4059.Full.Pdf

Published OnlineFirst June 11, 2014; DOI: 10.1158/1078-0432.CCR-13-1559

Clinical Cancer Cancer Therapy: Preclinical Research

Tyrosine Phosphoproteomics Identiﬁes Both Codrivers and Cotargeting Strategies for T790M-Related EGFR-TKI Resistance in Non–Small Cell Lung Cancer

Takeshi Yoshida1,5, Guolin Zhang1, Matthew A. Smith1, Alex S. Lopez2, Yun Bai1, Jiannong Li1, Bin Fang3, John Koomen3, Bhupendra Rawal4, Kate J. Fisher4, Ann Y. Chen4, Michiko Kitano5, Yume Morita5, Haruka Yamaguchi5, Kiyoko Shibata5, Takafumi Okabe5, Isamu Okamoto5, Kazuhiko Nakagawa5, and Eric B. Haura1

Abstract Purpose: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non–small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI– resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine- phosphorylated peptides and mass spectrometry–based identification/quantification. Profiles of erlotinib perturbations were examined. Results: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib–treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M- mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M. Clin Cancer Res; 20(15); 4059–74. 2014 AACR.

Introduction cell lung cancer (NSCLC) with TKI-sensitive EGFR muta- Despite the benefits shown with EGFR-tyrosine kinase tions (1, 2), acquired resistance is a critical clinical problem. EGFR inhibitor (EGFR-TKI) treatment in patients with non–small A secondary point mutation in exon 20 of that substitutes methionine for threonine at amino acid position 790 (T790M) was identified in patients with NSCLC who Authors' Affiliations: 1Department of Thoracic Oncology, 2Tissue Core, developed acquired resistance to gefitinib or erlotinib (3, 4). 3 4 Proteomics and Molecular Oncology Program, Biostatistics Program, H. Nearly 50% of patients with NSCLC with acquired resis- Lee Moffitt Cancer Center and Research Institute, Tampa, Florida; and 5Department of Medical Oncology, Kinki University Faculty of Medicine, tance to EGFR-TKIs have the T790M secondary mutation Osaka-Sayama, Osaka, Japan (5–7). Irreversible EGFR-TKIs, such as CL387,785 (8), Note: Supplementary data for this article are available at Clinical Cancer PF00299804 (9), BIBW-2992 (afatinib; ref. 10), and HKI- Research Online (http://clincancerres.aacrjournals.org/). 272 (11), are thought to be one strategy to overcome T790M-induced resistance. However, a number of studies T. Yoshida and G. Zhang contributed equally to this article. have shown their limited activity in cells with T790M Corresponding Author: Eric B. Haura, Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, mutations given the increased affinity of ATP binding to Tampa, FL 33612. Phone: 181-3745-6827; Fax: 181-3745-6817; E-mail: T790M EGFR proteins or through mechanisms affecting eric.haura@moffitt.org other pathways such as MET activation (8, 9, 12–18). doi: 10.1158/1078-0432.CCR-13-1559 Clinical studies have also highlighted the limited efficacy 2014 American Association for Cancer Research. of irreversible EGFR-TKIs. In the LUX-Lung 1 Trial,

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was independent of EGFR signaling and that cotargeting Translational Relevance SFKs with afatinib led to combined growth suppression in in Acquired resistance to EGFR-tyrosine kinase inhibitor vitro and in vivo in cells with T790M. Globally, our results (EGFR-TKI) is a critical clinical problem in patients with suggest that an unbiased mass spectrometry approach can non–small cell lung cancer (NSCLC) with TKI-sensitive identify codrivers of resistance that can be cotargeted to EGFR mutation. We applied mass spectrometry–based enhance efficacy of targeted agents. tyrosine phosphoproteomics to paired TKI-sensitive and resistant cell lines to visualize molecular networks relat- Materials and Methods ed to the acquired EGFR-TKI resistance. The results Reagents suggest that multiple receptors and signaling molecules Gefitinib, erlotinib, afatinib, and WZ4002 were pur- such as MET, AXL, and IRS2 can collaborate to drive chased from Chemie Tek (Indianapolis, IN). CL-387,785 resistance to EGFR-TKI. We also identified Src family was purchased from AXXORA (San Diego, CA). kinases (SFK) as a central signaling hub in TKI-resistant cells with T790M gatekeeper mutation. SFK phosphor- Cell culture ylation was also detected in human NSCLC samples with The human H1975, H460, A549, and H1299 NSCLC cell T790M. In vitro and in vivo experiments demonstrated lines were obtained from American Type Culture Collec- that irreversible EGFR-TKI (afatinib) or T790M-selective tion. The human HCC4006 NSCLC cells were kindly pro- EGFR-TKI (WZ4006) combined with the SFK inhibitor vided by Dr. Paul Bunn (University of Colorado, Aurora, dasatinib overcame T790M-mediated resistance, thereby CO). The human HCC827 NSCLC cells were provided by nominating a new strategy for translation into the clinic. Dr. Jon Kurie (MD Anderson Cancer Center, Houston, TX). The human PC9 NSCLC cell line was kindly provided by Dr. Hayata, Tokyo Medical University (Tokyo, Japan). PC9GR cells were generated by exposure of PC9 cells containing a conducted to compare afatinib treatment versus placebo in TKI-sensitive EGFR mutation (exon 19; E746-A750) to patients with advanced NSCLC whose disease progressed gradually increasing concentrations of gefitinib, beginning after receiving first-generation EGFR-TKIs (erlotinib, gefiti- at 3 nM and up to 2 mM, for 3 months. HCC4006-T790M nib), afatinib did not extend the primary endpoint of and HCC827-T790M cells were generated as previously overall survival despite significant improvements in pro- described (26). All cell lines have been maintained in a gression-free survival (19). These preclinical and clinical central repository at Moffitt since 2008. All cell lines had results suggest that irreversible EGFR-TKIs as single agents been authenticated by STR analysis (ACTG Inc, Wheeling, are insufficient to overcome resistance. IL) as of September 2010, and all cells had been routinely One strategy to improve on the limited efficacy of irre- tested and were negative for mycoplasma (PlasmoTest, versible EGFR-TKI is through combination with other path- InvivoGen, San Diego, CA). Cell viability was determined way inhibitors. For example, studies that combined afatinib using the CellTiter-Glo Luminescent Cell Viability Assay with the anti-EGFR monoclonal antibody cetuximab (20) (Promega, Madison, WI). Apoptosis assays were performed or the PI3K/mTOR inhibitor PI-103 (12) and HKI-272 using PE-conjugated monoclonal active caspase-3 antibody combined with mTOR inhibitor rapamycin (21) have apoptosis kit (BD Biosciences). Rescue experiments were shown promise in overcoming T790M resistance. Another done as previously described (27). reason for the limited efficacy of agents targeting T790M could be mediated through other tyrosine kinases, such as Genotyping receptor tyrosine kinases (RTK), which provide additional Total genomic DNA from parental and resistant cells protection against EGFR-TKIs (22). Recent studies have was prepared using the DNeasy Blood & Tissue Kit (Qia- shown that growth factor ligands can protect oncogene- gen, Valencia, CA) in accordance with the product man- addicted cells from molecularly targeted agents; thus, ual. Direct DNA sequencing was used to detect EGFR altered expression of these growth factor receptors could mutations as previously described (28). We also applied further identify resistance pathways (23–25). the PCR-invader assay to detect minor populations of We explored the underlying ability of some growth factor EGFR mutation, as previously described (29). MET gene ligands to drive resistance to TKIs by examining the basal copy number per cell was determined by fluorescence in tyrosine phosphoproteome and the effects of EGFR-TKIs on situ hybridization with the use of the LSI D7S522 (7q31) other RTKs. In this study, we tested the hypothesis that a Spectrum Orange and chromosome 7 centromere (CEP7) global evaluation of tyrosine phosphorylation (using mass Spectrum Green probes (Vysis; Abbott), as previously spectrometry) between the sensitive and resistant cells, described (30). along with EGFR perturbations, could identify additional resistance mechanisms that could give insight into cotarget- Tyrosine phosphoproteomics ing strategies. Our results identified numerous coexpressed Tyrosine phosphopeptides were purified according to the RTKs and non-RTKs that, under proper environmental manufacturer’s recommendations for the Cell Signaling circumstances, cooperate to drive resistance to EGFR-TKIs. PhosphoScan kit (P-Tyr-100) (Cell Signaling Technology). We further showed that Src family kinase (SFK) signaling Briefly, 2 108 cells were lysed in urea buffer; extracted

