Amplification of EGFR Wild-Type Alleles in Non–Small Cell Lung Cancer Cells Confers Acquired Resistance to Mutation-Selective

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Published OnlineFirst February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-2359 Cancer Therapeutics, Targets, and Chemical Biology Research

Ampliﬁcation of EGFR Wild-Type Alleles in Non–Small Cell Lung Cancer Cells Confers Acquired Resistance to Mutation-Selective EGFR Tyrosine Kinase Inhibitors Shigenari Nukaga1, Hiroyuki Yasuda1, Katsuya Tsuchihara2, Junko Hamamoto1, Keita Masuzawa1, Ichiro Kawada1, Katsuhiko Naoki3, Shingo Matsumoto2, Sachiyo Mimaki2, Shinnosuke Ikemura2, Koichi Goto4, Tomoko Betsuyaku1, and Kenzo Soejima1

Abstract

EGFR-mutated lung cancers account for a significant subgroup of wild-type alleles but not mutant alleles was sufficient to confer non–small cell lung cancers overall. Third-generation EGFR tyro- acquired resistance. These findings underscore the importance of sine kinase inhibitors (TKI) are mutation-selective inhibitors with signals from wild-type EGFR alleles in acquiring resistance to minimal effects on wild-type EGFR. Acquired resistance develops to mutant-selective EGFR-TKI. Our data provide evidence of wild- these agents, however, the mechanisms are as yet uncharacterized. type allele-mediated resistance, a novel concept of acquired resis- In this study, we report that the Src–AKT pathway contributes to tance in response to mutation-selective inhibitor therapy in cancer acquired resistance to these TKI. In addition, amplification of EGFR treatment. Cancer Res; 77(8); 1–12. 2017 AACR.

Introduction and erlotinib) and second- (afatinib) generation EGFR-TKIs has been repeatedly demonstrated by multiple clinical trials (11–13). EGFR-mutated lung cancers comprise a significant subgroup of However, lung cancer cells inevitably acquire resistance to these non–small cell lung cancer (NSCLC; refs. 1–3). In general, EGFR inhibitors after approximately one year (14–16). tyrosine kinase domain somatic mutations activate EGFR by Multiple mechanisms of acquired resistance to first- and sec- promoting the active conformation of EGFR (4–7). Activated ond-generation EGFR-TKIs have been identified to date—includ- EGFR transduces signals to downstream pathways, such as the ing the EGFR T790M gatekeeper mutation (17, 18), transforma- phosphoinositide 3-kinase (PI3K)–AKT and MEK–ERK mitogen- tion to small cell lung cancer (SCLC; refs. 19, 20), and adaptive activated protein kinase (MAPK) pathways. After the identifica- bypass pathway activation through the MET (21), AXL (22), and tion of EGFR-activating mutations and response to first-genera- FGFR1 (23) axes. Of these, the EGFR T790M mutation is respon- tion EGFR tyrosine kinase inhibitors (EGFR-TKI; refs. 8–10), the sible for acquired resistance in approximately 50% of cases. The treatment strategy for NSCLC patients harboring these mutations T790 residue is located at the entrance to a hydrophobic pocket on changed dramatically. The significant improvement of prognosis the posterior side of the ATP-binding cleft. As such, the T790M of lung cancer patients with EGFR mutations by first- (gefitinib mutation induces a conformational change within the EGFR ATP- binding pocket, resulting in the steric hindrance of first or second- generation EGFR-TKIs (17). Coincidentally, EGFR T790M also 1Division of Pulmonary Medicine, Department of Medicine, Keio University, fi 2 enhances the af nity between ATP and its binding pocket (24), School of Medicine, Tokyo, Japan. Division of Translational Research, Explor- thereby synergistically conferring resistance to EGFR-TKIs. To atory Oncology Research and Clinical Trial Center, National Cancer Center, Chiba, Japan. 3Keio Cancer Center, Keio University School of Medicine, Tokyo, address this issue, third-generation EGFR-TKIs, such as osimerti- Japan. 4Division of Thoracic Oncology, National Cancer Center Hospital East, nib (AZD9291; ref. 25), rociletinib (CO-1686; ref. 26), and Chiba, Japan. nazartinib (EGF816; ref. 27), have been developed that irrevers- Note: Supplementary data for this article are available at Cancer Research ibly bind the EGFR ATP-binding pocket by forming a covalent Online (http://cancerres.aacrjournals.org/). bond with the C797 residue at pocket periphery, simultaneously fi Corresponding Authors: Hiroyuki Yasuda, Keio University School of Medicine, blocking ATP binding due to the increased af nity. Interestingly, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 813-3353-1211; these third-generation EGFR-TKIs are also effective for the major- Fax: 813-3353-2502; E-mail: [email protected]; Kenzo Soejima, ity of EGFR-activating mutations, such as in-frame deletions in [email protected]; and Katsuya Tsuchihara, Division of Transla- exon 19 and the L858R point mutation in exon 21 (28). Further- tional Research, Exploratory Oncology Research and Clinical Trial Center, more, mutation selectivity of third-generation EGFR-TKIs has National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. been reported in preclinical and clinical models (25–27). The E-mail: [email protected] wide therapeutic window of third-generation EGFR-TKIs mirrors doi: 10.1158/0008-5472.CAN-16-2359 the significant efficacy and safety of these agents performed in 2017 American Association for Cancer Research. multiple clinical trials (29, 30). Third-generation EGFR-TKIs are

