Combining Onartuzumab with Erlotinib Inhibits Growth of Non

Published OnlineFirst December 18, 2014; DOI: 10.1158/1535-7163.MCT-14-0456

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Combining Onartuzumab with Erlotinib Inhibits Growth of Non–Small Cell Lung Cancer with Activating EGFR Mutations and HGF Overexpression Yuji Sano1, Eri Hashimoto1, Noriaki Nakatani2, Masaichi Abe3, Yasuko Satoh1, Kiyoaki Sakata1, Toshihiko Fujii1, Kaori Fujimoto-Ouchi1, Masamichi Sugimoto1, Shigehisa Nagahashi1, Masahiro Aoki1, Hiroshi Motegi2, Eiichi Sasaki4, and Yasushi Yatabe4

Abstract

Erlotinib, a tyrosine kinase inhibitor of the epidermal growth hHGF xenograft tumors, onartuzumab or erlotinib alone min- factor receptor (EGFR-TKI), benefits survival of patients with imally inhibited tumor growth; however, combining onartu- non–small cell lung cancer (NSCLC) who harbor activating EGFR zumab and erlotinib markedly suppressed tumor growth. The mutations. However, elevated expression of hepatocyte growth total MET protein level was decreased in PC-9/hHGF cells, factor (HGF), a ligand of the MET receptor tyrosine kinase, causes because MET is constitutively phosphorylated by autocrine erlotinib resistance. Because onartuzumab, a monovalent anti- HGF, leading to its ubiquitination and degradation. Onartu- body to MET, blocks HGF-induced MET activation, the addition zumab reduced phospho-MET levels, inhibited MET ubiquiti- of onartuzumab to erlotinib may improve therapeutic efficacy. nation, and consequently restored MET protein levels. More- We engineered the human NSCLC cell line PC-9 (MET-positive over, in NSCLC clinical specimens harboring activating EGFR cells harboring an exon 19 deletion of EGFR) to overexpress hHGF mutations, more than 30% of patients expressed high levels of and evaluated the effects of an onartuzumab and erlotinib HGF. Our findings raised the possibility that patients with combination in vitro and in vivo in xenograft models. A stable NSCLC with EGFR mutations who express high levels of HGF clone of PC-9/hHGF was less sensitive to erlotinib than the may benefit from onartuzumab and erlotinib combination parental PC-9, and the addition of onartuzumab to erlotinib therapy, and that HGF can be a novel biomarker for selecting suppressed the proliferation of these cells in vitro.InPC-9/ such patients. Mol Cancer Ther; 14(2); 533–41. 2014 AACR.

Introduction EGFR T790M mutation, which causes resistance to EGFR-TKI, has been seen in half of these treatment-resistant patients (7–9). Some Lung cancer, one of the leading causes of cancer-related mor- studies show that other than EGFR T790M, MET amplification tality, is classified into two histologic subgroups: non–small cell and high expression of hepatocyte growth factor (HGF) also cause lung cancer (NSCLC) and small-cell lung cancer (SCLC; ref. 1). treatment resistance (10–13). High HGF expression levels in The treatment of NSCLC, which accounts for approximately 85% tumors, even in patients with activating EGFR mutations, are of lung cancer, has improved with the development of molecular associated with a poor response to EGFR-TKI (14, 15) because targeted therapies (2). Tyrosine kinase inhibitors of epidermal the HGF–MET pathway, an alternative signaling route to EGFR, is growth factor receptor (EGFR-TKI), such as erlotinib and gefitinib, also activated (16). are efficacious in patients with NSCLC with activating EGFR MET belongs to the receptor tyrosine kinase family. It was mutations (3–6). However, continuous administration of the originally found as a fusion protein, TPR–MET, which was pro- drug decreases efficacy in once-responsive patients; the acquired duced by translocation in a sarcoma cell line treated with a carcinogen (17). MET and its ligand, HGF, are known to regulate numerous biologic activities, including cell proliferation, migra- 1Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., tion, invasion, angiogenesis, survival, and metastasis (18–21). Kamakura, Kanagawa, Japan. 2Project Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., Chuo-ku, Tokyo, Japan. 3Clinical MET is highly expressed in various cancers (20), including NSCLC, Development Division, Chugai Pharmaceutical Co., Ltd., Chuo-ku, and elevated MET and HGF levels in patients correlate with a Tokyo, Japan. 4Department of Pathology and Molecular Diagnostics, higher pathologic tumor stage (22). Aichi Cancer Center, Nagoya, Aichi, Japan. Unlike other anti-MET antibodies that may activate MET sig- Note: Supplementary data for this article are available at Molecular Cancer naling (23, 24), onartuzumab was designed to be monovalent, to Therapeutics Online (http://mct.aacrjournals.org/). avoid agonistic activity (25), and it blocks the binding between Corresponding Author: Yuji Sano, Discovery Pharmacology Department, MET and HGF (26, 27). Onartuzumab is produced in Escherichia Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247- coli and does not have antibody-dependent cellular cytotoxicity 8530, Japan. Phone: 81-467-47-6241; Fax: 81-467-47-2234; E-mail: (ADCC; ref. 28). Demonstrating the ligand blocking activity of [email protected] onartuzumab in xenograft models is problematic because mouse doi: 10.1158/1535-7163.MCT-14-0456 HGF does not activate human MET and thus has no effect on the 2014 American Association for Cancer Research. proliferation of grafted human tumors (29, 30). Therefore, the

