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The Epidermal Growth Factor Receptor-L861Q Mutation Increases Kinase Activity without Leading to Enhanced Sensitivity Toward Epidermal Growth Factor Receptor Kinase Inhibitors

      Introduction:

      Mutations in the epidermal growth factor receptor (EGFR) kinase domain such as EGFR-L858R and EGFR-G719S have been reported to activate the kinase and also sensitize a subset of patients with non-small cell lung cancer to EGFR kinase inhibitor treatment. Nevertheless, for other common point mutations such as EGFR-L861Q, it is unclear whether and to what extent they sensitize toward gefitinib and erlotinib. Thus far, there is no reliable cellular assay to compare in a ligand-independent manner intrinsic kinase activity and drug sensitivity of the unmutated (wild type) and mutated EGFR kinase domain.

      Methods:

      To overcome this obstacle, we introduced L858R, G719S, and L861Q into the backbone of EGFRvIII. EGFRvIII has a wild type-kinase domain but is activated in a ligand-independent manner through a deletion in the extracellular domain.

      Results:

      Using this tool, we show that the L861Q mutation displays enhanced kinase activity and transforming potential compared with L858R, G719S, and also to the wild type-EGFR kinase domain. Interestingly, L861Q does not increase drug sensitivity toward clinically used EGFR kinase inhibitors in contrast to the L858R and G719S mutation. In addition, we demonstrate that EGFR-L861Q could be effectively inhibited with the irreversible second-generation EGFR inhibitor WZ-4002.

      Conclusions:

      Thus, in the common EGFR-L861Q mutation, activation of the kinase domain is uncoupled from a sensitizing effect toward clinically approved kinase inhibitors. Therefore, patients with EGFR-L861Q may not have the same clinical benefit from gefitinib/erlotinib treatment as patients with EGFR-L858R and EGFR-G719S mutations. Treatment with irreversible second-generation kinase inhibitors such as WZ-4002 may be an attractive option in the future for patients with EGFR-L861Q.

      Key Words:

      A subset of patients with non-small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) kinase domain was shown to respond to targeted therapy.
      • Lynch TJ
      • Bell DW
      • Sordella R
      • et al.
      Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.
      Nevertheless, not all mutations in the EGFR kinase domain are activating and/or are drug sensitive.
      • Sharma SV
      • Bell DW
      • Settleman J
      • et al.
      Epidermal growth factor receptor mutations in lung cancer.
      • Kobayashi S
      • Boggon TJ
      • Dayaram T
      • et al.
      EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.
      • Greulich H
      • Chen TH
      • Feng W
      • et al.
      Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      Recently, we have established a comprehensive profile of kinase activity, transforming ability, and drug sensitivity of a panel of EGFR kinase domain mutations.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      We have shown that mutations L858R and G719S are very sensitive to all the drugs tested, whereas another common mutation L861Q is relatively insensitive towards inhibitor treatment.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      This is in agreement with a previous report.
      • Lee JC
      • Vivanco I
      • Beroukhim R
      • et al.
      Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.
      So far, it was not possible to compare the drug sensitivity of ligand-independent mutant EGFR with that of EGF-dependent wild-type EGFR in a ligand-independent cellular assay.
      • Jiang J
      • Greulich H
      • Janne PA
      • et al.
      Epidermal growth factor-independent transformation of Ba/F3 cells with cancer-derived epidermal growth factor receptor mutants induces gefitinib-sensitive cell cycle progression.
      Thus, we aimed to establish a cell-based system to compare the drug sensitivities of wild type (wt) and mutant receptor kinase domains.
      EGFRvIII is an active oncogenic kinase, which is frequently found in glioblastoma patients.
      • Pedersen MW
      • Meltorn M
      • Damstrup L
      • et al.
      The type III epidermal growth factor receptor mutation. Biological significance and potential target for anti-cancer therapy.
      EGFRvIII lacks part of the extracellular ligand-binding domain leading to constitutive, ligand-independent activation.
      • Pedersen MW
      • Meltorn M
      • Damstrup L
      • et al.
      The type III epidermal growth factor receptor mutation. Biological significance and potential target for anti-cancer therapy.
      In a recent report, we have successfully used EGFRvIII to demonstrate that certain EGFR mutants that were reported in patients with NSCLC in fact are kinase dead.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      Because EGFRvIII contains a wt-kinase domain and does not require ligand for activation, it can be used as test backbone to study the effect of mutations on kinase activity and inhibitor sensitivity in cells in a ligand-independent manner.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      Using this system in this study, we compared the kinase activity and transforming ability of common EGFR mutations.