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also subject to acquired resistance. It is important to fully clarify ing to the manufacturer's protocol with 150 mg of protein for each the mechanisms of acquired resistance to third-generation experiment. Signal intensity was calculated using LumiVision EGFR-TKIs to improve the prognosis of lung cancer patients Analyzer software. harboring EGFR mutations. The mechanisms clarified to date include C797S or L798I mutation (31, 32), constitutive MAPK Establishment of the H1975 EGFR stable cell line pathway activation by mutated KRAS or MEK (33), or bypass H1975 cells with stable EGFR wild-type or C797S overexpres- pathway activation via MET or ERBB2 (32, 34–36). However, sion were generated by retroviral infection using MigR1-EGFR the mechanisms underlying acquired resistance to third-gener- constructs that harbor a GFP as previously described (5). Briefly, ation EGFR-TKIs are not fully clarified. Phoenix-Ampho cells were transfected with 15 mg of wild-type or In this study, we elucidated novel mechanisms underlying C797S EGFR using Lipofectamine 2000. Viral supernatant was acquired resistance to third-generation EGFR-TKIs by whole- collected 2 days after transfection and applied to H1975 cells on exome sequencing analysis. Notably, we determined that Src– retronectin-coated plates (Takara) for 24 hours. EGFR-overexpres- AKT pathway activation and EGFR wild-type allele amplification sing H1975 cells were sorted by a FACS MoFlo XDP (Beckman can both contribute to EGFR-TKI acquired resistance, the latter of Coulter) based on GFP expression. Purified cells were then grown which might possibly be due to the decreased inhibitory pressure in RPMI with 10% FBS. for EGFR wild-type by mutation selective third-generation EGFR- fi EGFR TKIs. Our ndings highlight the importance of wild-type Western blot analysis allele-mediated signaling in acquired resistance to mutant-selec- Cells were treated with increasing concentrations of EGFR-TKIs tive EGFR-TKIs. (0.1–1 mmol/L). Cetuximab was used at concentrations of 10 mg/mL. Cells were lysed in Cell Lysis Buffer (Cell Signaling Materials and Methods Technology), and equivalent amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Reagents The membranes were incubated overnight with primary antibodies Erlotinib, afatinib, rociletinib, and wortmannin were purchased at 4 C and then with secondary antibodies for 1 hour. Immuno- from LC Laboratories. Osimertinib was purchased from Selleck reactive proteins were visualized with LumiGLO reagent and Chemicals (Houston). Nazartinib was purchased from ApexBio. peroxide (Cell Signaling Technology), then exposed to X-ray film. Bosutinib was purchased from Cayman Chemical). Cetuximab was purchased from Keio University Hospital (Tokyo, Japan).Total EGFR antibody (#2232), EGFR E746-A750del–specific antibody Apoptosis assay (#2085), total AKT antibody (#9272), phospho-AKT (S473/D9E) Cells were seeded in 6-well plates (50,000/well) and were m m antibody (#4060), total p44/42 MAPK antibody (#9102S), phos- treated with rociletinib (1 mol/L) and cetuximab (10 g/mL) pho-p44/42 MAPK (T202/204) antibody (#9101S), E-cadherin individually or in combination for 72 hours. Control cells were antibody (#3195S), vimentin antibody (#5741S), total Src anti- treated with DMSO. Apoptosis was monitored using the TACS – body (#2108), Phospho-(Tyr416)-Src antibody (#2101S), PTEN Annexin V FITC Apoptosis Detection Kit (R&D Systems) accord- antibody (#9559), and PI3 kinase p110 alpha antibody (#4255S) ing to the manufacturer's protocol. The proportion of apoptotic fl were purchased from Cell Signaling Technology). The HA-tag cells was evaluated by ow cytometric analysis using a Gallios (ab18181) and integrin beta 1 (ab52971) antibodies were pur- Flow Cytometer system (Beckman Coulter). chased from Abcam). Phospho-EGFR (Y1068) antibody (44788G) was purchased from Invitrogen/Life Technologies. Actin antibody Quantitative RT-PCR and quantitative PCR was purchased from Sigma-Aldrich. Total RNA was isolated from cultured cells using an RNeasy Mini Kit (Qiagen) and genomic DNA was isolated using a DNeasy Cell lines Blood & Tissue Kit (Qiagen). RNA was subjected to reverse The PC9 (EGFR E746-A750del) and H1975 (EGFR L858R þ transcription using the High-Capacity RNA-to-cDNA Kit (Life T790M) human NSCLC cell lines were obtained from Dr. Susumu Technologies) according to the manufacturer's protocol. Quan- fl Kobayashi (Beth Israel Deaconess Medical Center, Boston, MA) titative RT-PCR was performed using uorescent SYBR Green and and the ATCC, respectively. Cells were cultured in RPMI1640 an ABI Prism 7000 Sequence Detection System (Life Technolo- growth medium supplemented with 10% FBS at 37Cina gies). Human GAPDH was used to normalize input cDNA. The LINE1 repetitive element was used as a reference gene for EGFR humidified 5% CO2 incubator. Cell authentication for PC9 and H1975 was performed by the authors in June 2015 using genetic copy-number analysis. The primers used in this study are shown profiling of polymorphic short tandem repeat (STR) loci (Takara). in Supplementary Table S1.