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only paracrine models available to date are those that supply nologies), and resistant colonies were expanded. We detected human HGF via implanted osmotic pumps (26), by human HGF human HGF in the culture medium of clones by enzyme-linked transgenic mice (31), or by the coinoculation of cancer cells with immunosorbent assay (ELISA) and selected one clone for PC-9, HGF-producing human fibroblast (32). However, onartuzumab and two clones for HCC827 secreting a high level of HGF for has shown high efficacy in autocrine models that use the U-87MG further analysis. and KP4 cell lines, which produce human HGF (26, 27). In a phase II study of patients with second-line or third-line Flow cytometric analysis NSCLC, where most of the patients possess wild-type EGFR, Cells were suspended in phosphate-buffered saline (PBS) erlotinib combined with onartuzumab improved progression- with 0.5% bovine serum albumin (BSA). The anti-MET mono- free survival (PFS) and overall survival (OS) in MET-positive clonal antibody prepared in-house was added, and cells were patients, as determined by immunohistochemistry (IHC; refs. 33, then incubated on ice for 30 minutes. After washing with PBS 34). A phase III (MetLung) study was conducted with the same containing 0.5% BSA, FITC-conjugated anti-mouse secondary study design as the phase II study, where high HGF levels were not antibody (Dako) was added. After washing with PBS containing an inclusion criterion for patient selection. Interim analysis of the 0.5% BSA, the samples were analyzed with Epics XL flow phase III data could not confirm the efficacy observed in the phase cytometer (Beckman–Coulter), or BD FACSVerse (Becton Dick- II study (35). inson) using the CellQuest Pro software (Becton Dickinson), or Here, we modified EGFR-mutated cell lines to overexpress BD FACSuite software (Becton Dickinson). The antibody-bind- human HGF, and observed the additive effect of onartuzumab ing capacity of the cell surface MET was calculated using a QIFI and erlotinib on tumor growth inhibition. Our results suggested kit (Dako). the possibility that the combination of onartuzumab and erloti- fi EGFR nib may be more ef cacious in patients with activating Western blotting mutations who are erlotinib-resistant due to an elevated HGF Cells were lysed with the cell lysis buffer [100 mmol/L Tris-HCl expression. (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 10% glycerol, 1% Triton X-100). Tumor samples were homogenized with a multi- Materials and Methods beads shocker cell disruptor (Yasui Kikai), or a Biomasher (Funa- Cell lines koshi). Protein concentration in the lysates was determined using PC-9, a human lung adenocarcinoma cell line harboring an the Bio-Rad Protein Assay (Bio-Rad), and 50 mg of total protein activating EGFR mutation (deletion of E746–A750) was pur- was resolved by SDS-PAGE. After transfer of proteins onto nitro- chased from and characterized by Immuno-Biological Laborato- cellulose membranes, the membranes were probed with specific ries in 2009. HCC827, a human lung adenocarcinoma cell line antibodies against MET (Life Technologies), HGF (LifeSpan Bio- with an activating EGFR mutation (deletion of E746–A750), and Science), EGFR (1005), beta-actin (C-2; Santa Cruz Biotechnol- U-87MG, a glioblastoma cell line, were purchased from and ogy), phospho-EGFR (Y1068), phospho-MET (Y1234/1235), characterized by the American Type Culture Collection in 2006 ERK, phospho-ERK (T202/Y204), AKT and phospho-AKT and 2005, respectively. KP4, a pancreatic cancer cell line, was (S473), all from Cell Signaling Technology. After washing with purchased from and characterized by RIKEN Bio Resource Center Tris-buffered saline containing 0.05% Tween-20 (TBS-T), mem- in 2001. All cell lines were cultured according to suppliers' branes were probed with species-specific IR-dye 680- or 800CW- instructions. No further cell line authentication was conducted conjugated secondary antibodies (LI-COR Bioscience). After for all these cell lines. washing with TBS-T, membranes were scanned and analyzed with an Odyssey infrared imaging system (LI-COR Bioscience). Reagents Erlotinib was obtained from Hoffmann–La Roche. Onartuzu- Ubiquitination assay mab was obtained from Genentech, Inc. Gefitinib was purchased Cells were treated with 50 mmol/L of MG132, a proteasome from Kemprotec. Crizotinib, a MET and ALK dual inhibitor, was inhibitor (Sigma–Aldrich) and/or 30 mg/mL of onartuzumab for prepared in-house. 12 hours. Cell lysate protein (800 mg) was used for immunoprecipitation with 1.2 mg of in-house–prepared anti-MET chimeric Cell growth inhibition assay antibody (in which the Fc region was replaced with human Fc); Cells were plated in 96-well plates. After incubation for 1 day, immunoprecipitation proceeded on ice for 12 hours. After protein A sepharose was added to lysates, samples were rotated gently at various concentrations of erlotinib were added. For the combination assay, 30 mg/mL of onartuzumab was combined with 4 C for 2 hours. After extensive washing, immunoprecipitates various concentrations of erlotinib. To examine the effect of HGF were resolved by SDS-PAGE and proteins transferred onto on the sensitivity of PC-9 to erlotinib, 25 or 50 ng/mL of nitrocellulose membranes. Ubiquitinated MET was detected by recombinant human HGF (R&D Systems) was used together with Western blotting using anti-ubiquitin Ab (P4D1; Santa Cruz various concentrations of erlotinib. The cells were incubated for a Biotechnology). further 3 days, and viable cells were counted by WST-8 assay (Dojindo Laboratories). Xenograft models All animal studies were performed in accordance with the Establishment of stable PC-9/hHGF and HCC827/hHGF clones policies of the Institutional Animal Care and Use Committee We transfected human HGF expression plasmids into PC-9 or (IACUC) of Chugai Pharmaceutical Co., Ltd. PC-9, PC-9/hHGF, HCC827 cells by electroporation (Bio-Rad). Transfected cells were HCC827, or HCC827/hHGF cells were inoculated subcutaneous- selected in medium containing 300 mg/mL of zeocin (Life Tech- ly into female nude mice. After tumors reached approximately

