Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from Gefitinib in EGFR Wild-Type Non−Small Cell Lung Carcinoma Cell Lines

Published OnlineFirst January 31, 2013; DOI: 10.1158/1535-7163.MCT-12-0880

Molecular Cancer Companion Diagnostics & Biomarkers Therapeutics

Quantitative Chemical Proteomics Proﬁling Differentiates Erlotinib from Geﬁtinib in EGFR Wild-Type Non–Small Cell Lung Carcinoma Cell Lines

Angelique Augustin1, Jens Lamerz2,Hel ene Meistermann1, Sabrina Golling1, Stefan Scheiblich4, Johannes C. Hermann5, Guillemette Duchateau-Nguyen2, Manuel Tzouros1, David W. Avila1, Hanno Langen1, Laurent Essioux2, and Barbara Klughammer3

Abstract Although both erlotinib and gefitinib target the EGF receptor (EGFR), erlotinib is effective in patients with EGFR wild-type or mutated tumors, whereas gefitinib is only beneficial for patients with activating mutations. To determine whether these differences in clinical outcomes can be attributed to their respective protein interaction profiles, a label-free, quantitative chemical proteomics study was conducted. Using this method, 24 proteins were highlighted in the binding profiles of erlotinib and gefitinib. Unlike gefinitib, erlotinib displaced the ternary complex formed by integrin-linked kinase (ILK), a-parvin, and PINCH (IPP). The docking of erlotinib in the three-dimensional structure of ILK showed that erlotinib has the ability to bind to the ATP- binding site, whereas gefitinib is unlikely to bind with high affinity. As the IPP complex has been shown to be involved in epithelial-to-mesenchymal transition (EMT) and erlotinib sensitivity has been correlated with EMT status, we used a cellular model of inducible transition and observed that erlotinib prevented EMT in a more efficient way than gefitinib by acting on E-cadherin expression as well as on IPP levels. A retrospective analysis of the MERIT trial indicated that, besides a high level of E-cadherin, a low level of ILK could be linked to clinical benefit with erlotinib. In conclusion, we propose that, in an EGFR wild-type context, erlotinib may have a complementary mode of action by inhibiting IPP complex activities, resulting in the slowing down of the metastatic process of epithelial tumors. Mol Cancer Ther; 12(4); 520–9. 2013 AACR.

Introduction cially potent inhibitors of activating mutation-positive Over the past few years, several drugs targeting protein EGFR, erlotinib has also shown efficacy versus placebo kinases have shown clinical efficacy as cancer therapy (1). in EGFR wild-type NSCLC (2, 3, 7, 8). Among these, erlotinib and gefitinib are first-generation To determine whether differences in clinical outcomes tyrosine-kinase inhibitors (TKI) that directly inhibit the seen in patients with EGFR wild-type tumors receiving EGF receptor (EGFR; refs. 2–5). Recent clinical data have erlotinib or gefitinib can be attributed to differences in the shown that patients with non–small cell lung carcinoma protein interaction profiles of these 2 compounds, a quan- (NSCLC) whose tumors harbor an activating EGFR muta- titative chemical proteomics study was conducted. Chem- tion respond especially well to treatment with these TKIs ical proteomics is a mass spectrometry–based affinity (3, 4, 6). Although both TKIs target EGFR and are espe- chromatography approach, which uses immobilized small molecules as bait to capture and identify interacting protein complexes from an entire proteome. This tech- Authors' Affiliations: 1Protein and Metabolite Biomarkers, 2Bioinformat- nique has been successfully applied to explore the mode ics and Exploratory Data Analysis, Translational Research Sciences, and of action of kinase inhibitors and to assess naturally 3Tarceva Clinical Biomarker Subteam, Pharmaceuticals Division, F. Hoff- mann-La Roche Ltd., Basel, Switzerland; 4Discovery Research Oncology, occurring small molecules to identify their targets (9, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Penzberg, Ger- 10). To gain specificity by filtering out background binders many; and 5Discovery Chemistry, Pharmaceuticals Division, F. Hoff- generated by low-affinity abundant proteins, the work- mann-La Roche Ltd., Nutley, New Jersey flow is best accompanied with a competition-binding step Note: Supplementary data for this article are available at Molecular Cancer that involves free compound pretreatment of the probed Therapeutics Online (http://mct.aacrjournals.org/). mixture. Here, competition-binding experiments and Corresponding Authors: Angelique Augustin, Translational Research label-free liquid chromatography/mass spectrometry Sciences, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., Grenza- cherstrasse 124, 4070 Basel, Switzerland. Phone: 41-61-688-4050; Fax: (LC/MS) signal detection were used to identify protein 41-61-688-1929; E-mail: [email protected]; and Barbara complexes that can be specifically displaced from an Klughammer, [email protected] affinity matrix by unmodified erlotinib or gefitinib. This doi: 10.1158/1535-7163.MCT-12-0880 reveals a potential complementary mode of action for 2013 American Association for Cancer Research. erlotinib in the context of epithelial-to-mesenchymal