Cell proliferation assay FISH MTS cell proliferation assays were performed as previously EGFR and chromosome 7 FISH analysis was performed by described (37). Brieﬂy, 2 103 cells/well were seeded in 96-well Genetic lab Co., Ltd. using Vysis LSI EGFR SpectrumOrange and plates and treated with EGFR-TKI or dimethyl sulfoxide (DMSO) CEP 7 SpectrumGreen probes, respectively. vehicle 24 hours later. Absorbance was measured 72 hours after treatment. All experiments were performed at least three times. Standard Sanger sequencing of PIK3CA Isolated cDNA from H1975 cells was used as template in PCR Phospho-receptor tyrosine kinase array reactions for PIK3CA. Ampliﬁed PIK3CA was sequenced and The human phospho-receptor tyrosine kinase (phospho-RTK) compared with the National Center for Biotechnology Informa- array kit was purchased from R&D Systems) and screened accord- tion reference sequence NM_006218.3.

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EGFR Wild-Type Allele Ampliﬁcation Induces Resistance

PIK3CA siRNA knockdown rociletinib (CO-1686), we established third-generation EGFR- Cells were transfected with PIK3CA-specific siRNA (#S10520; TKI–resistant cells using a dose-escalation method. PC9 and Life Technologies) or negative control siRNA (Ambion Silencer H1975 cells were cultured with rociletinib or osimertinib. The Select Negative Control mix, Life Technologies) using siLentFect initial concentration was 0.03 mmol/L, and this was incremen- transfection reagent (Bio-Rad) according to the manufacturers' tally increased to 1 mmol/L. After several months of exposure, protocols. PIK3CA qRT-PCR was used to confirm gene knockdown. resistant cell lines, rociletinib-resistant PC9 (PC9-COR), osimertinib-resistant PC9 (PC9-AZDR), rociletinib-resistant Mouse xenograft model H1975 (H1975-COR), and osimertinib-resistant H1975 All animal experiments were approved by the Laboratory Ani- (H1975 AZDR) were established. Their resistance to a first- mal Center, Keio University School of Medicine (Tokyo, Japan). (erlotinib), third-generation (rociletinib, osimertinib, or nazar- Female BALB/c-nu mice were purchased from Charles River. Mice tinib) EGFR-TKIs was confirmed by the MTS cell proliferation were anesthetized with ketamine and PC9-COR#9 cells were assay (Fig. 1). The calculated IC50 values are summarized in subcutaneously injected in a Matrigel suspension. Calipers were Supplementary Table S2. Notably, the resistant cells displayed used to measure tumor volume. Once average tumor volume cross-resistance to all third-generation EGFR-TKIs examined in reached 150 mm3, mice were randomized to receive vehicle alone, this study, suggesting that the mechanisms of resistance to third- cetuximab (1 mg/mouse twice per week, intraperitoneally), roci- generation drugs may not be inhibitor specific. Further, resistant letinib (30 mg/kg daily, orally), or a combination of both. Animals cells also showed a diminished propensity to undergo treatment- were humanely sacrificed and tumor tissues were harvested. induced apoptosis (Supplementary Fig. S1), indicating that the cells acquired resistance to third-generation EGFR-TKIs. Whole-exome sequencing – Whole-exome sequencing libraries were prepared with 3 mg Whole-exome sequencing of parental and EGFR-TKI resistant of DNA. The exomes were captured using the SureSelect Human cells All Exon V5þUTRs Kit (Agilent Technologies) according to the Bypass pathway activation is consistently reported as one of the manufacturer's instructions and then sequenced using a HiSeq mechanisms by which cells acquire resistance to EGFR-TKIs. As fi 1500 system (Illumina) to generate 100-bp paired-end data. such, we rst examined the phosphorylation levels of multiple The whole-exome sequencing data were deposited in the DDBJ receptor tyrosine kinases in our