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proteins (40–80 mg) were then reduced by dithiothreitol, non-resistant cell line? To adjust for multiple hypothesis alkylated by iodoacetamide, and then digested by trypsin. testing, the resulting P values for the main effects of cell line Peptide mixture was isolated from lysate using Sep-Pak C18 and treatment as well as the cell line-by-treatment interac- columns and then lyophilizated. Phosphorylated peptides tion term were used to estimate false discovery rate. We used were immunoaffinity purified using phosphotyrosine anti- FDR 20% to declare statistical significance. We further body after lyophilizated peptide mixture was dissolved. performed network analysis based on these potential can- Volumes of phosphotyrosine peptides were then downsized didates. Interactions among all identified tyrosine phos- to 20 mL by vacuum drying for further experiments. The phorylated proteins were retrieved from the Molecular further peptide mixture separation and phosphosite assign- Interaction database (MINT) (32); the IntAct database ing have been previously described (31). To quantify each (33); the Database of Interacting Proteins (DIP) (34); the tyrosine-contained peptide, we calculated peak area [also General Repository for Interaction Datasets (BioGRID) (35) called extraction ion chromatography (EIC)] using Label- and the Biomolecular Interaction Network Database free strategy and xCalibur as the tools. Identification and (BIND) (36) using InnateDB (37) and visualized in Cytos- quantification of some obscure peptides were manually cape 2.8.3 (38). verified. After quantification, 774 phosphorylated tyrosine sites were identified. An in-house algorithm was implemen- Protein expression analysis ted to identify unique phosphorylation tyrosine (pY) sites, Western blot analysis of whole cell lysates was performed remove redundant sites, and merge miss-cleaved peptides as described previously (27). Primary antibodies to EGFR, by using protein ID, peptide sequence, and phosphoryla- MET, pTyr 1234/1235 MET, IRS2, pTyr 1131 IGF1R, AXL, tion start-site index, with quantification of peak areas. When pTyr 702 AXL, Src, pTyr 416 Src, Akt, pSer 473 Akt, Erk, only identifiable to the level of pairs of pYs (e.g., next to each pThr202/Tyr204 Erk, and PARP were obtained from Cell other or up to 11 amino acids apart), then the indepen- Signaling Technology. Primary antibodies to pY1068-EGFR dent unit for analysis was the unique pY pair (instead of were obtained from Invitrogen (Carlsbad, CA). Primary single site). Mis-cleaved phosphopeptides or fragments of antibodies to b-actin were purchased from Sigma-Aldrich the same phosphopeptides were merged. Peptides shared by (St. Louis, MO). multiple proteins were annotated. Among which, two pairs of sequences were potential results of co-elution and there- Assessment of tumor growth inhibition in vivo fore not included in further analyses. A total of 524 unique All animal procedures were approved by our Institutional phosphotyrosine units (pYs) or pY pairs were identified. Animal Care and Use Committee. PC9GR cells (2106) Quantification and stability of 5 MYG peptides across were injected subcutaneously into the flank of 7-week-old samples were examined, with the average of 3 of them used female athymic nude mice. The mice were divided into 4 for normalizing the peak ratio areas across 16 samples (8 treatment groups of 7 animals: those treated over 3 weeks by biological samples with technical duplicates) so that the daily oral gavage of vehicle, afatinib (10 mg/kg), dasatinib normalized quantities across samples were comparable. (15 mg/kg), or both afatinib and dasatinib; 0.5% (wt/vol) Reproducibility between technical replicates for each pY aqueous solution of hydroxypropylmethylcellulose was was estimated using Pearson correlation. The correspond- used as vehicle for afatinib, and 50% propylene glycol was ing P values were used to estimate false discovery rate. High used as vehicle for dasatinib. Treatment was initiated when technical reproducibility of FDR 1% was used in our tumors in each group achieved an average volume of 100 study. In addition, if the pY was detected in at least half mm3, with tumor volume being determined twice weekly of the samples in this study, i.e., at least 8 of 16 samples, it for 21 days after the onset of treatment from caliper mea- passed the QC criteria. Among the 524 unique pY units, 403 surement of tumor length (L) and width (W) according to of them passed the QC criteria and were included in the the formula LW2/2. analyses. 377 of them were unique pY sites while 26 of them were unique pY pairs. Averages of technical replicates from 8 Src-Tyr416 immunohistochemistry staining biological samples were used in the analyses. We used a Immunohistochemistry staining was performed to mea- simple imputation (i.e., when one of technical replicates is sure the expression of phosphor-Src (Tyr416) in paraffin missing, the detected value from the other remaining tech- tissues from 10 lung cancer patients with mutant-positive nical replicate was used). Data were analyzed in log2 scale EGFR T790M. prior to parametric analyses and also for ease of interpre- Slides were stained for phosphor-Src (Tyr416) (mouse tation. For example, the difference of 1 in log2 scale is a 2- monoclonal antibody; Millipore) using a Ventana Discovery fold change between two conditions. Two-way ANOVA XT automated system (Ventana Medical Systems, Tucson, with the interaction term was performed to answer the AZ) following the manufacturer’s protocol with proprietary following three research questions: 1) Which tyrosine sites reagents. Briefly, slides were deparaffinized on the automated are differentially phosphorylated between the cell lines with system with EZ Prep solution (Ventana). Enzymatic retrieval and without drug resistance? 2) Which tyrosine sites are method was used in protease 1 at 4 minute (Ventana), CC1 differentially phosphorylated between the control and erlo- Standard and CC2 standard conditions. The primary mono- tinib-treated groups? 3) Which pYs phosphorylation clonal antibody (Millipore) reacts to secondary antibody at response to treatment is different between the resistant and different dilution-titrations. Both primary and secondary