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transition (EMT). EMT is a known mechanism through instructions. Binding efficiency was estimated by com- which epithelial cells lose adhesion molecules such as E- paring the optical densities of the compound solutions cadherin and acquire a mesenchymal phenotype, which before and after coupling at the maximal absorbance allows them to migrate into the stroma and become wavelengths 247 and 330 nm. Cells were lysed in 50 invasive (11). The correlation between erlotinib sensitivity mmol/L HEPES pH 7.3 buffer containing 150 mmol/L and EMT has been shown previously in vitro and in vivo NaCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 1% NP-40, (12, 13) and suggests that patients with epithelial-type protease inhibitors (Complete EDTA free; Roche Applied NSCLC tumors, that is, which had not undergone the Sciences), and 1 mmol/L orthovanadate; samples were transition, could have a better outcome with an erlotinib- cleared by centrifugation at 12,000 g for 15 minutes at containing regimen (12). The complex formed by integrin- 4C. Cell lysates were preincubated with increasing con- linked kinase (ILK), a-parvin, and PINCH is bound to centrations (0, 0.0856, 0.2862, 0.957, 3.2, 10.7, 35.78, 119.6, erlotinib but not gefitinib in the chemical proteomics and 400 mmol/L final concentration) of unmodified erlo- profiling of several EGFR wild-type NSCLC cell lines. tinib or gefitinib for 1 hour at 4C under rotation. The This complex, also known as ILK–PINCH–Parvin com- pretreated samples were loaded onto Sepharose beads plex (IPP) complex, forms a structural and signaling entity immobilized with the respective compound (erlotinib or in the integrin pathway (14) and has been shown to gefitinib) for 3 hours at 4C under rotation. Beads were influence EMT. ILK and PINCH overexpression are able washed 3 times with lysis buffer, treated with SDS-PAGE to trigger EMT, by inducing loss of E-cadherin (15, 16). buffer for 5 minutes at 85C, and eluted proteins separated TGF-b-1–induced EMT in mammary epithelial cells on 4% to 20% Tris–glycine SDS-PAGE. After protein required functional ILK (17). The next steps were to fixation in 40% ethanol and 10% acetic acid, gels were investigate a potential structural explanation for this stained overnight with Coomassie Brilliant blue. For pro- binding discrepancy between the 2 compounds, as well tein in-gel digestion, an adapted protocol of Shevchenko as using an EMT-inducible cellular model to assess their and colleagues (21) was used. In brief, each gel lane was respective functional effect on EMT. These findings were cut into 5 slices between the 20 and 150 kDa region. further correlated with gene expression data from the Proteins were reduced with 50 mmol/L dithioerythritol MERIT open-label, multicenter, phase II clinical trial, for 45 minutes at 56C, alkylated with 55 mmol/L iodoa- which aimed to identify genes with potential as biomar- cetamide for 1 hour at room temperature in the dark, and kers for clinical benefit from erlotinib therapy (18). digested with trypsin (Promega) overnight at room temperature. Peptides were extracted twice with 1:2 (v/v) Materials and Methods acetonitrile/25 mmol/L ammonium bicarbonate, and 1:2 (v/v) acetonitrile/5% formic acid, respectively, for 15 Reagents and cell lines a minutes at 37 C, dried down using a speedvac, and stored Anti-ILK, E-cadherin, and -parvin were obtained from at 20C before analysis. Samples were reconstituted in Cell Signaling Technology, anti-PINCH1 and glyceralde- 2% acetonitrile/5% formic acid and run in triplicate by hyde-3-phosphate dehydrogenase–horseradish peroxi- LC/MS. Nanoflow liquid chromatography/tandem mass dase (GAPDH-HRP) from Santa Cruz Biotechnology, and spectrometry (LC/MS-MS) was carried out by coupling anti-vimentin from BD Biosciences. All cell lines were an Easy-nLC system (Proxeon) to an LTQ Orbitrap Velos obtained from the American Type Culture Collection (Thermo Fisher Scientific) equipped with a Proxeon (ATCC) without further authentication. Cell cultures nanoelectrospray and an active background ion reduction H292, H226, H322, H358, H441, H460, and H1299 were device (ABIRD; ESI Source Solutions). Peptides were maintained in RPMI-1640, and Calu6 in EMEM; both loaded on a 10-mm Aqua C18 trapping (packed in-house, supplemented with 10% FBS. 100 mm inner diameter) and separated on a 200-mm Reprosil-Pur C18-AQ analytic (Dr. Maisch GmbH) col- Chemical proteomics profiling in H292 cells umn (75 mm inner diameter, 3 mm particle size, 120 A˚ ). Erlotinib and gefitinib were modified with a 6-(3-ami- Individual raw data files were processed with SEQUEST nopropyloxy) side chain for covalent linking to (version 27.0, revision 12, Thermo Fisher Scientific) and epoxy-activated Sepharose 6B beads (GE Healthcare) to searches were conducted against a concatenated for- generate affinity matrices, as described by Brehmer and ward/reverse human UniProt/SwissProt database (June colleagues (19). Starting material 4-[(3-ethynylphenyl) 2009 release, 34,275 entries, containing splice-variants) amino]-7-(2-methoxyethoxy)-6-quinazolinol was pre- allowing for a spectrum false-discovery rate of 2.5%. A pared as described in WO9630347A1 (20). Coupling of mass tolerance of 5 ppm for precursor and 1.0 Da for the derivatives to the epoxy-activated beads was done in fragment ions was set. Oxidized methionines (þ15.9949 þ 50% dimethylformamide (DMF)/0.1 mol/L Na2CO3 pH Da) and carbamidomethylated cysteines ( 57.0215 Da) 11 in the presence of 10 mmol/L NaOH, overnight at room were considered as differential and fixed modifications, temperature in the dark. After washing with 50% DMF/ respectively. Only fully tryptic peptides with not more 0.1 mol/L Na2CO3, any remaining reactive groups were than one mis-cleavage were considered for data analysis. blocked with 1 mol/L ethanolamine. Subsequent washing Detailed LC/MS and data analysis procedure can be steps were conducted according to the manufacturer’s found in the Supplementary Data.

