concentrations of erlotinib for 3 hours without serum and lysates were subjected to immunoblotting. E. A total 1.5 × 104 of cells were plated in 6-well plates, and treated with 1μM erlotinib for 14 days. The number of colonies was analyzed using ImageJ. Each experiment was assayed in duplicate determinations and data are representative of three independent experiments (mean ± SE). F. Caspase 3/7 activity was analyzed in stable PC9-mTOR lines that were treated with increasing concentrations of erlotinib for 48 hours. Each experiment was assayed in duplicate determinations and data are representative of three independent experiments (mean ± SE). *p<0.05, **p<0.01 compared to PC9-mTOR WT group. G. PC9-mTOR WT and E2419K cells were implanted into a subcutaneous flank of athymic nude mice. When tumors reached approximately 100 mm3, mice were treated with vehicle or 25 mg/kg erlotinib daily. Tumor volume was determined on the indicated days after the onset of treatment. Data represent mean ± SE (n = 5). *p<0.05, compared to the respective vehicle-treated group. #p<0.05, compared to erlotinib-treated PC9-mTOR WT group. H. PC9-mTOR E2419K cells were treated with erlotinib (1μM), AZD8055 (500nM), or a combination of erlotinib (1μM) and AZD8055 (500nM) for 3 hours. Lysates were then subjected to immunoblotting. I. PC9-mTOR E2419K cells were implanted subcutaneously into the flank of athymic nude mice. Once tumors reached approximately 100 mm3, mice were treated with vehicle, 25 mg/kg erlotinib, 20 mg/kg AZD8055, or a combination of 25 mg/kg erlotinib and 20 mg/kg AZD8055 daily. Tumor volume was determined on the indicated days after the onset of treatment. Data represent mean ± SE (n = 5). *p<0.05, compared to the vehicle treated group. #p<0.05, compared to erlotinib only treated group.