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. 2015 Sep;5(9):960-971.
doi: 10.1158/2159-8290.CD-15-0063. Epub 2015 Jun 2.

Combined EGFR/MEK Inhibition Prevents the Emergence of Resistance in EGFR-Mutant Lung Cancer

Erin M Tricker #  1   2 , Chunxiao Xu #  1   2 , Sharmeen Uddin  3   4 , Marzia Capelletti  1 , Dalia Ercan  1 , Atsuko Ogino  1 , Christine A Pratilas  3   4 , Neal Rosen  4   5 , Nathanael S Gray  6   7 , Kwok-Kin Wong  1   2   8   9 , Pasi A Jänne  1   2   8   9
Affiliations

Combined EGFR/MEK Inhibition Prevents the Emergence of Resistance in EGFR-Mutant Lung Cancer

Erin M Tricker et al. Cancer Discov. 2015 Sep.

Abstract

Irreversible pyrimidine-based EGFR inhibitors, including WZ4002, selectively inhibit both EGFR-activating and EGFR inhibitor-resistant T790M mutations more potently than wild-type EGFR. Although this class of mutant-selective EGFR inhibitors is effective clinically in lung cancer patients harboring EGFR(T790M), prior preclinical studies demonstrate that acquired resistance can occur through genomic alterations that activate ERK1/2 signaling. Here, we find that ERK1/2 reactivation occurs rapidly following WZ4002 treatment. Concomitant inhibition of ERK1/2 by the MEK inhibitor trametinib prevents ERK1/2 reactivation, enhances WZ4002-induced apoptosis, and inhibits the emergence of resistance in WZ4002-sensitive models known to acquire resistance via both T790M-dependent and T790M-independent mechanisms. Resistance to WZ4002 in combination with trametinib eventually emerges due to AKT/mTOR reactivation. These data suggest that initial cotargeting of EGFR and MEK could significantly impede the development of acquired resistance in EGFR-mutant lung cancer.

Significance: Patients with EGFR-mutant lung cancer develop acquired resistance to EGFR and mutant-selective EGFR tyrosine kinase inhibitors. Here, we show that cotargeting EGFR and MEK can prevent the emergence of a broad variety of drug resistance mechanisms in vitro and in vivo and may be a superior therapeutic regimen for these patients.