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Chemical proteomics profiling of erlotinib and (fold changes and P values) are derived from the statistical gefitinib in other NSCLC cell lines analysis already reported (18). We specifically looked at H358, H322, H226, and Calu6 cells were lysed in 50 differences between gene expression profiles of patients mmol/L HEPES pH 7.3 buffer containing 150 mmol/L with and without clinical benefit from erlotinib. Clinical NaCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2, 1% NP-40, benefit was defined as an objective response (complete protease inhibitors (Complete EDTA free, Roche Applied responses or partial responses were confirmed by repeat- Sciences), 1 mmol/L orthovanadate, and samples were ed assessments 4 weeks apart at any time during the cleared by centrifugation at 12,000 g for 15 minutes at treatment period) or maintenance of stable disease for 12 4C. Cell lysates were loaded on erlotinib-Sepharose and weeks after study entry. gefitinib-Sepharose, using the same experimental condi- The following 21 proteins, potentially involved in tran- tions as in the profiling experiment described earlier scriptional regulation of EMT or in IPP complex, were but without competing with the unmodified erlotinib or mapped to the corresponding genes and microarray gefitinib. The elution fractions were analyzed by LC/MS. probe sets: Twist, Snail (Sma1), Zeb (TCF8), Lef-1, E12/ Spectral counts corresponding to EGFR, GAK, LOK E47 basic loop helix (bHLH), Slug (Sma2), E-cadherin (Stk10), Abl2, SLK, MEKK1 (M3K1), ILK, a-parvin (CDH1), N-cadherin (CDH2), Myc, vimentin, catenin-b, (PARVA), and PINCH1 (LIMS1) were extracted and nor- catenin-d, fibronectin, epimorphin, ILK, a-parvin, b-par- malized toward the sum of all spectral counts correspond- vin, PINCH 1, PINCH 2, metastasis-associated protein 3 ing to all proteins identified in the respective fractions. (MTA3), and Y box–binding protein 1 (YB-1).