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antibodies were incubated following Ventana’s instruction 100-fold increased compared with parent PC9 cells (Sup- and the antibody product recommendation. The intensity of plementary Fig. S1A). This resistance was stable as it was not phosphor-Src expression was scored from 0–3 (0 ¼ no reversed by culturing PC9GR cells for up to 6 months in expression, 1 ¼ weak expression, 2 ¼ moderate expression, gefitinib-free medium (data not shown). PC9GR cells and 3 ¼ high expression), while the cellularity was scored acquired T790M while retaining exon 19 E746-A750, as from 0–3 (0 ¼ 0%, 1 ¼ 1-33%, 2 ¼ 34-66%, and 3 ¼ >66%). determined by both direct DNA sequencing and PCR-invad- The H scores formed by intensity of immunoactivity timing er assay (Supplementary Fig. S2A). In addition, we did not cellularity were stratified as low (0–2), intermediate (3–4), find MET amplification by FISH analysis in PC9GR cells and high (6–9). (Supplementary Fig. S2B), which is another mechanism of acquired EGFR-TKI resistance in NSCLC (28). Erlotinib still MET-pY100 proximity ligation and total MET has partial inhibitory effects on EGFR phosphorylation in immunofluorescence PC9GR cells (Supplementary Fig. S1B), consistent with Slides containing 5-mm sections were rehydrated through prior studies that T790M typically emerges as a minor xylene and graded alcohols. Heat-induced epitope retrieval population and resistant cells retain drug-sensitive alleles was carried out in Tris-EDTA (pH 9) in a pressure cooker for (8, 33). However, erlotinib could not completely inhibit 20 minutes and then allowed to cool for 20 minutes. downstream pAkt and pErk in PC9GR cells, consistent with Nonspecific binding was blocked by incubation with resistance to EGFR-TKIs in the presence of T790M (Supple- 1.5% BSA, and primary antibodies were incubated over- mentary Fig. S1B). night in 1.5% BSA-PBST. For proximity ligation, antibodies were rabbit anti-MET System-level comparison of tyrosine phosphorylation (clone D1C2, Cell Signaling Technology) and mouse ant- identifies common RTK pathways associated with pY100 (Cell Signaling Technology). PLA probes were anti- erlotinib resistance rabbit (-) and anti-mouse (þ) and were incubated for 1 hour We hypothesized that erlotinib-resistant PC9GR cells in 0.15% BSA/PBST. Detection was carried out using the could collect additional mechanisms of resistance through DuoLink in situ PLA Far Red kit (O-Link Biosciences, acquired alterations in tyrosine kinase signaling that could Uppsala, Sweden). AlexaFluor 488-conjugated anti-cyto- collaborate with T790M to codrive resistance to EGFR-TKI. keratin was used to demarcate epithelial regions (clone We therefore profiled tyrosine kinase signaling by charting AE1/AE3, eBiosciences). tyrosine phosphorylated peptides in PC9 and PC9GR cells. For immunofluorescence, antibodies were rabbit anti- As shown in our schema (Fig. 1), tryptic peptides were MET (clone D1C2, Cell Signaling Technology) and detected derived from cellular protein lysates and enriched with via AlexaFluor 647-labeled anti-rabbit secondary antibodies anti-phosphotyrosine (pTyr) antibodies followed by iden- (Invitrogen). Murine pan-cytokeratin (clone AE1/AE3, tification and quantification using liquid chromatography Dako) was used to demarcate epithelial regions (tumor coupled with tandem mass spectrometry (LC/MS-MS; mask) and detected via AlexaFluor 555-labeled anti-mouse refs. 32, 34). Changes in peptides in PC9GR cells were secondary antibodies (Invitrogen). Images were acquired identified and compared with PC9 cells, thus allowing us on a PM2000. to determine additional changes beyond T790M that could be codrivers of TKI resistance. We perturbed EGFR-driven Statistical methods signaling in erlotinib-sensitive PC9 and erlotinib-resistant Anderson-Darling statistics and normal curves were PC9GR cells to identify EGFR-dependent pathways/net- examined to assess whether tumor measurements were works and potential pathways/networks independent of normally distributed. A square-root transformation was EGFR signaling that could play a role in EGFR-TKI resis- performed on the tumor measurements to make them tance. After 1-hour erlotinib treatment, cell pellets were approximately normal. ANOVA test was used to assess collected and pTyr peptides were identified in untreated whether there was a statistically significant difference on and treated PC9 and PC9GR cells. Changes in peptides were tumor sizes measured across treatment groups at each time identified compared with control vehicle-treated cells in point. Tukey-Kramer method was used to perform all pair- each of the two cell lines. We hypothesized that this wise group comparisons. All statistical analyses were per- approach would identify downstream signaling events driv- formed using SAS (version 9.2; SAS Institute; Cary, NC). en by mutated EGFR but could also potentially identify proteins or pathways activated by TKI or unaltered by TKI Results that could, under the correct circumstances, potentiate drug Chronic gefitinib exposure of PC9 cells generates resistance. In total, between the two cell lines, we identified stable cell-autonomous resistance to EGFR-TKIs with 403 pTyr peptides corresponding to 265 unique phospho- T790M proteins. Examples of extracted ion chromatograms for pTyr After generation of PC9GR cells, we identified single-cell peptides corresponding to EGFR and MET are shown in clones of PC9GR cells that were highly resistant to erlotinib Supplementary Figs. S3 and S4. (Supplementary Fig. S1A). Although PC9GR cells are par- We next compared changes in pTyr abundance between tially sensitive to the irreversible EGFR-TKI CL387,785 as PC9 and PC9GR cells (Fig. 2A; Supplementary Table S1). expected from a previous report (8), IC50 for CL387,785 was We found 110 unique pTyr peptides (76 proteins) that were

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DMSO Drug DMSO Drug

Figure 1. Phosphoprotein network associated with mutant EGFR and PC9 parental PC9GR T790M tyrosine kinase signaling. Workflow of quantitative Drug treatment Lysate Digestion Anti-pTyr100 Ab phosphoproteomics analysis. EIC, extracted ion chromatography (used to quantify peptide abundance for each of the identified tyrosine-containing Drug response Quantification (EIC) LC/MS-MS Tyrosine proteome peptides). 2.5 2.0 1.5 DMSO 1.0 0.5 0.0 –0.5 –1.0 Drug –1.5 –2.0 –2.5 more abundant in the PC9GR cells, whereas 77 unique pTyr signaling is hyperactivated, in this context, it is not peptides (55 proteins) were less abundant in PC9GR cells responsible for affecting cell survival. than in PC9 cells. Compared with PC9 cells, PC9GR cells To examine whether changes of MET, IRS2, or AXL are demonstrated increased amounts of pTyr peptides corre- driven specifically by T790M, we examined phosphoryla- sponding to numerous RTKs, some of which could be tion of these molecules in lung cancer cell lines (HCC4006 potential codrivers of resistance in PC9GR cells under the and HCC827) engineered to express an exon 19 E746-A750 correct environmental circumstances. We observed a clear þ T790M allele. (Fig. 2B). We observed less pMET, less total subnetwork characterized by hyperactive MET signaling MET, slightly more abundant pAXL, and similar total AXL in (Fig. 2A, right) despite the lack of MET gene amplification. HCC4006-T790M cells compared with parent HCC4006 We observed nearly 11-fold more MET pTyr peptides cells. We found equivalent pMET and total MET, less pAXL in PC9GR than in PC9 cells. Similarly, we observed nearly and equivalent total AXL in HCC827-T790M cells compared >10-fold more pTyr peptides corresponding to ROR1 or with parent HCC827 cells. The levels of IRS2 protein were neurotrophic tyrosine kinase receptor related-1 (pTyr-789, unchanged across these HCC4006 and HCC827 cell lines 13-fold; pTyr-828, 34-fold). ROR1 is a pseudokinase unlike in PC9 and PC9GR cells. These results suggest that that cooperates with MET to promote tumorigenesis (35). changes of MET, IRS2, or AXL are not dependent on EGFR- Tyrosine phosphorylation of the MET adaptor proteins T790M but rather are likely to occur on a cell by cell basis. Gab1 and Gab2 were also more abundant in PC9GR cells. In addition, pTyr peptides corresponding to the AXL Perturbations by EGFR-TKI identify downstream RTK were increased approximately 8-fold in PC9GR cells. proteins and proteins involved in adaptive and AXL upregulation has recently been shown to be a mech- microenvironment-derived responses anism of acquired resistance of lung cancer cells to EGFR- We next compared alterations in pTyr peptide abundance TKI (36). Finally, increased abundance of multiple pep- in both cell lines following erlotinib exposure (Supplemen- tides corresponding to IRS2 (pTyr-675, 4.97-fold; pTyr- tary Table S1). We identified pTyr peptides with >1.5-fold 598, 5.47-fold; pTyr-823, 9.55-fold; pTyr-653, 19.93-fold; change differences from control (P < 0.05). In PC9 cells, we and pTyr-742, 21.29-fold), an adaptor protein linking observed 31 less abundant and 45 more abundant unique insulin and insulin-like growth factor (IGF) signaling to pTyr peptides following 1 hour of erlotinib treatment (Fig. PI3K signaling, was observed in PC9GR cells compared 3A). As expected, PC9GR cells displayed a more blunted with parent PC9 cells. This suggested that either more response to erlotinib than PC9 cells; nonetheless, we did insulin or IGF signaling exists in these cells or more IRS2 observe congruent changes in most pTyr peptides, thus protein is expressed. We confirmed higher levels of tyro- increasing our confidence that these pTyr peptides and sine-phosphorylated MET and AXL in PC9GR than in PC9 pathways are downstream of mutant EGFR given the cells and also found more total IRS2 protein in PC9GR expected biologic responses with cells harboring T790M than in PC9 cells (Fig. 2B). Despite the increased levels of mutations. Among the reduced pTyr peptides, we observed MET signaling, we found minimal effects of combined MK01, SHC1, GAB1, EGFR, and ERBB3 consistent with MET-TKI (PHA665752) and EGFR-TKI (erlotinib or irre- their known roles in ERBB signaling. Interestingly, we also versible CL387,785) in PC9GR cells (Fig. 2C). While MET observed reductions in peptides corresponding to Ras