      MATERIALS AND METHODS

      Constructs, Cell Lines, and Reagents

      Cloning of MiGR1-EGFRvIII was described previously.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      Point mutations were introduced into MiGR1-EGFRvIII using the Quickchange Site-Directed Mutagenesis kit (Fermentas, St. Leon-Rot, Germany) and confirmed by sequencing. HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (PAA, Pasching, Austria) supplemented with 10% fetal calf serum (PAA). Ba/F3 cells were cultured in Roswell Park Memorial Institute 1640 (Life Technologies) supplemented with interleukin-3 (IL-3; R&D, Wiesbaden, Germany). Stable Ba/F3 cell lines were established by retroviral infection with MiGR1-EGFRvIII constructs followed by IL-3 withdrawal. Gefitinib was provided by AstraZeneca, and AEE 788 was a kind gift from Novartis Pharma. Erlotinib was purchased from pharmacy. WZ-4002 was purchased from Axon Medchem BV. Each compound was dissolved in dimethyl sulfoxide to make an initial stock solution of 10 mmol/liter (gefitinib, AEE 788 and WZ-4002) and 2.5 mmol/liter (erlotinib).

      Proliferation Assay, Soft Agar Assay, and Western Blotting

      For proliferation analysis, Ba/F3-EGFRvIII cells (1 × 104) were plated into 96-well plates, and inhibitors were added at indicated concentrations. Cell proliferation was measured at 48 hours using CellTiter96 Proliferation Assay (Promega, Madison, WI).
      To test anchorage-independent growth in soft agar, 1 ml of 0.75% agar (Difco, Lawrence, KS) solution in Iscove's Modified Dulbecco's Medium (Life Technologies) was first placed as a bottom layer in six-well plates; 2.5 × 104 cells were seeded in 3 ml containing 0.3% agar. Colonies were stained with 3-(4,5-dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide (Sigma, Taufkirchen, Germany) and counted.
      HEK293 cells were transiently transfected with wild-type or mutant MiGR1/EGFRvIII using Lipofectamine 2000 reagent. Cell lysates were prepared for biochemical analysis. Cell lysates of stable Ba/F3 cell lines expressing wild-type or mutant EGFRvIII were prepared after treatment with inhibitors at indicated concentrations for 30 minutes. Cells lysis, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blotting were performed as described previously.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      Following antibodies were used for analysis: phosphorylated EGFR-Tyr1068 (Cell Signaling, Danvers, MA), p-EGFR-Tyr845 (Santa Cruz Biotechnoloy, Heidelberg, Germany), EGFR (Santa Cruz Biotechnology), pStat5-Tyr694 (Cell Signaling), Stat5 (Santa Cruz Biotechnology), pAkt-Ser473 (Cell Signaling), and Akt1/2 (Santa Cruz Biotechnology). Blots were scanned and subjected to analysis using ImageJ software.