resistant PC9 and H1975 cells database. Accession numbers: DRA004904, PRJDB5021, and with a human phospho-RTK array kit, but did not observe any SAMD00056514-SAMD00056521 activation of reported bypass pathways (HER2, HGFR, IGF-1R, and AXL signaling; Supplementary Fig. S2). Variant calling To clarify the heterogeneity of potential resistance mechan- Sequence reads were aligned to the human reference genome isms, the PC9-COR, PC9-AZDR, H1975-COR, and H1975- UCSC hg19 using the Burrows-Wheeler Aligner program (BWA, AZDR lines were subcloned to isolate resistant clones (Supple- http://bio-bwa.sourceforge.net/). Single-nucleotide variants (SNV) mentary Fig. S3). DNA isolated from the parental and resistant and insertions and deletions (INDEL) were called and annotated cell clones was then subjected to whole-exome sequencing. using the Genome Analysis Toolkit software package (GATK, http:// Quality of the whole-exome sequencing results and copy num- www.broadinstitute.org/gatk/). Sequencing artifacts were filtered ber alterations are summarized in Supplementary Table S3 and out using custom filters (GATK confidence score, 50; number of Supplementary Fig. S4. Several genetic alterations potentially variant reads in each direction, 1; variant allele frequency, 10%). relevant to EGFR-TKIs sensitivity were detected (Table 1), Known germline variants were filtered out using data from dbSNP including some already reported. For instance, osimertinib KRAS build 131, the 1,000 Genomes Project (Phase 1 exome data, resistance can occur via oncogenic signaling (33). In our KRAS fi released May 21, 2011), 1 Japanese genome, and 299 in-house model, G13D was identi ed in PC9-AZDR clones (Sup- fi Japanese exomes. plementary Fig. S5), supporting the validity of our ndings. EGFR C797S or L798I was not found in the resistant cells. Copy number analysis Epithelial-to-mesenchymal transition phenotype and AKT CNVs in resistant cells were analyzed from exome sequence pathway activation in third-generation EGFR-TKI–resistant data. The log ratio of depth coverage between parental and cells resistant cells was calculated using the GATK Depth of Coverage Parental H1975 cells harbor a heterozygous PIK3CA G118D tool. CNV segments were then called from the log ratio of depth of mutation. Interestingly, the PIK3CA wild-type allele was lost in coverage using the ExomeCNV package (38). three of the resistant clones—H1975-AZDR#1 and #3, and H1975-COR#3, leading to a loss of heterozygosity (Supplemen- Statistical analysis tary Table S4; Supplementary Fig. S6A and S6B). The activating Statistical analysis was performed using GraphPad Prism soft- PIK3CA G118D mutation is found in various cancer types, ware, version 4.0 (GraphPad Software). Two-sided Student t tests including endometrium (39) and colorectal (40, 41) cancers. In were used for comparisons, with P < 0.05 regarded as statistically order to investigate whether acquired homozygosity of PIK3CA significant. G118D could functionally induce resistance to EGFR-TKIs, we examined the impact of PIK3CA inhibition on EGFR-TKI resistant Results cells using the PIK3CA inhibitor wortmannin. Although effective Establishment of third-generation EGFR-TKI–resistant cells when used in combination with EGFR-TKI in parental H1975 To clarify the mechanisms underlying acquired resistance to cells, wortmannin was unable to restore EGFR-TKI sensitivity in third-generation EGFR-TKIs, such as osimertinib (AZD9291) and the H1975-resistant clones (Supplementary Fig. S6C and S6D).

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Figure 1. Establishment of third-generation EGFR-TKI–resistant cells. MTS cell proliferation assays for PC9 and H1975 parental and resistant cells treated with increasing concentrations of the indicated EGFR-TKIs for 72 hours. Error bars, SD.