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signaling, including SYGP1 or SynGAP, which can affect rescue effects compared with HGF in these cells (see Fig. 3C). ERK and p38 MAPK functions in neurons (37, 38). Tyro- These results suggest that altered RTK identified by phospho- sine-phosphorylated Rab7A (also known as Ras-related proteomics can be codrivers of resistance under specific en- protein Rab-7a) was likewise reduced by erlotinib and has vironmental circumstances. Furthermore, the increased levels been linked to EGFR trafficking in endosomes (39). of multiple RTKs in response to erlotinib suggest innate Interestingly, we observed that nearly equal amounts of priming of RTK, where RTKs are primed to cooperate with pTyr peptides were increased by erlotinib compared with growth factor ligands through intracellular mechanisms. peptides reduced by erlotinib (45 up and 31 down in PC9; 26 up and 30 down in PC9GR). We found more abundant Afatinib combined with dasatinib inhibits EGFR pTyr peptides for IRS2, MET, YES, AXL, FAK, ERBB2, and BRK signaling more efficiently than either agent alone in (PTK6) following erlotinib treatment, and this pattern was TKI-resistant NSCLC cells with T790M consistent across both PC9 and PC9GR cells (Fig. 3B). We We reexamined our data for pTyr peptides that were not hypothesized that the increased levels of RTK identified perturbed by EGFR-TKI and were not different between through our approach could cooperate with exogenous the PC9 and PC9GR cell lines. We hypothesized that this ligands and promote EGFR-TKI resistance. Recent studies analysis may uncover parallel signaling pathways that have highlighted the ability of growth factor ligands to pro- cooperate with EGFR to maintain cellular growth and/or mote resistance to targeted agents (23–25). We therefore survival. We identified 31 proteins that fulfilled this tested if the increased pTyr in key RTK could cooperate with criterion,includingmultipleSFKsaswellasCSK,PKCD, growth factor ligands to drive resistance to EGFR-TKI. We MAPK3, PIK3R2, SYK, TNK2, EPHB2, EPHA4, FAK, and incubated PC9 and PC9GR cells with cognate ligands corre- PTK2B. We observed no changes in pTyr peptides corresponding to the upregulated RTK in PC9GR, including IGF1, sponding to SFKs, including the pTyr peptide LIEDNEy- hepatocyte growth factor (HGF), and GAS6 (the ligand for TAR corresponding to the common autocatalytic site AXL RTK), and determined the effects on erlotinib sensitivity in c-SRC, YES, and FYN, following EGFR-TKI, suggesting in PC9 cells and afatinib sensitivity in PC9GR cells (Fig. 3C). this as an EGFR-independent pathway. We linked SFK Interestingly, HGF and IGF but not GAS6 had protective proteins to other proteins found in our entire dataset effects on both PC9 cells exposed to erlotinib and PC9GR through interaction databases (Fig. 4A), identifying a cells exposed to afatinib. In PC9 cells, the shift in IC50 was large group of proteins (N ¼ 28) with reported interac- rather modest; however, in PC9GR cells, the effect was more tions with SFK proteins (gray circles) that were also dramatic. This shift pattern was consistent between both cell unchanged by erlotinib. In addition, we identified poten- lines, with HGF having more of an effect than IGF1, whereas tial interactions between SFK and proteins either altered noeffectwasseenwithactivationofAXLbyGAS6inthese by erlotinib (gray parallelogram and diamond) or altered cells. Using Western blotting, we examined the effects of these in PC9GR compared with PC9 cells (gray V and dia- ligands on EGFR signaling with or without EGFR-TKI in both mond). For example, SRC can cooperate with EGFR, MET, PC9 and PC9GR cells. HGF activated pMET in both PC9 and ERBB3,SHC1,CBL,andSTAT3signalingnodes(gray PC9GR cells (Supplementary Fig. S5A and S5B). This HGF- parallelogram) that we previously identified as being induced activation of pMET and downstream pAkt and pErk altered by erlotinib and different between PC9 and were not inhibited by erlotinib in PC9 or by afatinib in PC9GR cells. PC9GR cells (Supplementary Fig. S5A and S5B). These results On the basis of this observation, we hypothesized that also suggest that MET activation in PC9 and PC9GR cells is not cotargeting SFKs and EGFR T790M with dasatinib and dependent on EGFR signaling. On the other hand, we did not afatinib, respectively, may produce additive or synergistic observe clear ligand-dependent activation of corresponding anti-tumor effects. Furthermore, our previous studies sug- RTKs or sustained activation ofpAktandpErkinthepresence gested that the antitumor effects of dasatinib are mediated of EGFR-TKIs in IGF1-induced or Gas6-induced PC9 and in part by direct EGFR inhibition that is mitigated by gain of PC9GR cells (Supplementary Fig. S5C–S5F). These results are T790M in EGFR (32). However, these studies also suggested consistent with our data showing that IGF1 and Gas6 had less that irreversible EGFR-TKIs combined with dasatinib could

Figure 2. Phosphoproteins associated with T790M-mediated resistance. A, connectivity of MET protein was determined using protein–protein interaction databases to better aid in visualizing differentially expressed proteins that may be associated with PC9GR cells. The left histograph shows change of pTyr sites in PC9GR cells compared with in PC9 cells. The fold change (P < 0.05, fold change > 1.5) of all tyrosine peptides were presented in log2 scale. Red bar shows the tyrosine phosphosites of MET network proteins in PC9GR cells. Right, the MET network. Statistically decreased or increased pTyr peptides were input into Cytoscape 2.8.3, and protein–protein interactions were identified using InnateDB based on molecular interactions and functional relations from public sources. Shapes reflect types of proteins shown in figure. Pink circle represents the pTyr peptides significantly different between PC9 and PC9GR cells and different between erlotinib-treated and control cells (P < 0.05; fold change >1.5). Color scale corresponds to fold change in Log2 scale. The yellow lines represent the direct interaction with MET. B, Western blotting of selected proteins in PC9, PC9GR, HCC4006, HCC4006-T790M, HCC827, HCC827-T790M cells. Membranes were blotted with pTyr 1234/1235 MET, total MET, pTyr 702 AXL, total AXL, and total IRS2 antibodies in PC9, PC9GR, HCC4006, HCC4006-T790M, HCC827, HCC827-T790M cells with actin confirming equal protein loading. C, PC9GR cells were treated for 72 hours with increasing concentrations of erlotinib alone, CL387,785 alone, PHA665752 alone, erlotinib þ PHA665752, or CL387,785 þ PHA665752. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. Please view online version for full details.

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A PTK6 pY114 Erlotinib-PC9 MAGED1 pY92 Erlotinib-PC9GR TNS1 pY1404 STAT3 pY705 TNS1 pY1254

pY886 pY1234 NEDD9 pY317 pY1404 pY1254 pY268 pY1254 pY114 ERBB2 pY1023 INPPL1 NEDD9 pY166 TNS1 pY1404 pY234 MET EPS8 pY774 pY166 TNS1 NEDD9 pY345 SHB STAT3 pY345 BCAR1 pY705 pY91 BCAR1 NUP205 pY902 pY317 PTK2 EPS8 NEDD9 pY861 ACTB pY234 PXN YES1 pY222 PXN pY88 NEDD9 BCAR1 pY234 pY118 ACTB pY1023 PXN pY118 pY774 pY88 pY91 ERBB2 HIPK3 pY359 pY345 PTK2 pY861 PTK6 ANXA2 pY317 pY166 ANXA2 pY24 TAGLN2 pY114 pY1234 INPPL1 pY886 pY268 pY24 pY192 pY705 pY92 STAT3 MET pY359 pY185 GLUL pY185 pY774 HIPK3GLUL TAGLN2 pY192 MAGED1 pY114 SHB EPS8 pY902 ACTB pY91 YES1 pY222 NUP205 MET pY1234 SHB pY114

SHB pY268 –6.81 –0.58 1.63 4.84

GAB1 pY259

RAB7A pY183 EGFR pY1197 CBL pY674 SHC1 pY427 pY259 pY1307 RAB7A pY659 pY1276 pY1197 pY1092 pY1172 pY1307 pY1197 GAB1 ERBB3 pY1276 ERBB3 pY1092 pY1138 pY1159 pY1159 ERBB3 EGFR pY1092 pY1172 CBLB pY1138 EGFR ERBB3 pY1159 pY1276 pY998 EGFR pY1172 MAPK1 EGFR EGFR pY998 SHC1 GAB1 CBL CBLB pY889 pY187 SHC1 pY659 ERBB3 pY1307 pY349 pY674 pY998 pY427 GAB1 pY659 pY427 CBL MAPK1 pY349 pY259 EGFR pY1138 SHC1 pY349 pY187 pY674