Alignment of EGFR and ILK crystal protein structures Results Publicly available crystal structure protein data bank Interaction profile of erlotinib and gefitinib in H292 (pdb) files of EGFR-bound erlotinib or gefitinib and the NSCLC cell line crystal structure of ILK complexed with ATP were down- A quantitative chemical proteomics method was loaded from the crystallographic database [ref. 22; pdb applied to erlotinib and gefitinib (Fig. 1A) using the same codes 1M17, 2ITY, 3KMW; (23–25)]. The 3 kinase struc- wild-type EGFR-expressing NSCLC cell line H292 as tures were aligned using the coordinates from Ca atoms of biological target to identify the interaction profiles of the a subset of the residues forming the ATP-binding site compounds. Cell lysates were preincubated with increas- [residue numbers based on EGFR numbering 691–693, ing concentrations of unmodified erlotinib or gefitinib 701–703, 719–72 (21) 1, 751, 766–772, 820–823, 831–832]. before fractionation on the respective affinity matrix, Alignment and distance measurements were conducted captured proteins were eluted, separated by SDS-PAGE using the PyMOL visualization package (25). and identified by LC/MS and protein database mining (Supplementary Fig. S1). A total of 1,285 and 1,303 protein Testing EMT on H358 NSCLC cell line groups were identified in the bound fractions to erlotinib H358 cells were treated with recombinant 10 ng/mL and gefitinib, respectively (Supplementary Table S1). A TGF-b3 (Peprotech) and 5 mmol/L erlotinib or gefitinib for linear model (27, 28) was applied on mass spectrometry 3, 6, 10, and 13 days. Cell culture medium was renewed signals to derive concentration-dependent signal decrease every 3 days. Cell lysis was conducted in PBS containing of specific binders. Comparison of the binding profiles of 1% NP-40 and protease inhibitors. After clearing by cen- the 2 compounds was carried out using a robust, simple, trifugation at 12,000 g for 15 minutes at 4C, proteins statistical procedure adapted from Bretz and Hothorn (29) were separated by SDS-PAGE and processed for Western on peptide signals and extended to multiple testing of blot analysis. Images were acquired using a compact corresponding protein groups. Nine proteins were found digital camera (Luminescent Image Analyzer; Fujifilm) with an adjusted P value below 5% in the samples dis- and quantified with ImageQuant TL (GE Healthcare). placed with erlotinib (Fig. 1B, Regions A, B and x-axis; Statistical analysis was conducted using JMP 8.0.2 (SAS Supplementary Table S2), and 19 in those displaced with Institute Inc.). To determine the influence of erlotinib and gefitinib (Fig. 1B; regions A, C, and y-axis; Supplementary gefitinib on ILK and PINCH after TGF-b3 stimulation, the Table S2), including EGFR, which was specifically dis- following model was applied: ExpressionD13 Treatment placed by both compounds, confirming the validity of the þ ExpressionD3. For analysis of IPP expression in NSCLC approach. The presence of 2 other proteins, GAK and cell lines, simple t tests were conducted. Stk10, was also significant in both compound profiles (Fig. 1B; region A; Supplementary Table S2), whereas NUD12, Analysis of MERIT data K0564, SN1L1, KC1E, and EPHA1 were only identified in Gene expression profiles were measured in tumor biop- the gefitinib-bound fraction (Fig. 1B, y-axis; Supplemen- sy samples from 102 patients with stage IIIB/IV NSCLC, tary Table S2); M3K1 and LIMS1 only in the erlotinib- being treated with erlotinib (150 mg/day). Tumor biop- bound fraction (Fig. 1B, x-axis; Supplementary Table S2). sies were analyzed using gene expression profiling with While the majority of proteins displaced by either Affymetrix GeneChip Human Genome U133A Array erlotinib or gefitinib were protein kinases (7 for erlotinib, (Affymetrix). All gene expression results described here i.e., EGFR, GAK, STK10, ILK, SLK, ABL2, and M3K1; and

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of the other cell lines. In addition, we observed that the spectral counts of Abl2, Slk, MEKK1 (M3K1), ILK, a-parvin (PARVA), and PINCH1 (LIMS1) in the erlotinib- bound fraction were higher than those in the geﬁtinib- bound fraction. This was particularly noticeable for the IPP, which was enriched on the erlotinib but not the geﬁtinib column from these EGFR wild-type NSCLC cell lysates.