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200 mm3 in volume, onartuzumab in PBS (30 mg/kg body surface of PC-9 cells by flow cytometric analysis (Supplemen- weight, intraperitoneally, once) was administered alone or in tary Fig. S1B). combination with erlotinib in captisol (Cydex; 50 mg/kg body weight, orally, daily). Control animals received PBS and captisol Effect of HGF on erlotinib sensitivity of NSCLC cell lines as vehicle. Each group comprised 5 mice. Tumor volume was Next, we examined the effect of HGF on the erlotinib sensitivity measured twice per week. of PC-9 cells. As shown in Fig. 1A, HGF addition reversed the efficacy of erlotinib in PC-9 cells in a dose-dependent manner. To Quantitative reverse transcription polymerase chain reaction further assess the effect of HGF on the phosphorylation status of For quantitative reverse transcription polymerase chain reac- downstream molecules in the HGF–MET pathway, we analyzed tion (qRT-PCR), cultured cells and 124 NSCLC specimens, which lysates of cells treated with erlotinib and/or HGF by Western were comprised of 123 adenocarcinomas and one squamous cell blotting. Treatment of PC-9 cells with erlotinib decreased phos- carcinoma, were examined. Total RNA was extracted from snap- pho-EGFR levels and subsequently decreased phospho-ERK and frozen tissues using an RNeasy kit (Qiagen), and cDNAs were phospho-AKT levels. However, cotreatment of PC-9 cells with synthesized using a SuperScriptIII First-strand Synthesis System HGF and erlotinib reversed the effect of erlotinib, and restored (Life Technologies). The synthesized cDNA was used for HGF, or phospho-ERK and phospho-AKT levels. Because the phospho- MET qRT-PCR with SYBR Green (Life Technologies); levels were EGFR levels were still reduced and the phospho-MET levels were normalized to those of a housekeeping gene (GAPDH). Primer increased by this cotreatment, we assumed that activation of the sequences were as follows: HGF forward: 50-CTGCTCCCCA- HGF–MET pathway compensates for erlotinib-induced inhibi- TCGCCATCCCCTATG-30, HGF reverse: 50-TAGGGTAGTCTT- tion of the EGFR pathway (Fig. 1B). TGCTGATTTTTG-30, MET forward: 50-CAGTGAGAAGGCTAAAG- We examined the effect of HGF on erlotinib-sensitivity of GAAACGAAAGATG-30, MET reverse: 50- GGACCGTCAAGAAG- HCC827 cells, another cell line harboring an activating EGFR TAAATAAAATTGTTGC-30, GAPDH 50-forward: GTGAAGGTCGG- mutation, to confirm that PC-9 is not the only cell line to show AGTCAACG-30, GAPDH reverse: 50-TGAGGTCAATGAAGGG- erlotinib resistance upon treatment with HGF. Similar results were GTC-30. PCR was performed on an ABI PRISM 7900HT system obtained in HCC827 cells in growth inhibition and Western (Life Technologies) or ViiA7 real-time PCR system (Life Technol- blotting assays (Supplementary Fig. S1C and S1D). We also ogies), according to the manufacturers' instructions. examined the effect of HGF on erlotinib sensitivity of EGFR wild-type cell lines such as NCI-H292, NCI-H322, and Calu-3, Immunohistochemical analysis which show relatively high sensitivity to erlotinib (Supplemen- For immunohistochemical analysis of MET expression, we used tary Fig. S1A). As shown in Supplementary Fig. S1E, HGF had a a tissue microarray, which comprised a consecutive series of moderate effect on erlotinib resistance in these EGFR wild-type surgically resected lung cancer tissues, including 160 adenocar- cells. cinoma, 36 squamous cell carcinoma, 5 adenosquamous carcinoma, 4 small cell carcinoma, 4 large cell neuroendocrine carci- Establishment of PC-9/hHGF cell line noma, 4 adenocarcinoma in situ, 4 large cell carcinoma, and 2 To establish both an intrinsic and an acquired EGFR-TKI carcinoid tumor specimens. In this cohort, EGFR mutation status resistance model, a human HGF expression plasmid was trans- had already been assessed and has been reported (36). The fected into PC-9 cells. After zeocin selection, we performed HGF sections were stained with anti-MET antibody (SP44; Ventana ELISA and detected increased HGF levels in the culture media of Medical Systems) using a Bench Mark XT system (Ventana) according to the manufacturer's protocol. MET expression was evaluated according to the criteria used in the onartuzumab clinical trials (33, 34). This analysis, together with RT-PCR analysis of the clinical samples, formed part of a comprehensive lung cancer research program that had been approved by an institutional review board in the Aichi Cancer Center. Written informed consent for partic- ipation in the program was obtained from each patient.