MAPK1 pY187

–7 –6 –5 –4 –3 –2 –1 0 1 2 3 4675

Fold change (log2) BC 25 PC9 100 Ctr PC9-Erlotinib HGF 20 PC9GR 75 IGF1 Erlotinib PC9GR-Erlotinib GAS6 IC nmol/L 50 50 PC9 6.94 25 + HGF 50 ng/mL 28.74 15 + IGF1 50 ng/mL 14.09 0 + Gas6 200 ng/mL 6.99

Relative cell vialbility 0.1 1 10 100 1,000 Erlotinib (nmol/L)

Fold change Fold 10 PC9GR

100 Ctr 5 HGF Afatinib 75 IGF1 IC50 nmol/L 50 GAS6 PC9GR 193.00 + HGF 50 ng/mL >1000 25 0 + IGF1 50 ng/mL 422.30 MET pY1234 AXL pY702 IRS2 pY675 IRS2 pY823 0 + Gas6 800 ng/mL 174.00 Relative cell vialbility 0.1 1 10 100 1,000 Afatinib (nmol/L)

Figure 3. Erlotinib perturbations in PC9 and PCGR cell lines. A, effects of erlotinib on tyrosine containing phosphoproteomes in PC9 and PC9GR cell lines. Left, erlotinib-induced changes (P < 0.05; fold change >1.5) of pTyr sites in PC9 (bars) and PC9GR () cells. Four subnetworks were created within different catalog proteins (blue circle) based on increased or decreased pTyr sites in PC9 and PC9GR cells. Color scale represents the fold change of each pTyr site. B, pTyr peptide abundance measured by EIC across erlotinib-treated PC9 and PC9GR cells for MET, AXL, and IRS2 pTyr peptides. Y-axis indicates fold change above PC9 untreated pTyr abundance. C, PC9 or PC9GR cells were seeded in 96-well plates for 24 hours and then exposed to HGF or IGF1 (50 ng/mL) or GAS6 (200 ng/mL) and concomitantly exposed to increasing concentrations of relevant kinase inhibitor. After 72 hours, cell viability was assessed. IC50 was calculated for each condition. Please view online version for full details.

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inhibit EGFR T790M. Thus, we examined the effects of lentiviral vectors, we investigated whether dasatinib-resis- erlotinib, afatinib, dasatinib, or combined afatinib and tant forms of key SFKs could rescue effects of dasatinib (32). dasatinib on EGFR signaling in PC9GR and H1975 cells, To test which SFK is critical as a dasatinib target in NSCLC both resistant cell lines against EGFR-TKI because of T790M cells harboring T790M when combined with afatinib, we (Fig. 4B). In both cell lines, erlotinib inhibited neither EGFR infected cells with lentivirus expressing either wild-type nor SFKs, and no complete suppression of pAkt and pErk kinases or kinase alleles with drug-resistant gatekeeper (both essential downstream molecules of EGFR) was mutations of SFKs (SRC, LYN, FYN, and FRK) and examined observed. We found that afatinib could inhibit pEGFR; cell viability in response to increasing concentrations of however, SFKs were again unaltered. These results with dasatinib plus afatinib (Fig. 4E). Our results show that SRC erlotinib and afatinib are consistent with our pTyr mass and FYN were able to rescue PC9GR cells from dasatinib spectrometry results that detected no changes in pTyr SFKs plus afatinib. However, no effects were observed with LYN following erlotinib exposure. We also found that combin- and FRK, suggesting that SRC and FYN are essential SFKs as ing afatinib and dasatinib resulted in more efficient inhi- dasatinib targets in NSCLC cells with T790M. bition of pAkt and pErk in PC9GR and H1975 cells than each agent alone, suggesting that SFKs are cosignals related Dasatinib enhances the antitumor activity of afatinib to EGFR T790M and that irreversible EGFR-TKIs combined in vitro and in vivo with SFKs more efficiently block EGFR signaling in NSCLC To evaluate more formally whether our combination cells harboring T790M. effects were because of additional cell death, we measured caspase-3–positive cells following TKI treatment (Fig. 5A). Afatinib combined with dasatinib effectively inhibits In PC9GR cells, both erlotinib and dasatinib had no cell growth and significantly increases apoptotic cells effects on apoptosis, but the combination had modest in TKI-resistant NSCLC cells with T790M effects. Afatinib led to more apoptosis, which was further Given that the combination of afatinib and dasatinib increased when combined with dasatinib. Similar effects efficiently inhibited EGFR signaling in NSCLC cells harboring of afatinib plus dasatinib on apoptosis were observed in T790M, we examined the effects of this combination on cell HCC4006-T790M and HCC827-T790M cells (Supple- proliferation and apoptosis in PC9GR and H1975 cells. We mentary Fig. S10). WZ4002 also induced apoptosis as a found reduced IC50 levels in PC9GR and H1975 cells versus single agent, which was potentiated when combined with either agent alone (Fig. 4C; Supplementary Tables S2 and S3). dasatinib. In H1975 cells, similar effects were observed, We examined the effects of combining afatinib plus dasatinib with dasatinib increasing apoptosis when added to afa- in other NSCLC cells with T790M (HCC4006-T790M and tinib and WZ4002. These results indicate that Src inhi- HCC827-T790M cells), cells with TKI-sensitive EGFR muta- bitors enhance antiproliferative and proapoptotic effects tion only (PC9, HCC4006, and HCC827 cells), and cells with of irreversible or T790M-selective EGFR-TKIs in NSCLC wild-type EGFR (H460, A549, and H1299 cells). We observed cells with T790M. reduced IC50 for afatinib plus dasatinib versus either agent We hypothesized that the enhanced apoptosis with alone in HCC4006-T790M and HCC827-T790M cells (Sup- combined afatinib and dasatinib would translate into plementary Fig. S6), whereas curves for this combination improved in vivo effects on tumor growth. We examined were mostly overlapped with those for afatinib or dasatinib the antitumor effects of this combination in mouse xeno- alone in PC9, HCC4006, and HCC827 cells with TKI-sensi- graft models with PC9GR cells. As single agents, afatinib tive EGFR mutation only (Supplementary Fig. S7) or H460, (10 mg/kg) or dasatinib (15 mg/kg) had modest effects A549, and H1299 cells with wild-type EGFR (Supplementary on inhibiting tumor growth in PC9GR xenografts; Fig. S8). We also found that dasatinib combined with however, when combined, we observed significantly great- CL387,785, another irreversible EGFR-TKI, reduced IC50 er inhibition of growth, including tumor regression con- versus either agent alone in PC9GR cells (Supplementary sistent with our apoptosis results (Fig. 5B). These results Fig. S9). Furthermore, we also detected more apparent PARP demonstrate that Src inhibitors effectively enhance anti- cleavage when agents were combined than when used alone tumor effects of irreversible EGFR-TKI in gefitinib-resistant or when cells were erlotinib treated (see Fig. 4B). Similar to NSCLC xenografts with T790M, providing a rationale to results with afatinib, when we examined the effects of com- evaluate this strategy in patients with NSCLC who have bined dasatinib with WZ4002, another T790M-specific acquired EGFR-TKI resistance related to T790M. EGFR-TKI (40), IC50 was reduced versus when agents were used alone (Fig. 4D; Supplementary Tables S2 and S3). Tyrosine phosphoproteomes in lung adenocarcinoma samples with TKI-sensitive EGFR mutations Rescue experiments revealed that dasatinib enhanced To validate that our cell line models and data are appli- antitumor effects of afatinib by inhibition of SRC and cable to human lung cancer tissues, we conducted a mass FYN spectrometry tyrosine phosphoproteomics analysis on four Although our clustering approach and dasatinib results NSCLC tumor samples with TKI-sensitive EGFR mutations. strongly implicated SFKs as the key target, we examined this In total, we identified 279 unique pTyr sites corresponding in more detail given the extensive promiscuousness of to 189 unique proteins across all four tumor samples. For dasatinib. Using dasatinib-insensitive alleles expressed in each tumor, we identified 158, 153, 157, and 109 unique