Interaction profile of erlotinib and gefitinib in the ILK DVK loop region Erlotinib and gefitinib have a high degree of similarity in their chemical structure (Fig. 1A). Knowing the resolved crystal structures of EGFR and ILK (24, 25), we examined whether the binding of erlotinib to ILK was different from gefitinib, which might explain the discre- pancies in their binding profiles toward the IPP complex. As expected, when erlotinib or gefitinib were in a complex with EGFR, they displayed the same mode of binding to the ATP-binding sites. The overall protein conformation of EGFR-erlotinib, EGFR-gefitinib, and the ATP-binding pocket was very similar, resulting in an exact overlay of the 2 structures after aligning (data not shown). The pseudokinase ILK shows some crucial differences in fold and conformation to other kinases, including EGFR Figure 1. A, chemical structures of erlotinib and gefitinib. B, binding (25). However, when ignoring sequence differences, the profiles of erlotinib and gefitinib in H292. P values of erlotinib versus buried part of the ILK ATP-binding site is very similar to fi ge tinib after log10 transformation. Lines indicate 5% probability of error. the ATP-binding site of EGFR and allows a reliable struc- Region A shows proteins significantly binding to erlotinib and gefitinib; regions B and C show proteins significantly binding to erlotinib or tural alignment of ILK with EGFR when in a complex with gefitinib, respectively. Proteins in region D did not show significant either erlotinib or gefitinib (Fig. 3). A crucial difference in binding to either erlotinib or gefitinib. If a protein was identified in one the ATP-binding site is the conformation of the DFG loop compound and not in the other, it could not be quantified, and hence no and Ala338, the residue upstream of the DVK loop in ILK P P value is available ( value, NA). These proteins are displayed outside (residues 339–341, which form the typical DFG). This the 2-dimensional plot for completeness (x- and y-axis). Note: all cell lines were obtained from ATCC without further authentication. residue is part of a turn that is not observed in EGFR and is close to the space occupied by the aniline portions of erlotinib and gefitinib. 11 for gefitinib, i.e., EGFR, GAK, STK10, MKNK2, RIPK2, The different substitutions in the erlotinib and gefitinib ADCK3; MK09; EPHB2, EPHB4, KC1E, and EPHA1), a aniline rings (the 3 position is substituted with chlorine in notable difference between the 2 compounds was seen gefitinib but with acetylene in erlotinib and the 4 position in the non-protein kinase binding profile. In this sub- is unsubstituted in erlotinib but substituted with fluorine group, erlotinib only displaced 2 proteins (PARVA and in gefitinib) and the resulting differences in atom proxi- LIMS1), whereas gefitinib displaced 8 (NUD12, K0564, mities suggest that erlotinib is more likely than gefitinib to SN1L1, AP1G2, BLMH, HEAT3, SAAL1, and DFNA5). bind favorably to the ATP-binding site of ILK. Interestingly, LIMS1 (also known as PINCH1) forms an active complex together with PARVA (a-parvin) and ILK, Potential influence of erlotinib on the integrin another protein displaced by erlotinib. signaling pathway, connecting to EMT The IPP complex functions as a hub in the integrin Interaction profile of erlotinib and gefitinib in other signaling pathway (14). It plays a structural role by con- NSCLC cell lines necting integrins to the cytoskeleton and also transduces We next addressed the question of whether the chem- signals from the extracellular matrix to various intracel- ical proteomics profile was cell line–specific by testing 4 lular pathways involved in cell proliferation, migration, additional EGFR wild-type NSCLC cell lines (H358, H322, and survival. In addition, overexpression of both ILK and H226, and Calu6) for the 9 erlotinib-specific binders PINCH seems not only to follow the occurrence of EMT (EGFR, STK10, GAK, ILK, LIMS1, PARVA, SLK, M3K1, (15, 16), but ILK activity is also required for TGF-b1– and ABL2). Figure 2 shows the normalized spectral counts mediated EMT in mammary epithelial cells (17). On the of these 9 proteins, including EGFR, in the bound frac- basis of the fact that erlotinib binds to the IPP complex, as tions. All 9 proteins found in the erlotinib profile in H292 shown in our chemical proteomics approach, we hypoth- were also identified in the protein purifications from all 4 esized that in NSCLC cell lines that have not undergone

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Figure 2. Purification of erlotinib binders in other NSCLC cell lines. Additional chemical proteomics experiments were carried out using H226, Calu6, H358, and H322 cell lysates, loaded on erlotinib-Sepharose and gefitinib-Sepharose. Elution fractions from each purification were analyzed by LC/MS. The averaged normalized spectral counts out of 2 biological replicates, corresponding to the 9 proteins previously identified as binders to erlotinib, are represented from the erlotinib-Sepharose and gefitinib-Sepharose eluted fractions. Note: all cell lines were obtained from ATCC without further authentication.

EMT, erlotinib could potentially slow down the transition, by inhibiting the IPP-mediated E-cadherin pathway. To test this hypothesis, we used a model NSCLC cell line, H358, which had been previously described as switching from epithelial-to-mesenchymal state upon TGF-b3 treatment (30). The results show a clear decrease of E-cadherin after 13 days of TGF-b3 treatment, indicating that the cells are transitioning (Fig. 4A). Treatment with erlotinib prevents this, whereas gefitinib fails to inhibit the TGF-b3–mediated decrease of E-cadherin levels. We also examined the effects of the transition on ILK and PINCH expression (a-parvin could not be tested because of low detection by Western blot analysis). Figure 4B and C shows the average quantified Western blot analysis signals from both proteins, for 3 and 13 days of Figure 3. ATP-binding site of aligned and overlaid ILK and EGFR crystal treatment. The expression of both ILK and PINCH protein structures showing potential interactions of erlotinib and gefitinib was increased by the transition. Both proteins gave a with the area of the ILK DFG loop. Oxygen atoms are colored in red, lower signal when cells were treated with TGF-b3 and nitrogen in blue, chlorine in green, and fluorine in light cyan. Carbon atoms of the erlotinib/EGFR complex (1M17) are colored in salmon, carbon of erlotinib. the gefitinib/EGFR (2ITY) complex in yellow, and carbon atoms of ILK are Thus, this EMT model indicates that erlotinib reduces colored in white. Picture was generated with PyMOL (25). the extent of EMT by inhibiting the loss of E-cadherin and