Further, the siRNA-mediated knockdown of PIK3CA gene expres- not contribute to the acquired resistance to EGFR-TKI in H1975- sion in the H1975-resistant clones also failed to restore EGFR-TKI resistant cells. sensitivity (Supplementary Fig. S6E and S6F). These data indicate While performing these experiments, we noticed a change in that acquired homozygosity of the PIK3CA G118D mutation did cell morphology where the H1975-resistant cells exhibited a spindle cell-like morphology (Fig. 2A). Epithelial-to-mesenchymal transition (EMT) is reported to occur in EGFR-resistant Table 1. Results from whole-exome sequencing show several genetic NSCLC (42). To confirm whether H1975 cell clones acquired a alterations potentially relevant to EGFR-TKI sensitivity more mesenchymal phenotype, we analyzed EMT markers by Cell line Genetic alterations Western blotting. Consistently, expression of the mesenchymal EGFR PC9 E746_A750del marker vimentin and the epithelial E-cadherin was upregulated PC9-COR#9 EGFR E746_A750delþEGFR wild-type amplification and downregulated in the resistant clones at the protein level, PC9-AZDR#4 EGFR E746_A750delþKRAS G13D PC9-AZDR#5 EGFR E746_A750delþKRAS G13D respectively (Fig. 2B). Moreover, both total and phosphorylated H1975 EGFR L858R/T790MþPIK3CA G118D (heterozygosity) Src were elevated in H1975-resistant cells. Next, we examined the H1975-COR#3 EGFR L858R/T790MþPIK3CA G118D (homozygosity) effect of EGFR-TKI treatment on EGFR downstream pathway H1975-AZDR#1 EGFR L858R/T790MþPIK3CA G118D (homozygosity) activation, including AKT and MAPK, in H1975-resistant cells. EGFR þPIK3CA H1975-AZDR#3 L858R/T790M G118D (homozygosity) EGFR and ERK1/2 phosphorylation were efficiently inhibited by

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Figure 2. EMT and Src–AKT pathway activation in third-generation EGFR-TKI– resistant cells. A, Representative pictures of parental and resistant H1975 cells. B, Western blot analysis of H1975 parental and resistant (COR#3, AZDR#1) cells for E-cadherin, vimentin, integrin b1, total (t-) and phosphorylated (p-) Src, PTEN, and b-actin. C, Western blot analysis for total (t-) and phosphorylated (p-) EGFR, AKT, ERK1/2, and b actin in H1975 parental and resistant (COR#3, AZDR#1) cells. D, Integrin b1(ITGB1) gene expression relative to that of GAPDH in H1975 parental cells and resistant clones. Error bars, SD. E, PTEN gene expression relative to that of GAPDH in H1975 parental cells and resistant clones. Error bars, SD. F, MTS cell proliferation assays following treatment with the indicated EGFR- TKI concentrations with or without bosutinib or dasatinib in H1975 parental cells and resistant clones (H1975-COR#3, H1975-AZDR#1). Error bars, SD. G, Western blot analysis for total (t-) and phosphorylated (p-) Src and b-actin in H1975 parental cells treated with the indicated concentrations of bosutinib or dasatinib.

rociletinib and osimertinib treatment; however, limited effects H1975-resistant clones (Fig. 2B and D). A loss of PTEN and were observed in AKT phosphorylation in H1975-resistant cells subsequent AKT pathway activation facilitate EGFR-TKI resis- when compared with H1975 parental counterparts (Fig. 2C). tance in mutant lung cancer (44); however, PTEN expression was Integrin b1/Src/AKT-mediated bypass signaling has previously intact in H1975-resistant cells (Fig. 2B and E). To examine been reported in erlotinib-resistant NSCLC (43). Consistent with whether Src–AKT pathway activation contributed to the acquired this report, we found that integrin b1 and phospho-Src were resistance to third-generation EGFR-TKI resistance, we per- increased at the RNA and protein levels, respectively, in formed MTS proliferation assays on H1975 clones treated with

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Figure 3. EGFR wild-type allele amplification in rociletinib-resistant clones. A, Copy- number variants (CNV) in PC9-COR#9 cells analyzed from exome sequence data. B, Two-color FISH analysis of PC9 parental and PC9-COR#9 cells. Green and red signals indicate CEP7 and EGFR, respectively. C, Relative gene copy number of EGFR when compared with LINE1. D, Western blot analysis of total (t-) EGFR, exon 19 deletion-specific EGFR (EGFRdel19), and b-actin in PC9 parental and resistant cells. E, Relative gene expression in PC9 parental and resistant cells. Error bars, SD. F, Western blot analysis for total (t-) and phosphorylated (p-) EGFR, exon 19 deletion–specific EGFR (EGFRdel19), total (t-), and phosphorylated (p-) AKT, ERK1/2, and b-actin in PC9 parental and resistant cells.

third-generation EGFR-TKIs in the presence or absence of the Src confirm this finding, chromosome 7 and the EGFR gene were inhibitors dasatinib or bosutinib. Interestingly, both dasatinib analyzed by FISH and scored using the ratio EGFR signal to and bosutinib partially restored sensitivity to third-generation that of chromosome 7 (Fig. 3B). PC9 parental cells harbor EGFR-TKIs (Fig. 2F and G), suggesting that Src–AKT pathway EGFR gene amplification; however, the ratio of signals became activation may contribute to the acquired resistance to third- higher in PC9-COR#9 clones when compared with parental generation EGFR-TKIs. counterparts (Supplementary Table S5). In addition, genomic DNA qPCR for the EGFR loci in the PC9-COR clones confirmed EGFR wild-type allele amplification induces resistance to third- the amplification(Fig.3C).Furthermore,theEGFR wild-type generation EGFR-TKIs copy number and total EGFR expression were markedly Notably, our exome sequencing data revealed an amplifi- increased in PC9-COR#9 cells (Supplementary Table S6; Fig. cation of the EGFR gene locus on chromosome 7 (Fig. 3A). To 3D). The relative number of tags for the wild-type EGFR allele