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pY292 pY46 A pY313 pY88 PRKCDTYK2SDCBP pY783 pY397 PXN ITGB1 pY576 pY508 LYN PTK2 OCLN pY421 CTTN pY227 pY 287

pY702 AXL CTNNB1 pY194 pY30 YES1 pY341 GAB2 pY222 pY 334 pY904 pY417 pY780 SYK pY293 pY446 pY193CTNND1 PAG1 pY296 TNS3 pY334 PTK2B TGM2 pY228 pY163 pY601pY359

pY579 pY369 pY584 pY802 pY 317 pY345 ASAP2 SRC pY204 pY 345 ERBB2 EPHB2 pY 204 pY214 Figure 4. Effects of dasatinib combined with afatinib MAPK3 NEDD9 pY177 WASL PTPN6 pY349 pY317 on EGFR signaling and cell growth in geﬁtinib- pY427 SHC1 pY629 pY166 pY241 CSK SHANK2 pY 427 pY114 resistant NSCLC cells with T790M. A, pTyr proteins pY106 pY261 MET SHB ﬁ pY1003 pY774 corresponding to pTyr peptides identi ed in PC9 pY268 CDCP1 MCAM pY1234 and PC9GR cells are linked to SFK using InnateDB EPS8 CBL pY491 BCAR1 EPHA4 pY 674 pY 114 to capture literature reports and displayed in

pY525 pY674 pY869 Cytoscape. Pink circle, pTyr sites upregulated by STAT3 pY 774 pY287 FYN pY1138 PTPN11 pY 705 EGFR ELF2 erlotinib or higher expression in PC9GR than in PC9 pY1092 pY1307 PTPRA TNK2 pY705 PIK3R2 MPZL1 cells. Blue circle, pTyr sites downregulated by pY998 pY1197 ERBB3 ACTB CDKL5 pY1172 erlotinib or less expression in PC9GR than in PC9 pY1159 pY91 pY1276 cells. Yellow circles or V, SFK (SRC, YES1, FYN). Gray circle, pTyr proteins showing no difference pTyr sites up-regulated by erlotinib or higher expression in PC9GR than PC9 between PC9 and PC9GR cells and no change with pTyr sites down-regulated by erlotinib or lower expression in PC9GR than PC9 erlotinib treatment. Gray parallelogram, pTyr Proteins: altered by erlotinib only Proteins: altered in PC9GR compared to PC9 proteins altered by erlotinib across PC9 and PC9GR

Proteins: no change in the PC9GR compared to PC9, also no response to erlotinib datasets. Gray V, pTyr proteins different between Proteins: altered by erlotinib and in PC9GR compared to PC9 PC9 and PC9GR cells. Gray diamond, pTyr proteins different between PC9 and PC9GR cells and

PC9GR (Ex19del + T790M) H1975 (L858R + T790M) showing changes with erlotinib treatment. B, B PC9GR and H1975 cells were incubated for 6 hours (or 24 hours for PARP) in the absence or presence of Afatinib Dasatinib Combination Afatinib Dasatinib Combination erlotinib (100 nmol/L), afatinib (10 and 100 nmol/L), mol/L mol/L mol/L dasatinib (10 and 100 nmol/L), or afatinib and mol/L mol/L mol/L dasatinib in combination (10 and 100 nmol/L), as Cont Erlotinib 100 nmol/L Cont 10 n 10 n 10 n Erlotinib 100 nmol/L 10 nmol/L 10 nmol/L 10 nmol/L 100 n 100 n 100 n 100 nmol/L 100 nmol/L 100 nmol/L indicated. Cell lysates were subjected to protein p-EGFR p-EGFR expression analysis with antibodies to pEGFR, EGFR EGFR EGFR, pAkt, Akt, pErk, Erk, pSrc family, Src, or PARP along with antibodies to b-actin as a loading p-Src p-Src control. (Continued on the following page.) Src Src

p-Akt p-Akt

Akt Akt

p-Erk p-Erk

Erk Erk

PARP PARP

Actin Actin

pTyr sites corresponding to 117, 95, 111, and 87 proteins, suggesting the potential of clinical application of our find- respectively (Supplementary Table S4). Importantly, 83 ings in this study. unique pTyr sites on 65 proteins identified from mass spectrometry experiments in PC9 cells were also observed Activation of Src or MET in human lung tumor samples active in human patient tissues (Supplementary Fig. S11 with T790M gatekeeper EGFR mutations and Supplementary Table S5). This included: EGFR To validate that Src phosphorylation is indeed observed (pTyr1172, 1197), MET (pTyr1234), SFKs including SRC as a target in human NSCLC samples with T790M gate- (pTyr 419), FYN (pTyr 214, 420), LYN (pTyr 32, 193, 104, keeper mutation, we examined the expression of phos- 397), YES1 (pTyr223, 426), MK01 (pTyr 187), MK03 (pTyr phorylated Src (Tyr 416) in tumor samples with T790M 204), STAT3 (pTyr 705), and AXL (pTyr 886). These results using immunohistochemical staining. We found pSrc in in human tumor tissues from patients with EGFR mutations all EGFR T790M-positive tissue specimens, including 2 well validate our findings gained from cell line model, paired samples of pre- and post-EGFR-TKI treatment, with

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C Drug Sensitivity in PC9GR Drug Sensitivity in H1975

100 100 Erlotinib Erlotinib Afatinib Afatinib 75 Dasatinib 75 Dasatinib Afatinib + Dasatinib Afatinib + Dasatinib 50 50

Cell viability (%) 25 Cell viability (%)

0 0 1 10 100 1,000 10,000 1 10 100 1,000 10,000 Drug concentration (nmol/L) Drug concentration (nmol/L)

D Drug sensitivity in PC9GR Drug Sensitivity in H1975

100 100 Erlotinib Erlotinib WZ4002 WZ4002 75 Dasatinib 75 Dasatinib WZ4002 + Dasatinib WZ4002 + Dasatinib 50 50

Cell viability (%) 25 25 Cell viability (%)

0 0 1 10 100 1,000 10,000 1 10 100 1,000 10,000 Drug concentration (nmol/L) Drug concentration (nmol/L) E

100 100

75 SRC-WT vs. Afatinib + Dasatinib 75 FYN-WT vs. Afatinib + Dasatinib

50 SRC-MT vs. Afatinib + Dasatinib 50 FYN-MT vs. Afatinib + Dasatinib Cell viability (%)

Cell viability (%) 25 25

0 0 1 10 100 1,000 10,000 1 10 100 1,000 10,000 Drug concentration (nmol/L) Drug concentration (nmol/L)

100 100

LYN-WT vs. Afatinib + Dasatinib 75 75 FRK-WT vs. Afatinib + Dasatinib

LYN-MT vs. Afatinib + Dasatinib 50 50 FRK-MT vs. Afatinib + Dasatinib Cell viability (%) Cell viability (%) 25 25

0 0 1 10 100 1,000 10,000 1 10 100 1,000 10,000 Drug concentration (nmol/L) Drug concentration (nmol/L)

Figure 4. (Continued.) C, PC9GR and H1975 cells were treated for 72 hours with increasing concentrations of erlotinib alone, afatinib alone, dasatinib alone, or afatinib þ dasatinib. D, PC9GR and H1975 cells were treated for 72 hours with increasing concentrations of erlotinib alone, WZ4002 alone, dasatinib alone, or WZ4002 þ dasatinib. Data generated by cell viability assay (CellTiter-Glo) are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. E, PC9GR cells were infected with lentivirus expressing wild-type and mutant gatekeeper forms of each indicated Src family kinase for 48 hours. Subsequently, cells were exposed to increasing concentrations of afatinib plus dasatinib for 72 hours, after which cell viability was assessed by cell viability assay (CellTiter-Glo). Data are expressed as a percentage of the value for untreated cells. Determinations were done in triplicate. Please view online version for full details.