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Figure 4. Erlotinib slows down the transition of H358 by stabilizing E-cadherin. H358 cells were treated for 3, 6, 13, and 20 days with TGF-b3 alone or together with erlotinib and gefitinib. The levels of expression of E-cadherin (A), ILK, and PINCH were analyzed by Western blot analysis. For ILK (B) and PINCH (C), Western blot analysis signals were quantified and averaged from 2 biological replicates. The effect of 13 days of treatment with erlotinib and gefitinib was estimated on TGF-b3–stimulated cells after adjustment to the level at 3 days of respective treatment. Compared with TGF-b3–stimulated untreated cells, a downregulation (in the sense of a "non-upregulation") by gefitinib on ILK (one-sided P ¼ 0.085) and PINCH-1 (one-sided P ¼ 0.12) did not reach significance. In contrast, there was a significant downregulation by erlotinib on ILK (one-sided P ¼ 0.03) and PINCH-1 (one-sided P ¼ 0.02). Note: all cell lines were obtained from ATCC without further authentication. also seems to prevent a TGF-b3–mediated increase of both mesenchymal marker. All the IPP proteins were detected, members of the IPP complex, ILK and PINCH. with varying intensities, in all 8 cell lines except for a-parvin in H358 (Fig. 5A). After quantification and nor- Expression of IPP complex in NSCLC cell lines malization, the overall sum of IPP complex intensities was ILK and PINCH overexpression has been correlated almost 2 times higher in mesenchymal versus epithelial with tumor progression and/or aggressiveness in several cells (Fig. 5B). human malignancies (14). At present, the 3 components of the complex are studied mainly on an individual basis, not ILK expression in erlotinib-treated patients—data addressing the possibility of the expression of the entire from the MERIT trial complex being correlated with EMT status. Eight EGFR Taking into account the data from NSCLC cell lines, and wild-type NSCLC cell lines were selected on the basis of considering that the EMT status of biopsies from patients this status (13, 30), which was assessed by the expression with NSCLC has been previously correlated with clinical of E-cadherin as an epithelial marker and vimentin as a benefit from erlotinib treatment (12), we evaluated the

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(0.93-fold; P ¼ 0.011) and upregulated (1.38-fold; P ¼ 0.033), respectively, in patients with clinical beneﬁt. It should be noted that these genes exhibited only small trends of regulation that were not signiﬁcant when a procedure of P value adjustment for multiple testing was applied, as in the initial work from Tan and colleagues (ref. 18; Fig. 6).