Statistical analysis All preclinical data were analyzed by the Student t test. For prevalence studies of MET-IHC and HGF qRT-PCR results, the x2 test or Fisher exact test was used (two-tailed). P values < 0.05 were considered as statistically significant. Figure 1. HGF reverses the growth-inhibitory effect of erlotinib in PC-9 cells, an erlotinib-sensitive NSCLC cell line harboring an activating EGFR mutation. A, Results HGF lowered the sensitivity to erlotinib in PC-9 cells. Cells were treated with Erlotinib sensitivity of PC-9 cells various concentrations of erlotinib with or without 20 or 50 ng/mL of recombinant human HGF for 3 days. Viable cells were counted by WST-8 PC-9 is a representative NSCLC cell line harboring a deletion n ¼ EGFR assay ( 3 wells; error bars, SD). B, HGF restored phospho-AKT (pAKT) and of exon 19 of , and is well known to be particularly phospho-ERK (pERK) levels that were downregulated by erlotinib treatment. fi sensitive to EGFR-TKI (37). We con rmed that PC-9 is more PC-9 cells were treated with 1 mmol/L of erlotinib and/or 50 ng/mL of HGF for sensitive than other NSCLC cell lines to erlotinib (Supplemen- 1 hour. Cell lysates were prepared, and phosphorylated protein levels were tary Fig. S1A). We also confirmed MET expression on the examined by Western blotting.