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Figure 4. EGFR wild-type allele ampliﬁcation contributes to third-generation EGFR-TKI resistance. A, Western blot analysis of H1975 cells transduced with empty vector (mock) or HA-tagged EGFR. B, MTS cell proliferation assays for H1975 mock and H1975 EGFR cells treated with the indicated EGFR-TKI concentrations with or without EGF (10 ng/mL, 100 ng/mL) for 72 hours. Error bars, SD. C, Western blot analysis of H1975 mock and H1975 EGFR cells treated with the indicated concentrations of EGF and rociletinib for phosphorylated (p-) and total (t-) EGFR, AKT, ERK1/2, and b-actin.

(31 tags) was about one fourth (0.25 times) that of the mutant ERK by rociletinib was attenuated in the PC9-COR#9 clone allele (127 tags) in PC9 parental cells; however, the wild-type when compared with the PC9 parental cells (Fig. 3F). These allele was approximately 8.5-fold more prevalent in PC9- results indicated that EGFR wild-type allele amplification likely COR#9 cells (512 vs. 60 tags for the wild-type and mutant induces the acquired resistance to third-generation EGFR-TKIs alleles, respectively). These data indicate that the amplified through EGFR ligand-induced activation. EGFR wild-type allele likely conveyed resistance to third-gen- To ascertain the plausibility of the aforementioned situation, eration EGFR-TKIs. we expressed HA-tagged wild-type EGFR in H1975 parental cells We next examined EGFR ligand expression to evaluate the and evaluated their sensitivity to rociletinib and osimertinib in the possibility of ligand-induced activation of EGFR (45). Until presence of EGF. When compared with mock-transfected H1975 now, seven EGFR ligands have been identified: epithelial cells, wild-type EGFR-overexpressing H1975 cells developed resis- growth factor (EGF), amphiregulin, heparin-binding EGF-like tance to rociletinib and osimertinib upon EGF administration growth factor (HB-EGF), epiregulin, TGFa, epigen, and beta- (Fig. 4A and B), and rescued EGF-induced EGFR, AKT, and ERK1/2 cellulin. Quantitative RT-PCR analysis revealed an increased phosphorylation (Fig. 4C). Thus, these data indicate that EGFR expression of all seven ligands in PC9-COR cells and PC9- wild-type allele amplification and concomitant EGFR ligand COR#9 cells compared with PC9 parental cells (Fig. 3E). As expression contribute to acquired resistance to third-generation expected, the inhibition of phosphorylation of EGFR, AKT, and EGFR-TKIs.

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Figure 5. Rociletinib and cetuximab combination treatment overcomes EGFR wild–type–mediated acquired resistance in vitro. A, MTS cell proliferation assays for PC9 and PC9-COR#9 cells treated with the indicated concentrations of cetuximab for 72 hours. Error bars, SD. B, MTS cell proliferation assays of PC9-AZDR#5 cells treated with the indicated concentrations of cetuximab and osimertinib for 72 hours. Error bars, SD. C, MTS cell proliferation assays for PC9-COR#9 cells treated with the indicated concentrations of rociletinib and cetuximab for 72 hours. Error bars, SD. D, Western blot analysis of phosphorylated (p-) and total (t-) EGFR, AKT, ERK1/2, and b-actin in PC9-COR#9 cells treated with the indicated concentrations of rociletinib and cetuximab for 8 hours. E, Flow cytometric data for PC9-COR#9 cells treated with DMSO, rociletinib, cetuximab, and rociletinb/cetuximab in combination for 72 hours. The numbers (%) indicate the proportion of Annexin V–FITC- and/or propidium iodide-stained cells. F, MTS cell proliferation assays for PC9-COR#9 cells treated with the indicated concentrations of rociletinib, nazartinib, or afatinib for 72 hours. Error bars, SD.