Discussion In tumors dependent on an EGFR-mutated receptor, the mutation triggers a decreased affinity of the kinase for ATP but its affinity to erlotinib is kept intact. This, taken together, leads to an increased sensitivity to the TKI. The difference in efficacy between erlotinib and gefitinib has been at least partially attributed to a difference in phar- macokinetic properties, that is, erlotinib at 150 mg has an area under the curve 7 times higher than gefitinib at 250 mg. In this study, we hypothesize that an additional activity besides the inhibition of EGFR could explain the difference in clinical benefit seen between erlotinib and gefitinib in EGFR wild-type tumors, for example, inhibition of ILK. Indeed, the chemical proteomics profiling of erlotinib and gefitinib has highlighted differences in the binding profiles of the 2 compounds in at least 5 different EGFR wild-type NSCLC cell lines. Many kinase inhibitors, which have been approved for clinical use, have been profiled with this method leading to the discovery of new activities, for example, on DDR1 for imatinib (31) or CAMK2G for bosutinib (32). Given the conservation of the ATP-binding site among human protein kinases, the focus had been placed on potential binding activity with Figure 5. IPP expression in NSCLC cell lines. A, Western blot analyses of other protein kinases. Indeed, Abl2, Slk, and STK10 (LOK) a EGFR ILK, -parvin, PINCH, E-cadherin, vimentin, and GAPDH on wild- have been confirmed to bind to erlotinib in vitro with at type NSCLC epithelial cell lines H292, H441, H358, H322, and mesenchymal cell lines H460, H1299, H226, and Calu6. B, quantified least 25-fold higher affinity than gefitinib (33). However, Western blot analysis signals normalized on GAPDH levels and averaged these assays revealed also at least 30-fold lower affinity of to determine the level of expression of the IPP complex. A significant erlotinib for Abl2, Slk, and STK10 by comparison with upregulation in the GAPDH normalized expression was observed for EGFR. A recent study using native kinase binding profil- a-parvin (one-sided P ¼ 0.045), ILK (one-sided P ¼ 0.007), and PINCH-1 (one-sided P ¼ 0.011) from epithelial-to-mesenchymal cell lines. Note: all ing in HL60 and PC3 cell lines also reported inhibitory cell lines were obtained from ATCC without further authentication. activities of erlotinib on STK10 (LOK), MAP3K1 (M3K1), Slk, and ILK, which correlates well with our findings (34). potential relationship between IPP, a subset of genes Further experiments would be required to confirm the involved in EMT, and response to erlotinib. Specifically, effect of erlotinib on these kinases in the appropriate in we looked at the differences between their expression vivo background. Our data show that substantial infor- profiles in patients with and without clinical benefit from mation can also be deduced from the nonkinase proteins erlotinib. that form complexes with the direct binders. Unlike gefi- In total, 16 of 21 preselected genes were identified nitib, erlotinib interacted with the ternary complex with detected probe sets and statistically analyzed [Twist, formed by ILK, a-parvin, and PINCH in our assay. Snail (Sma1), Zeb (TCF8), Lef-1, E12/E47 basic loop helix We propose a structural explanation for the difference (bHLH), Slug (Sma2), E-cadherin, Myc, vimentin, catenin- in binding to IPP between erlotinib and gefitinib. The b, catenin-d, fibronectin, epimorphin, ILK, a-parvin, and docking of erlotinib in the 3-dimensional structure of ILK b-parvin]. Data for the following 5 genes: PINCH 1, shows that erlotinib is able to bind to the ATP-binding site PINCH 2, metastasis-associated protein 3 (MTA3), Y box– of the pseudokinase, as opposed to gefitinib, which is less binding protein 1 (YB-1), and N-cadherin were not available. likely to bind with high affinity. Indeed, when the EGFR Among the 31 probe sets (corresponding to 16 unique crystal structures, complexed either with erlotinib or genes), only 2 were differentially expressed in tumor gefitinib, are aligned with the ILK structure, the different samples from patients with and without clinical benefit: substitutions of the aniline moieties of the compounds ILK and CDH1 (E-cadherin), which were downregulated seem to have crucial implications for their potential

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Figure 6. E-cadherin (CDH1) and ILK expression in patients with versus without clinical benefit. Each dot represents the gene signal (log2 transformed) measured in one sample. For each group of patients (without or with clinical benefit), a boxplot is displayed where the thick black horizontal line (within a box) represent the median gene signal for that group. The top and bottom boundaries of the box correspond respectively to the 25th percentile and 75th percentile of the gene signals. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. binding to ILK. Acknowledging that the distance mea- tions between ligands and proteins. Measurements of in sured between overlaid structures depends on the align- vitro binding affinity of the compounds to the purified ment method used and may vary slightly, a few observa- complex will be required to experimentally validate the tions can be made from our structural alignment. Gefitinib results of this in silico approach. has 2 substitutions on the aniline ring, whereas erlotinib Our study shows that erlotinib prevents EMT induced has only 1, which points inside the ATP-binding site, close by TGF-b3 in a more efficient way than gefitinib in the to the DVK loop. The 4-position is not substituted in H358 cellular model. As expected, this does not influence erlotinib, whereas in gefitinib it features fluorine. Com- cellular proliferation in our in vitro assays, where the pared with hydrogen, the van der Waal’s radius of the different cell lines used in this study were growth inhib- fluorine atom is larger and the carbon–fluorine bond is ited to a varying extent but equally by both compounds as significantly longer than the carbon–hydrogen bond (35). previously reported (12, 36). EMT will be more relevant This hydrogen-to-fluorine substitution seems to be impor- for invasive tumor growth in vivo, and clinical studies tant, as the fluorine sits in an environment where it clashes have already shown the link between E-cadherin (CDH1) and causes steric repulsions with the Cb-methyl and the levels and response to erlotinib (12). The retrospective carbonyl oxygen of Ala338 (1.5 and 2 A˚ , respectively; analysis presented here on data from the MERIT trial Fig. 5). Furthermore, the fluorine also creates an unfavor- confirms that patients with higher expression of E-cad- able electrostatic interaction with the carbonyl oxygen of herin (CDH1) have an increased clinical benefit from Ala338. In contrast, erlotinib projects a slightly polarized treatment with erlotinib, as previously published (18). hydrogen atom from the aniline 4-position that can estab- A similar analysis has been conducted on gefitinib by lish an attractive interaction with the carbonyl oxygen of Kook and colleagues and did not show any correlation Ala338. The repulsion from the fluorine atom in gefitinib of E-cadherin expression with response rate or with could either prevent it from binding to ILK with reason- progression-free survival and overall survival in patients able affinity or it could trigger a rearrangement of the loop with inoperable stage IIIB/IV NSCLC treated with containing the DVK motif upon binding. Because this loop gefitinib (37). Interestingly, a recent study has shown a is connected to the activation loop of ILK that is crucial for causative relation between loss of E-cadherin and over- binding a-parvin (25), such a rearrangement is likely to expression of ILK in squamous cell carcinoma and ade- have an influence on the ability of ILK to form a complex nocarcinoma of the lung (38). with a-parvin. More sophisticated methods such as In addition, in our cellular model, erlotinib showed a molecular dynamics simulations could be applied to tendency to prevent the overexpression of ILK and further elucidate the favorable and unfavorable interac- PINCH induced by TGF-b3. It should be noted that neither