      RESULTS AND DISCUSSION

      The most common point mutations reported in patients with NSCLC, EGFR-L858R, EGFR-G719S, and EGFR-L861Q, were selected for this study. Our aim was to determine kinase activity and transforming potential of these mutants compared with the wt-EGFR kinase domain. Using the wt EGFR as reference has several limitations: ligand stimulation and the required serum starvation of cells may alter cellular responses. In addition, the wt receptor cannot be used as comparison for the transforming potential of certain EGFR mutants. The use of EGFRvIII as reference abrogates several of these limitations. EGFRvIII contains a wt-kinase domain and, thus, can be used to study the impact of mutations on the kinase activity in an unaltered cellular setting.
      Therefore, we first cloned all three point mutations into the EGFRvIII backbone. For analysis of kinase activity and signaling, we chose HEK293 cells, which lack endogenous EGFR.
      • Kancha RK
      • von Bubnoff N
      • Peschel C
      • et al.
      Functional analysis of epidermal growth factor receptor (EGFR) mutations and potential implications for EGFR targeted therapy.
      • Sakai K
      • Arao T
      • Shimoyama T
      • et al.
      Dimerization and the signal transduction pathway of a small in-frame deletion in the epidermal growth factor receptor.
      Both EGFRvIII-G719S and EGFRvIII-L858R showed a two- to four-fold increased autophosphorylation compared with EGFRvIII containing a wt-kinase domain (Figures 1A, B). EGFRvIII-L861Q showed the strongest autophosphorylation, which was more than 10-fold higher than EGFRvIII (Figures 1A, B). This data are in agreement with studies using the wt-EGFR receptor with ligand stimulation.
      • Chen YR
      • Fu YN
      • Lin CH
      • et al.
      Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants.
      EGFRvIII-L861Q was also the strongest activator of Stat5 indicating that not only autophosphorylation but also substrate phosphorylation is enhanced by this mutation. None of the EGFRvIII constructs increased Akt activity above levels observed under normal serum conditions (Figure 1A).
      Figure thumbnail gr1
      FIGURE 1EGFRvIII-based assay identifies L861Q as hyperactive kinase with strongest oncogenic potential. A, Clinically relevant point mutations in EGFRvIII backbone were transiently overexpressed in HEK293 cells and analyzed for autokinase activity and Stat5 activation. B, Comparative activation of EGFR and Stat5 was quantified and plotted as fold increase in activity with respect to wild type. Analysis was done in triplicates, and measured “p values” were shown. C, Ba/F3 cells stably expressing EGFRvIII mutants were tested for their ability to outgrow untransduced cells on IL-3 withdrawal. Percentage of eGFP-positive cells was measured in triplicates at indicated time points by FACS analysis. D, NIH/3T3 cells stably expressing wild-type or mutant EGFRvIII were plated in soft agar assay at a density of 2.5 × 104 cells/well in triplicates in a six-well plate. Three weeks after plating, colonies were stained with MTT solution and counted. E, Average number of colonies for each cell line was shown. EGFR, epidermal growth factor receptor; IL, interleukin; eGFP, enhanced green fluorescent protein; FACS, fluorescence activated cell sorting; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide.
      To compare the transforming abilities of these mutants, we performed a competitive growth assay, in which the outgrowth of oncogene-transduced cells under growth factor withdrawal in a mixed population is measured. To this end, newly transduced (transduction efficiency ∼20%), unselected stable Ba/F3 cell lines expressing EGFRvIII mutants together with enhanced green fluorescent protein were deprived of IL-3. Outgrowth of enhanced green fluorescent protein-positive cells was measured by fluorescence activated cell sorting analysis over time. EGFRvIII-L861Q expressing cells showed the strongest proliferation advantage in this competition assay (Figure 1C). EGFRvIII-G719S and EGFRvIII-L858R expressing cells were also selected more efficiently than cells expressing unmutated EGFRvIII (Figure 1C). After prolonged IL-3 deprivation, however, all EGFRvIII constructs finally conferred IL-3 independent growth. Thus, mutation L861Q leads to the strongest gain in kinase activity and most rapid induction of cytokine independent growth compared with the wt-kinase domain. To test the transforming potential of EGFRvIII-based mutants in an independent assay, NIH/3T3 cells were transduced with wild-type or mutant EGFRvIII kinase, and stable cell lines were used for a soft agar assay. EGFRvIII with wild-type kinase induced anchorage- and growth factor-independent colony formation, whereas no colonies were detected in untransduced or vector-transduced cells (Figures 1D, E). Moreover, cells expressing EGFRvIII + G719S, EGFRvIII + L858R, and EGFRvIII + L861Q formed higher numbers of colonies compared with cells expressing unmutated EGFRvIII kinase (Figures 1D, E).