Rociletinib/cetuximab cotreatment overcomes EGFR wild- ligand binding would be sufﬁcient to restore sensitivity to roci- type–mediated acquired resistance in vitro and in vivo letinib in PC9-COR#9 cells. For this, we performed MTS assays in Concomitant EGFR wild-type ampliﬁcation and ligand expres- the presence of cetuximab, an anti-EGFR antibody that blocks sion promoted us to determine whether the inhibition of EGFR ligand binding. As a single agent treatment, cetuximab had no

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Figure 6. Overcoming EGFR wild-type–mediated acquired resistance by rociletinib and cetuximab combination treatment in vivo. A and B, PC9-COR#9–derived tumor-bearing mice were randomized into control, rociletinib, or rociletinib/cetuximab combination treatment groups. The body weight of the mice was monitored. Tumor size was measured to calculate tumor volume. Values indicate average tumor volume in each group. , P < 0.01 for the combination of rociletinib/ cetuximab combination versus rociletinib alone. Error bars, SD. C, Representative images of tumor-bearing mice and tumors. D, Proposed model of EGFR wild-type–mediated acquired resistance.

effect on cell proliferation in PC9 parental cells and PC9- eration EGFR-TKIs. We evaluated the effect of afatinib on PC9- COR#9 cells (Fig. 5A), nor did rociletinib/cetuximab combi- COR#9 cells by MTS assay because we previously reported the nation therapy show a synergistic effect in PC9 parental cells decreased mutation specificity of afatinib (28). The prolifera- and PC9-AZDR#5 cells that harbor a KRAS G13D mutation tion of PC9-COR#9 cells was inhibited by lower concentrations (Fig. 5B). However, combination therapy effectively restored of afatinib than rociletinib or nazartinib (Fig. 5F). These find- rociletinib sensitivity in PC9-COR#9 cells (Fig. 5C). We used ings also indicate that EGFR wild-type mediated signaling Western blotting to investigate whether rociletinib/cetuximab contributes to PC9-COR#9 cell resistance. cotreatment suppressed pathway activation downstream of In addition, we evaluated the efficacy of rociletinib and cetuximab EGFR in PC9-COR#9 cells and found that it efficiently inhibited or afatinib cotreatment with H1975 cells expressing wild-type EGFR both AKT and ERK1/2 phosphorylation(Fig.5D).Analysisof (H1975 EGFR) or C797S EGFR (H1975 C797S; Supplementary Fig. apoptosis by FACS revealed an increase in Annexin V–positive S7). As observed previously, wild-type EGFR overexpression cells with the subsequent addition of cetuximab (12.5 % with induced rociletinib resistance following EGF stimulation. The addi- 1 mmol/L rociletinib alone, 32.7% with rociletinib/cetuximab tion of afatinib or cetuximab restored rociletinib sensitivity to both combination; Fig. 5E), indicating that ligand-mediated EGFR transduced lines. However, the restoration of afatinib was more activation contributed to the acquired resistance to third-gen- significant in H1975 EGFR cells compared with H1975 C797S cells.