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Figures

Figure 1
Figure 1
ERK reactivation following EGFR TKI treatment. (A) Cells were treated with DMSO (Media), 100 nM WZ4002, 1 μM of WZ4002 or 1 μM gefitinib weekly (mean +/− SD, n = biological triplicates, 60 wells each). (B) PC9 cells were treated with 1 μM WZ4002 or 1 μM gefitinib and AKT and ERK1/2 phosphorylation was assessed. Hsp90 was assessed as a loading control. Image is representative of three independent experiments. (C) Cell lines were treated with 100 nM WZ4002 (W), 30 nM trametinib (T) or a combination thereof (C) for the indicated time and phosphorylation of AKT and ERK1/2 was assessed by immunoblotting. Image is representative of two independent experiments.
Figure 2
Figure 2
Acquired resistance is prevented by WZ4002 + trametinib combination treatment in TKI naive and T790M+ cell lines. (A) Cell lines were treated with increasing doses of WZ4002 alone or in combination with 30 nM trametinib (Tram) and viability was assessed by MTS (n = 3 independent experiments, mean +/− SD). TKI naive cell lines (B), T790M+ (C) and cell lines harboring non-T790M mechanisms of TKI resistance (D) were treated weekly with 100 nM WZ4002, 30 nM trametinib (Tram) or a combination thereof for 6 weeks. Arrows indicate wells did not achieve 50% confluence by six weeks. Data is representative of 2-3 independent experiments (180 wells per condition). (E) Cell lines were treated with DMSO (Media), 100 nM WZ4002, 30 nM trametinib or a combination thereof and the percentage of wells at 50% or greater confluence were assessed at 6 weeks (n = biological triplicates, 1 plate (60 wells) each). Graph is representative of two or three independent experiments per cell line. (F) PC9 cells were treated with DMSO control (Media), 100 nM WZ4002, 30 nM trametinib (Tram) or 100 nM IGF-1R inhibitor BMS754807 (BMS) or WZ4002 combinations weekly (top panel). HCC827 cells were treated with DMSO (Media), 100 nM WZ4002, 30 nM trametinib, 100 nM crizotinib (Criz) or WZ4002 combinations weekly (bottom panel). Positive wells (n = three biological replicates, 1 plate each) were assessed weekly and graphed mean +/− SD. Each experiment was repeated two times.
Figure 3
Figure 3
Effect of WZ4002 + trametinib treatment on signaling and apoptosis. (A) Cell lines were treated with DMSO (-), 100 nM WZ4002 (W), 30 nM trametinib (T) or the combination (C) for eight hours and phosphorylation of EGFR, AKT, ERK1/2 and S6 was assessed. Hsp90 was assessed as a loading control. Long term sensitivity or resistance of each cell line to W+T treatment is indicated below the figure. Image is representative of three independent experiments. (B) TKI sensitive and resistant cell lines were treated with 100 nM WZ4002 (W), 30 nM trametinib (T) or a combination thereof (C) for 24 hours and Bim levels were assessed. Hsp90 was used as a loading control. Image is representative of three independent experiments. (C) Cell lines were treated with DMSO control (Media) 100 nM WZ4002, 30 nM trametinib (Tram) or a combination thereof for 72 hours. Caspase 3 activity of lysates was measured and fold change was calculated relative to DMSO control (n = 3 to 4 independent experiments, mean +/− SD). Significance was calculated by one way ANOVA (p < 0.05).
Figure 4
Figure 4
Curative effect of WZ4002 + trametinib treatment in vivo. (A) PC9 GR4 xenograft tumors were treated with vehicle control, WZ4002, trametinib (Tram) or the combination thereof (mean +/− SEM, 5 mice per condition). Treatment cessation is indicated by the arrow. Tumor cure is defined as no residual disease beyond treatment cessation. (B) WZ4002 + trametinib treatment is also effective in the TKI naïve PC9 xenograft model (mean +/− SEM, n = 5). (C) WZ4002 + trametinib treatment prevents tumor growth in the H1975 xenograft model but does not result in tumor cures (mean +/− SEM, n = 5).
Figure 5
Figure 5
Co-targeting EGFR and MEK prolongs effective treatment duration in EGFR L858R/T790M genetically engineered mice. (A) Tumor volume after two weeks of vehicle, WZ4002, trametinib or a combination thereof. Significance was assessed by two-tailed t test, p = 0.0041. A subset of the WZ4002 alone treated mice are published in a previous report (13). (B) Phosphorylation of EGFR, AKT, ERK1/2, S6 and 4EBP1 were assessed by immunohistochemistry after two doses of vehicle, trametinib, WZ4002 or combination. H&E, hematoxylin and eosin. (C) Analysis of apoptosis by TUNEL staining after two doses of WZ4002, trametinib or a combination thereof. H&E, hematoxylin and eosin. (D) Quantification of TUNEL positive cells. Significance was assessed by one way ANOVA, p < 0.001. (E) Tumor volume after W+T treatment. Upon tumor growth, mice treated with trametinib (purple line) were treated with a combination of WZ4002 and trametinib (green line). A subset of single agent WZ4002 treated mice was previously reported (13) (n = 5, mean +/− SEM). (F) Survival of WZ4002 + trametinib treated mice compared to WZ4002 alone. Significance was determined by log-rank test, p = 0.0097. One mouse in the combination treatment group was euthanized at 12.3 weeks for a leg problem unrelated to treatment. A necropsy was performed and no tumor was observed (data not shown).
Figure 6
Figure 6
Heterogeneous resistance mechanisms develop in response to WZ4002 + trametinib combination treatment in the EGFR L858R/T790M genetically engineered mice. (A) Phosphorylation of EGFR, ERK1/2, S6 and 4EBP1 were assessed by immunohistochemistry and instances of positive nodules aside negative nodules (highlighted with dotted lines) were observed. (B) Total number of tumor nodules IHC positive (red) or negative (black) for EGFR, ERK, S6 and 4EBP1 phosphorylation. Percentage of positive cases is listed above each bar (n = 27). (C) Tumor volume of WZ4002 + trametinib resistant tumors after treatment with a combination of WZ4002, trametinib and Torin2. Yellow T indicates tumor mass. (D) Quantification of tumor volume for each mouse after initiation of WZ4002 + trametinib + Torin2 combination treatment. (E) PC9 derived cell lines tolerant to WZ4002 + trametinib (WT1, 3 and 4) were treated with 100 nM WZ4002 + 30 nM trametinib and phosphorylation of EGFR, AKT, ERK1/2 and S6 were assessed. Image is representative of two independent experiments. (F) Viability of WZ4002 + trametinib tolerant cell lines were assessed after the addition of 10 nM Torin2 to WZ4002 + trametinib co-treatment at 72 hours (n = 3 independent experiments, mean +/− SD).
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