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transfectants; one such clone (PC-9/hHGF) was chosen for further on PC-9 cells, but had a minimal effect on PC-9/hHGF cells analysis (Supplementary Fig. S2A). (Fig. 2B). We then verified that the mature form of HGF was expressed in Then, we investigated whether a combination of onartuzumab these cells by Western blotting (Fig. 2A). and erlotinib increased the growth-inhibitory effect of erlotinib. PC-9/hHGF cells were cotreated with various concentrations of EGFR-TKI sensitivity of PC9-/hHGF cells erlotinib and 30 mg/mL of onartuzumab. When combined with We treated PC-9/hHGF cells with various concentrations of onartuzumab, erlotinib showed increased growth inhibition of gefitinib or erlotinib to examine whether PC-9/hHGF showed PC-9/hHGF cells, compared with the effects of onartuzumab resistance to EGFR-TKI. As expected, the sensitivity of PC-9/hHGF alone (25%–30% growth inhibition; Fig. 2C). Next, we cotreated to EGFR-TKI was decreased compared with that of the PC-9 parent PC-9/hHGF cells with various concentrations of onartuzumab cells (Supplementary Fig. S2B). and 1 mmol/L of erlotinib and also found increased growth inhibition compared with the effect of erlotinib alone (20% Effect of onartuzumab and erlotinib on PC-9/hHGF cell growth inhibition; Supplementary Fig. S2C). Similar results were proliferation obtained when replacing onartuzumab with crizotinib, a MET/ To confirm whether HGF overexpression renders PC-9 ALK-TKI that interferes with the MET–HGF pathway (Fig. 2D, dependent upon HGF–MET signaling in addition to EGFR Supplementary Fig. S2D). We also established HGF-overexpres- signaling, we examined the effect of onartuzumab on cell sing clones of HCC827 cells and confirmed the high HGF expres- growth. Onartuzumab alone had no growth-inhibitory effect sion level by qRT-PCR (Supplementary Fig. S2E). We examined

Figure 2. Establishment of a stable PC-9 cell line expressing human HGF. A, PC-9 cells were transfected with a human HGF-expression vector. These PC-9/hHGF cells overexpressed mature human HGF, as detected by Western blotting. B, onartuzumab inhibited growth of PC-9/hHGF, but not PC-9 cells. Cells were treated with 30 mg/mL of onartuzumab for 3 days. Viable cells were counted with WST-8 assay, and growth inhibition activity was calculated (n ¼ 6 wells; error bars, SD). C, increased growth-inhibitory effect of erlotinib in combination with onartuzumab in PC-9/hHGF cells. A constant concentration of onartuzumab (30 mg/mL) was combined with various concentrations of erlotinib and used for a growth inhibition assay in PC-9 and PC-9/hHGF (n ¼ 3 wells; error bars, SD). D, MET-TKI in combination with erlotinib showed a synergistic effect on growth inhibition of PC-9/hHGF cells. Cells were treated with 1 mmol/L of crizotinib, a MET/ALK dual inhibitor (as a MET-TKI), and/or 1 mmol/L of erlotinib for 3 days. Viable cells were measured by WST-8, and growth inhibition was calculated (n ¼ 4 wells; error bars, SD). E, combination of onartuzumab and erlotinib decreased phospho-AKT (pAKT) and phospho-ERK (pERK) levels in PC-9/hHGF cells. Cells were treated with 30 mg/mL of onartuzumab and/or 100 nmol/L of erlotinib for 6 hours. Cell lysates were prepared, and the phospho-AKT and phospho-ERK levels were detected by Western blotting.