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A PC9GR (Ex19del +T790M) H1975 (L858R +T790M) PC9GR H1975 40 40

30 30 Figure 5. Effects of dasatinib combined with afatinib on apoptosis and in vivo tumor 20 20 regression in geﬁtinib-resistant NSCLC cells with T790M. A, apoptosis assay was carried out 10 10 using PE-conjugated caspase-3 antibody, following incubation of

Caspase-3–positive cells (%) Caspase-3–positive cells (%) PC9GR or H1975 cells for 72 hours 0 0 with DMSO, erlotinib (E), dasatinib (D), erlotinib þ dasatinib, afatinib DMSO DMSO (A), afatinib þ dasatinib, WZ4002 (W), and WZ4002 þ dasatinib. E 100 nmol/LD 100 nmol/L A 100 nmol/L W 100 nmol/L E 100 nmol/LD 100 nmol/L A 100 nmol/L W 100 nmol/L Values are expressed as a percentage of caspase-3-positive cells. Determinations were done in E 100 nmol/L + DA 100100 nmol/Lnmol/L + WD 100 nmol/L + D 100 nmol/L E 100 nmol/L + DA 100100 nmol/Lnmol/L + WD 100 nmol/L + D 100 nmol/L triplicate. Bars, SD. , P < 0.001 Treatment (48 h) Treatment (48 h) versus DMSO or each single agent (Student t test). B, Nude mice with tumor xenografts established by B In vivo activity in PC9GR cells subcutaneous implantation of PC9GR cells were treated daily for 21 days with vehicle (control), 700 Control 650 afatinib (10 mg/kg), dasatinib (15 mg/kg), or afatinib þ dasatinib by 600 Afatinib oral gavage. Tumor volume was ) 550 3 Dasatinib determined at the indicated times 500 after the onset of treatment. Points, 450 Afatinib + Dasatinib mean of values from 5 mice/group; 400 bars, SE. , P < 0.05 for afatinib 350 combined with dasatinib versus control or each agent alone by 300 ANOVA (Tukey–Kramer 250 comparison). (Continued on the 200 Tumor volume (mm following page.) 150 100 50 0 0 2 4 6 8 10 12 14 16 18 20 22 Days

varied intensities and cellularity (Table 1; Fig. 5C). These Discussion results confirmed results from cell lines that Src activity persists in EGFR T790M-positive tumor tissues. We fur- We applied tyrosine phosphorylation profiling using ther examined changes in tyrosine phosphorylated MET LC/MS-MS to directly compare an EGFR-TKI–sensitive expression in matched pre- and post-EGFR-TKI treatment cell line versus its acquired resistance counterpart to patient tumor tissue specimens. We found evidence for uncover additional resistance mechanisms and propose increased tyrosine phosphorylated MET in one patient cotargeting strategies to enhance the effects of agents (patient 10 in Table 1) that was not due to increased total specifically targeting the T790M EGFR allele. To our MET protein (Fig. 5D), whereas no evidence was found knowledge, this is the first such report to apply a mass in the second patient (patient 9 in Table 1) for which pre- spectrometry–based phosphoproteomics approach to and posttreatment biopsy tissues were available for study. compare the molecular networks between EGFR-TKI– These results provide further support using tumor tissues sensitive and -resistant pairs. The driving force behind that MET tyrosine phosphorylation can occur in T790M- this approach is the limited efficacy of irreversible EGFR- containing tissues and this can be independent of total TKIs in targeting T790M, as shown in both preclinical MET expression. and clinical studies (8, 9, 12, 14–19), and the ability of

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tinib sensitivity. In PC9GR cells, we identified higher C levels of pTyr peptides corresponding to MET signaling, including more MET, ROR1, and Gab1/2 proteins. Inter- estingly, this coordinated activation of MET signaling was not secondary to MET gene amplification, as our FISH results revealed no amplification of the MET gene. The results with the basal phosphoproteome corre- sponded to HGF’s strong effects in protecting both PC9 H score: 9 H score: 6 and PC9GR cells from erlotinib or afatinib, respectively. In addition, these results suggest a form of "lineage" addiction, whereby resistant cells with T790M can carry forward RTKs that can cooperate to drive resistance. Importantly, these results suggest that interrogating protein activation status or network signaling may highlight proteins that play a role in protecting cells against EGFR- H score: 3 TKI, especially when in a microenvironment rich with cognate growth factor ligands. Despite observing more AXL pTyr peptides in PC9GR cells, we demonstrated no D Pretreatment Posttreatment ability of AXL pathway activation by Gas6 ligand to drive resistance to either erlotinib or afatinib. The reasons for thisarenotclear,butonelimitofourapproachwasthe lack of absolute measurements of pTyr peptides. It is possible that, compared with MET or IRS2 pTyr peptides, pTyr peptides corresponding to AXL are far lower in absolute amount and thus are inefficient to compete for

MET-pY100 PLA downstream signaling effectors. It will be interesting and important to determine how basal phosphoproteome measurements can predict the effects of growth factor protection against targeted agents. As AXL signaling still remains poorly understood, another explanation for our results could be the limited or absence of key adaptors or other effector proteins involved in AXL signaling. Using pTyr peptide data obtained from both PC9 and Total MET IF PC9GR cells exposed to erlotinib, we identified proteins downstream of EGFR in these cells with mutant gain-of- function EGFR proteins. One of the more interesting findings was that nearly half of the statistically significant Figure 5. (Continued.) C, evidence for activation of Src in the T790M- pTyr peptides were increased in abundance following positive biopsy specimens. Immunohistochemical staining was used to erlotinib treatment. It is increasingly recognized that detect expression of tyrosine phosphorylation Src (Tyr416) in 10 EGFR signaling pathways display large amounts of crosstalk T790M-positive tissue specimens. H score (intensity cellularity) was andthatadaptiveresistancemechanismshavebeen calculated for each sample, with scores 0 and 9 representing the lowest and highest expression, respectively. Three representative 200 images observed in cells exposed to targeted agents (41). Our show the different levels of pSrc expression in EGFR T790M-positive results match our investigations using purified Src human lung specimen. D, evidence for increased MET tyrosine homology-2 domains to profile tyrosine kinase signaling phosphorylation in posttreatment T790M biopsy specimens. Proximity in lung cancer cells, where we observed increased pTyr ligation assays (PLA) for MET and pY100 were performed to assess MET signaling in multiple lung cancer cells exposed to TKIs phosphorylation in serial biopsy specimens obtained from an adenocarcinoma patient (right lung, lower lobe, 33 months apart). Original (42). Similar events have also been observed in crizoti- biopsy confirmed EGFR exon21 L858R mutation; rebiopsy confirmed nib-treated EML4-ALK cells and dasatinib-treated DDR2- L858R/T790M mutation. Top, evidence of MET-pY PLA signal in mutant lung cancer cells, arguing that these paradoxical pretreatment biopsy, while clusters of highly phosphorylated MET are changes are consistent across multiple tumor types and observed in the posttreatment biopsy. MET-pY PLA was localized to cytokeratin (þ)-staining regions (data not shown). Bottom, increased kinase inhibitors (unpublished observations). The MET-pY PLA signal is not due to increased total MET protein. underlying mechanisms of these changes require additional study, as they could be important in promoting adaptive resistance to targeted agents and could in some other tyrosine kinases, especially RTKs, to limit EGFR-TKI cases cooperate with microenvironmental factors, such efficacy. Through a systematic interrogation of pTyr pep- as growth factors, to limit TKI efficacy. Collectively, these tides and proteins using LC/MS-MS, we identified both results suggest that cell intrinsic (receptors, signaling RTKs and non-RTKs able to be recruited to confer erlo- proteins) and extrinsic (ligands) factors can collaborate

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Table 1. Src phosphorylation detected in human NSCLC samples with T790M gatekeeper mutation

Patient ID EGFR mutant H score Intensity Cellularity 1 T790M/L858R 3 1 3 2 T790M/19del(E746-A750) 4 2 2 3 T790M/19del(E746-A750) 6 2 3 4 T790M/L858R 6 2 3 5 T790M/19del(E746-A750) 6 2 3 6 T790M/19del(E746-A750) 9 3 3 7 T790M/19del(E746-A750) 9 3 3 8 T790M/L858R 9 3 3 9 (Pre-TKI) 19del(E746-A750) 9 3 3 9 (Post-TKI) T790M/19del(E746-A750) 9 3 3 10 (Pre-TKI) L858R 4 2 2 10 (Post-TKI) T790M/L858R 9 3 3