      We then determined the effect of oncogenic EGFR kinase domain mutations on drug sensitivity. We used stably transduced Ba/F3 cell lines expressing wild-type or mutant EGFRvIII and the EGFR kinase inhibitors gefitinib, erlotinib, and AEE788. Gefitinib and erlotinib are selective EGFR inhibitors already approved in the clinic. AEE788 is a compound in development inhibiting both EGFR and Her2. EGFRvIII-L858R expressing cells were very sensitive to all drugs tested with an half maximal inhibitory concentration (IC50) value of less than 50 nM (Figures 2AC). EGFRvIII-G719S expressing cells displayed an intermediate sensitivity. Both mutations led to lower IC50 values compared with wild-type EGFRvIII, thus sensitizing cells to EGFR kinase inhibitors (Table 1). In contrast, EGFRvIII-L861Q expressing cells displayed IC50 values identical to wt EGFRvIII (Figures 2AC). Although this mutation induced the strongest proliferation advantage and highest increase in kinase activity among all mutations tested, it did not lead to enhanced kinase inhibitor sensitivity (Figure 2D). Western blot analysis after treatment of Ba/F3 cells expressing EGFRvIII constructs with gefitinib or erlotinib showed that EGFRvIII-G719S and EGFRvIII-L858R but not EGFRvIII-L861Q displayed enhanced inhibition of downstream Stat5 signaling compared with the wild-type EGFRvIII (Figure 2E). Thus, in this common EGFR mutation, activation of the kinase domain seems to be uncoupled from sensitizing effects toward kinase inhibitors. In a recent report, L858R and G719S mutants were shown to have a reduced binding affinity toward adenosine triphosphate compared with wild-type EGFR, making them more accessible for ATP competitive EGFR inhibitors.
      • Yun CH
      • Boggon TJ
      • Li Y
      • et al.
      Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity.
      In contrast, EGFR-L861Q was demonstrated to retain high binding affinity for ATP.
      • Carey KD
      • Garton AJ
      • Romero MS
      • et al.
      Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib.
      This may explain the lack of enhanced sensitivity toward reversible inhibitors of this mutant. Thus, these results suggest that NSCLC patients with the EGFR-L861Q mutation may not benefit as much from EGFR inhibitor treatment with gefitinib or erlotinib as patients with the EGFR-L858R, EGFR-G719S, or EGFR exon 19 deletions.
      Figure thumbnail gr2
      FIGURE 2EGFR-L861Q is not a drug-sensitizing mutation unlike L858R and G719S. Stable Ba/F3 cell lines expressing wild-type or mutant EGFRvIII were tested for their sensitivity toward (A) gefitinib, (B) erlotinib, and (C) AEE788 at indicated concentrations. D, Table representing the effect of individual mutations on intrinsic properties of the kinase. E, Ba/F3 cells expressing wild-type or mutant EGFRvIII were treated with increasing concentrations (50, 100, 250, 500, or 1000 nM) of gefitinib or erlotinib for 30 minutes and analyzed for the inhibition of EGFR autophosphorylation and Stat5 phosphorylation. EGFR, epidermal growth factor receptor.
      TABLE 1IC50 Values (nM) of Wild-Type and Mutant EGFRvIII Kinases against Gefitinib, Erlotinib, AEE788, and WZ-4002 Calculated from Figures 2 and 3
       MutationsGefitinibErlotinibAEE788WZ-4002
       EGFRvIII498409198138
       EGFRvIII + G719S155571766
       EGFRvIII + L858R55161010
       EGFRvIII + L861Q45836616074
      EGFR, epidermal growth factor receptor.
      Variation in drug response toward different activating mutations in oncogenic tyrosine kinases has been reported in various cancers, and accumulating evidence indicates that this may have impact on the clinical outcome on inhibitor treatment.
      • Kancha RK
      • Grundler R
      • Peschel C
      • et al.
      Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations.
      Therefore, it may be beneficial to test alternative EGFR inhibitors toward less sensitive activating EGFR mutations such as EGFR-L861Q. WZ-4002 is a novel irreversible inhibitor of EGFR kinase that was recently shown to have significant activity both in vitro and in vivo.
      • Zhou W
      • Ercan D
      • Chen L
      • et al.
      Novel mutant-selective EGFR kinase inhibitors against EGFR T790M.
      Both EGFRvIII-L858R and EGFRvIII-G719S showed lower IC50 values toward WZ-4002 treatment compared with wild-type EGFRvIII (Figure 3, Table 1). Interestingly, EGFRvIII-L861Q also showed significantly more sensitivity against WZ-4002 compared with wild-type EGFRvIII (Figure 3, Table 1). WZ-4002 is an irreversible inhibitor and binds to the active conformation of the EGFR kinase.
      • Zhou W
      • Ercan D
      • Chen L
      • et al.
      Novel mutant-selective EGFR kinase inhibitors against EGFR T790M.
      Because all mutations tested in this study are activating, they may be more sensitive to WZ-4002. Therefore, irreversible second-generation kinase inhibitors such as WZ-4002 may offer a more potent alternative treatment for patients with the EGFR-L861Q mutation in the future.
      • Zhang Q
      • Liu Y
      • Gao F
      • et al.
      Discovery of EGFR selective 4,6-disubstituted pyrimidines from a combinatorial kinase-directed heterocycle library.
      The Ba/F3-EGFRvIII-based system described in this report will be a valuable tool to test novel compounds and strategies.
      Figure thumbnail gr3
      FIGURE 3EGFR-L861Q is more sensitive to WZ-4002 compared with wild-type EGFRvIII. A, Ba/F3 cells stably expressing either wild-type or mutant EGFRvIII were treated with the indicated concentrations of WZ-4002 for 48 hours and tested for cell proliferation inhibition. B, Wild-type or mutant Ba/F3-EGFRvIII cell lines were treated with increasing concentrations of WZ-4002 (50, 100, 25, 500, or 1000 nM) for 30 minutes and analyzed for inhibition of EGFR, Stat5, and extracellular signal-regulated kinase 1/2 phosphorylation. EGFR, epidermal growth factor receptor.

      ACKNOWLEDGMENTS

      Supported by a grant from the Wilhem Sander Stiftung (to J.D.).
      The authors thank Madhavi Kancha and Natalie Bartosch for technical support.

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