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Augustin et al.

erlotinib nor gefitinib have been shown to bind the TGF-b in deciphering the details of the signaling pathway lead- receptors in vitro (33), excluding the possibility of a direct ing to hindrance of EMT. effect in our model on TGF-b receptor signaling. Over- expression of both ILK and PINCH has been shown to Disclosure of Potential Conflicts of Interest induce EMT by lowering E-cadherin levels (16, 39, 40). All authors were employees of F. Hoffmann La Roche Ltd. at the date of the study. Interestingly, while ILK is captured in our chemical proteomics experiments to the same extent in all the cell lines, Authors' Contributions PINCH is most enriched in H226 and Calu6 fractions (Fig. Conception and design: A. Augustin, J. Lamerz, B. Klughammer 2), which are mesenchymal cell lines (30). Overall, these Development of methodology: A. Augustin, J. Lamerz, H. Meistermann, S. Golling, G. Duchateau-Nguyen, L. Essioux, B. Klughammer observations indicate that erlotinib may exert a long-term Acquisition of data (provided animals, acquired and managed patients, effect on the expression of ILK and PINCH, which could provided facilities, etc.): A. Augustin, H. Meistermann, S. Golling, S. explain the observed consequences on the EMT. Because Scheiblich, M. Tzouros, D.W. Avila, B. Klughammer Analysis and interpretation of data (e.g., statistical analysis, biostatis- the expression of both proteins is regulated, at least in tics, computational analysis): A. Augustin, J. Lamerz, S. Golling, J.C. part, by proteasomal degradation (41), we could hypoth- Hermann, G. Duchateau-Nguyen, L. Essioux Writing, review, and/or revision of the manuscript: A. Augustin, J. esize that erlotinib would accelerate this degradation and Lamerz, H. Meistermann, S. Golling, J.C. Hermann, G. Duchateau- therefore counteract the EMT process. Nguyen, M. Tzouros, D.W. Avila, H. Langen, L. Essioux, B. Klughammer The respective overexpression of ILK and PINCH has Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): A. Augustin, S. Golling been associated with poor prognosis in NSCLC (42–44). Study supervision: A. Augustin, H. Langen, B. Klughammer Although PINCH could not be monitored in the MERIT trial, low expression of ILK seems to indicate a better Acknowledgments benefit with erlotinib. This strengthens our theory that in The authors thank Nikos Berntenis for providing automated sequence interpretation platform; Michel Petrovic for automating the quantitative patients with a low expression of IPP, the remaining free interpretation platform; and Peter Berndt and Wolfgang von der Saal for erlotinib can still act on the complex, whereas in cases of seminal discussions. overexpression, this effect would not be significant. In conclusion, we propose that, in a wild-type EGFR Grant Support Support for third-party writing assistance for this article was provided context, unlike gefitinib, erlotinib targets both EGFR and by F. Hoffmann-La Roche Ltd. the IPP complex activities, resulting in the slowing down The costs of publication of this article were defrayed in part by the pay- of the metastatic process of epithelial tumors. Further ment of page charges. This article must therefore be hereby marked adver- tisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. investigations on additional models will be necessary to confirm this observation and explore the early effects of Received September 4, 2012; revised January 17, 2013; accepted January erlotinib on the activities of the IPP complex, for example 25, 2013; published OnlineFirst January 31, 2013.

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Quantitative Chemical Proteomics Profiling Differentiates Erlotinib from Gefitinib in EGFR Wild-Type Non−Small Cell Lung Carcinoma Cell Lines

Angélique Augustin, Jens Lamerz, Hélène Meistermann, et al.

Mol Cancer Ther 2013;12:520-529. Published OnlineFirst January 31, 2013.

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