NOTE: Patient 9 was treated with "geﬁtinib and erlotinib." Patient 10 was treated with "geﬁtinib and erlotinib þ ARQ197 (MET-TKI)."

to drive resistance to kinase inhibitors in a systems-level can detect serine/threonine phosphopeptides (44), could manner. identify other proteins and pathways that may play roles Our phosphoproteomics analyses in PC9 and PC9GR in EGFR TKI resistance. cells demonstrated that SFKs are also critical as an EGFR- Although single-agent dasatinib has no activity in independent cosignal in NSCLC cells with T790M. These patients with NSCLC with TKI-sensitive EGFR mutation results were enabled by tyrosine phosphorylation profil- whoacquiredresistancetoEGFR-TKI(45),ourresults ing combined with analysis of proteins based on known suggest a role for SFKs in maintaining downstream sig- protein–protein interactions. We validated the inferences naling despite irreversible EGFR-TKIs and support further derived from the phosphoproteomics by showing studies of irreversible EGFR-TKIs combined with dasati- that afatinib combined with dasatinib resulted in antitu- nib in patients with NSCLC who acquire resistance to mor activity regarding cell proliferation and apoptosis in EGFR-TKI. Src is known to be both an upstream activator PC9GR, H1975, HCC4006-T790M, and HCC827-T790M and a downstream mediator of EGFR, and its phosphor- cells (each harboring T790M). These results appear to be ylation is detected in about one-third of lung cancer generalized to additional T790M EGFR-TKIs, as dasatinib tumors (46, 47). Although MET activation might not be demonstrated similar combination effects with the always observed in the presence of T790M based on our in T790M-selective EGFR-TKI WZ4002 (40). We did not vitro and tumor tissue analysis, pSrc seems to be generally observe combination effects with afatinib plus dasatinib detected in our NSCLC tumor samples harboring T790M, compared with either agent alone in cells with TKI-sen- consistent with our results of cell models. In addition, our sitive EGFR mutation only (PC9, HCC4006, and HCC827 mass spectrometry data from tumor samples with TKI- cells) or wild-type EGFR (H460, A549, and H1299 cells). sensitive EGFR mutation demonstrated a high degree of The enhanced apoptosis with combined afatinib and overlap with results from cell models, thereby validating dasatinib in the cells with T790M translated into the overall approach. These results from tumor samples improved in vivo effects on tumor growth in PC9GR cells. suggestthatourresultsfromlung cancer cell line models Collectively, our results suggest that dasatinib can be are applicable to translate in to the clinic. Our previous generally used as a combination therapy with irreversible chemical and phosphoproteomic characterization iden- or T790M-selective EGFR-TKIs for patients with NSCLC tified nearly 40 different kinase targets of dasatinib and whoacquiredEGFR-TKIresistanceassociatedwith showed that SRC, FYN, and EGFR are relevant targets for T790M. dasatinib action in NSCLC (32). Our recent phase I/II As our approach was limited to examining the tyrosine study showed that dasatinib combined with erlotinib is phosphoproteome, we were unable to detect serine/thre- tolerable, with 63% of patients with advanced NSCLC onine signaling including mTOR/AKT or MEK/Erk path- showing disease control, including two having partial ways both of which are also essential for carcinogenesis. response and one having bone response (48). Another Previous studies have indicated that mTOR inhibitor group also showed that dasatinib combined with erloti- combined with MEK inhibitor or irreversible EGFR-TKI nib is safe and feasible in NSCLC (49). is potential strategy to overcome T790M (21, 43). Further On the basis of these clinical studies along with the studies examining the global phosphoproteome, such as experiments reported here, dasatinib has potential clinical with immobilized metal affinity chromatography which activity in NSCLC treatment, but this is limited to

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Phosphoproteomics of EGFR-TKI Resistance

combinations with T790M-targeted agents and in genotype- Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): T. Yoshida, G. Zhang, M.A. Smith, specific patients. This will be formally tested in a phase I trial A.S. Lopez, Y. Bai, J. Li, M. Kitano, Y. Morita, H. Yamaguchi, K. Shibata, of afatinib and dasatinib (NCT01999985). Our results also T. Okabe, I. Okamoto, K. Nakagawa, E.B. Haura highlight the ability of phosphoproteomics to identify other Study supervision: K. Nakagawa, E.B. Haura important mediators of drug sensitivity, and examination of these proteins may be important in clinical studies of Acknowledgments T790M-targeting agents. The authors thank the Moffitt pY Group, the Kinki University Medical Oncology Research Group, Tsutomu Iwasa, and Kunio Okamoto for helpful discussions, Rasa Hamilton for editorial assistance, Fumi Kinose for assis- Disclosure of Potential Conflicts of Interest tance with cell culture, and Linda Ley and Carol Ulge for administrative E.B. Haura reports receiving a commercial research grant from Boehrin- assistance. The authors also thank BML, Inc and SRL, Inc for technical ger-Ingelheim. No potential conflicts of interest were disclosed by the other assistance. authors. Grant Support Authors' Contributions The work was partially funded by grants from the Moffitt Cancer Center SPORE in Lung Cancer (P50-CA119997), the V Foundation for Cancer Conception and design: T. Yoshida, G. Zhang, E.B. Haura Research, and in part by the National Cancer Institute, part of the NIH, Development of methodology: T. Yoshida, G. Zhang, M.A. Smith, through grant number 2 P30-CA76292-14, which provide support to the A.S. Lopez, Y. Bai, J. Koomen, E.B. Haura Proteomics Core, the Flow Cytometry Core, the Tissue Core, and the Animal Acquisition of data (provided animals, acquired and managed patients, Facility at the H. Lee Moffitt Cancer Center and Research Institute, an NCI- provided facilities, etc.): T. Yoshida, G. Zhang, M.A. Smith, B. Fang, designated Comprehensive Cancer Center. J. Koomen, K. Nakagawa, E.B. Haura The costs of publication of this article were defrayed in part by the Analysis and interpretation of data (e.g., statistical analysis, biosta- payment of page charges. This article must therefore be hereby marked tistics, computational analysis): G. Zhang, M.A. Smith, A.S. Lopez, Y. Bai, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate B. Fang, B. Rawal, K.J. Fisher, A.Y. Chen, I. Okamoto, K. Nakagawa, E.B. this fact. Haura Writing, review, and/or revision of the manuscript: T. Yoshida, G. Zhang, M.A. Smith, B. Fang, J. Koomen, B. Rawal, A.Y. Chen, K. Nakagawa, Received June 13, 2013; revised April 9, 2014; accepted April 23, 2014; E.B. Haura published OnlineFirst June 11, 2014.

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4074 Clin Cancer Res; 20(15) August 1, 2014 Clinical Cancer Research

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Correction Clinical Cancer Correction: Tyrosine Phosphoproteomics Research Identiﬁes Both Codrivers and Cotargeting Strategies for T790M-Related EGFR-TKI Resistance in Non–Small Cell Lung Cancer

In this article (Clin Cancer Res 2014;20:4059–74), which was published in the August 1, 2014, issue of Clinical Cancer Research (1), an author's name was misprinted. The corrected name should read as follows: "Y. Ann Chen." The authors regret this error.

Reference 1. Yoshida T, Zhang G, Smith MA, Lopez AS, Bai Y, Li J, et al. Tyrosine phosphoproteomics identiﬁes both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non–small cell lung cancer. Clin Cancer Res 2014;20:4059–74.

Published online August 3, 2015. doi: 10.1158/1078-0432.CCR-15-1183 Ó2015 American Association for Cancer Research.

www.aacrjournals.org 3571 Published OnlineFirst June 11, 2014; DOI: 10.1158/1078-0432.CCR-13-1559

Tyrosine Phosphoproteomics Identifies Both Codrivers and Cotargeting Strategies for T790M-Related EGFR-TKI Resistance in Non−Small Cell Lung Cancer

Takeshi Yoshida, Guolin Zhang, Matthew A. Smith, et al.

Clin Cancer Res 2014;20:4059-4074. Published OnlineFirst June 11, 2014.

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