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Finally, mouse xenograft models were used to examine whether specificity of circulating tumor DNA, it will become commonly the roclietinib/cetuximab combination treatment could suppress used as reliable samples in the clinics. However, the contam- EGFR wild-type-mediated resistance in vivo. During treatment, the ination of nonmalignant cells derived DNA is inevitable and body weight of the mice and PC9-COR#9 cells derived tumor may limit the accuracy of gene copy-number analysis in cancer volumes were monitored (Fig. 6A–C). Notably, tumor volumes cells. Thus, to identify the EGFR wild-type allele amplification, increased in the control and rociletinib groups; however, roclie- we believe biopsies from either primary tumors or metastatic tinib/cetuximab cotreatment significantly inhibited tumor lesions are necessary. growth. Collectively, these data support the possibility that com- In summary, the present study characterized novel mechan- bination therapy may be an effective approach to overcome EGFR isms of acquired resistance to mutant-selective third-genera- wild-type-dependent acquired resistance. tion EGFR-TKIs. We propose a novel concept of acquired resistance to mutation selective inhibitors, wild-type allele Discussion mediated resistance. In addition, we propose a preclinical rationale for the use of more promiscuous EGFR-TKI, afatinib, In NSCLC, EGFR mutations activate EGFR, which induce or cetuximab combination therapy in EGFR inhibitor–resistant the transformation of lung epithelial cells. EGFR is ubiqui- cancers. While these findings underscore the importance of tously expressed in organ epithelial cells, including lung, EGFR wild-type–mediated signals in acquired resistance, addi- gastrointestinal tract, and skin. As such, it is essential to tional preclinical and clinical trials are necessary to evaluate selectively inhibit mutant EGFR-derived signals to minimize the efficacy and safety of these treatments to avoid adverse side adverse effects of EGFR-TKIs, such as diarrhea and skin rash. effects related to EGFR pathway inhibition in nonmalignant Third-generation EGFR-TKIs selectively inhibit EGFR mutants cells. and minimally affect the wild-type EGFR (25, 26). This mirrors the significant efficacy and safety of third-generation Disclosure of Potential Conflicts of Interest EGFR-TKIs observed in recent clinical trials. Specifically, osi- K. Soejima has received speakers bureau honoraria from Chugai, Ono, Taiho, mertinib and rociletinib showed significant efficacy and safety Eli Lilly, AstraZeneca, Pfizer, and Shionogi. No potential conflicts of interest for previously treated NSCLC patients harboring EGFR-acti- were disclosed by the others. vating mutations, although rociletinib is no longer in development. The response rate and progression-free survival of Authors' Contributions osimertinib for EGFR T790M-positive patients were 61% and Conception and design: S. Nukaga, H. Yasuda, K. Tsuchihara, J. Hamamoto, 9.6 months, respectively (29). The response rate of rociletinib I. Kawada, S. Ikemura, K. Goto, K. Soejima Development of methodology: S. Nukaga, H. Yasuda, K. Tsuchihara, J. Hama- for EGFR T790M-positive patients was 45% (46). Neither moto, I. Kawada, S. Matsumoto, S. Ikemura, K. Soejima osimertinib nor rociletinib exhibited a dose-limiting toxicity; Acquisition of data (provided animals, acquired and managed patients, therefore, a maximum tolerated dose was not determined provided facilities, etc.): S. Nukaga, H. Yasuda, K. Tsuchihara, J. Hamamoto, (29, 30). Thus, mutation-selective inhibitors offer a clinically K. Masuzawa, K. Naoki, S. Mimaki, S. Ikemura relevant advantage of EGFR-TKI treatment with minimal Analysis and interpretation of data (e.g., statistical analysis, biostatistics, adverse effect. computational analysis): S. Nukaga, H. Yasuda, K. Tsuchihara, J. Hamamoto, fi K. Masuzawa, K. Naoki, S. Ikemura In this study, we identi ed several novel mechanisms of Writing, review, and/or revision of the manuscript: S. Nukaga, H. Yasuda, acquired resistance to third-generation EGFR-TKIs, including K. Tsuchihara, I. Kawada, K. Naoki, S. Matsumoto, S. Ikemura, K. Goto, Src–AKT pathway activation and EGFR wild-type allele amplifi- T. Betsuyaku, K. Soejima cation (Fig. 6D and Supplementary Fig. S8). Most cancer-specific Administrative, technical, or material support (i.e., reporting or organizing somatic driver mutations, such as those in KRAS and EGFR, exist data, constructing databases): H. Yasuda, J. Hamamoto, S. Ikemura, in heterozygous. Until today, a limited number of studies have T. Betsuyaku Study supervision: H. Yasuda, K. Tsuchihara, I. Kawada, S. Ikemura, K. Goto, focused on the function of the wild-type allele in cancer cells. T. Betsuyaku, K. Soejima Here, we show that cancer cells exploit this decreased inhibitory pressure for the wild-type allele. Acknowledgments The mechanisms underlying acquired resistance to third-gen- We thank Ms. Mikiko Shibuya for her excellent technical assistance. We also eration EGFR-TKIs have already been partially clarified. These thank the Collaborative Research Resources, Keio University, School of Med- include the C797S (26) and L798I (32) mutations that presum- icine, for cell sorting. ably prevent the covalent binding of EGFR-TKIs to EGFR, bypass pathway activation (33–35, 47), and mutant EGFR gene ampli- Grant Support fi fication (48, 49). Specifically, Piotrowska and colleagues reported This work was supported in part by Grants-in-Aid for Scienti c Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and EGFR T790M allele amplification in rociletinib-resistant clones fi Technology of Japan to S. Nukaga (Grant #16K19465), T. Betsuyaku (Grant (49). To our knowledge, this is the rst report describing a role for #15H04833), K. Soejima (Grant #22590870), H. Yasuda (Grant #25860656, wild-type EGFR in acquired resistance to mutant-selective third- 15H05666, and 15K14398) and the Practical Research for Innovative Cancer generation EGFR-TKIs. Control from Japan Agency for Medical Research and Development to S. Recently, circulating tumor DNA in blood is becoming a Matsumoto (16ck0106012h0003). study sample for clarification of mechanisms underlying The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked acquired resistance to EGFR-TKIs (26, 32). By using recent advertisement fi in accordance with 18 U.S.C. Section 1734 solely to indicate next-generation sequencing technology, the identi cation of this fact. cancer cells derived genetic alterations including mutations and gene copy-number alterations become possible. Considering Received August 30, 2016; revised January 4, 2017; accepted January 23, 2017; the convenient access to blood samples and sensitivity and published OnlineFirst February 15, 2017.

OF10 Cancer Res; 77(8) April 15, 2017 Cancer Research

EGFR Wild-Type Allele Ampliﬁcation Induces Resistance

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OF12 Cancer Res; 77(8) April 15, 2017 Cancer Research

Amplification of EGFR Wild-Type Alleles in Non−Small Cell Lung Cancer Cells Confers Acquired Resistance to Mutation-Selective EGFR Tyrosine Kinase Inhibitors

Shigenari Nukaga, Hiroyuki Yasuda, Katsuya Tsuchihara, et al.

Cancer Res Published OnlineFirst February 15, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-2359

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