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whether the combination of onartuzumab and erlotinib increased Effects of onartuzumab and erlotinib in xenograft models growth inhibition of HCC827/hHGF cells and found the same Next, we examined the in vivo efficacy of the combination of results as for PC-9/hHGF cells (Supplementary Fig. S2F). These onartuzumab and erlotinib. We confirmed that HGF levels in the results indicated that autocrine HGF overexpression contributes sera of PC-9/hHGF-engrafted mice whose tumor size was around to EGFR-TKI resistance in cells harboring activating EGFR muta- 200 mm3 were comparable with those of HGF-high patients in the tions (Fig. 2B–2D and Supplementary Fig. S2B–S2D and S2F). onartuzumab phase I study (Supplementary Fig. S3A; ref. 38). Consistent with the in vitro data, erlotinib, but not onartuzumab, Effect of onartuzumab and erlotinib on phosphorylation of showed strong growth inhibition of xenograft tumors derived AKT and ERK from the parent PC-9 line (Fig. 3A). On the other hand, erlotinib To confirm this additive effect of onartuzumab and erlotinib, was less efficacious in PC-9/hHGF xenograft models than in PC-9 we analyzed the phosphorylation status of signaling molecules in models, and onartuzumab demonstrated minimal effects. How- cells treated with onartuzumab and/or erlotinib by Western ever, a combination of the two drugs significantly increased the blotting. In PC-9 cells, treatment with erlotinib alone caused a efficacy of tumor growth inhibition (Fig. 3B). Similar results were significant reduction of both phospho-AKT and phospho-ERK obtained in the HCC827 and HCC827/hHGF clone 9 xenograft levels, but onartuzumab alone had no impact on the phosphor- model (Fig. 3C and D). Surprisingly, the efficacy of the drug ylation status of these molecules, and combining onartuzumab combination in the PC-9/hHGF xenograft model lasted for more with erlotinib caused no further reduction. On the other hand, in than 100 days, showing that the onartuzumab and erlotinib PC-9/hHGF cells, erlotinib treatment could not reduce phospho- combination has a marked and durable effect (Supplementary AKT or phospho-ERK levels as much as it could in PC-9 cells, Fig. S3B). whereas a combination of the two drugs resulted in an additive or We also examined the phosphorylation status of downstream synergistic effect on phospho-AKT and phospho-ERK reduction molecules in PC-9/hHGF tumors and found that the combination (Fig. 2E). of onartuzumab and erlotinib decreased phospho-ERK and

Figure 3. In vivo efficacy of onartuzumab and erlotinib combination in xenograft models. A, adding onartuzumab to erlotinib did not increase therapeutic efficacy in a PC-9 xenograft model. Onartuzumab was administered at 30 mg/kg body weight intraperitoneally by injection once. Erlotinib was administered daily at 50 mg/kg orally (n ¼ 5 mice in each group; error bars, SD). B, additive efficacy of combination therapy in the PC-9/hHGF xenograft model. Dosing and administration schedule were the same as described for the PC-9 xenograft model (n ¼ 5 mice per group; error bars, SD). C, absence of an additive efficacy of combination therapy in the HCC827 xenograft model. Dosing and administration schedule were as described for the PC-9 xenograft model (n ¼ 5; error bars, SD). D, additive efficacy of combination therapy in the HCC827/hHGF clone 9 xenograft model. Dosing and administration schedule were as described for the PC-9 xenograft model (n ¼ 5; error bars, SD).

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Table 1. MET-diagnostic positive ratio in Japanese lung cancer patients onartuzumab and erlotinib are required to inhibit the prolifera- MET diagnosis Positive tion of PC-9/hHGF and HCC827/hHGF tumors (Supplementary EGFR status Positive Negative Total ratio (%) Fig S3E). Mutation 29 53 82 35.4 Wild-type 20 109 129 15.5 Total 49 162 211 23.2 MET expression in Japanese lung cancer patients with activating EGFR NOTE: Statistical significance between mutation and wild-type, P ¼ 0.00135. mutations No tumor was detected in 8 patients. MET-IHC was performed with FFPE To assess the ratio of MET-positive Japanese NSCLC patients sections of lung cancer specimens. with activating EGFR mutations, we performed MET-IHC on their FFPE lung cancer tissue sections, using the same method and phospho-AKT levels more than the administration of the single scoring system as in the onartuzumab clinical trials (Table 1, drugs did (Supplementary Fig. S3C). We also performed Ki-67 Supplementary Fig. S4). According to an onartuzumab phase II immunostaining in formalin-fixed paraffin-embedded (FFPE) clinical trial that was conducted mainly in the United States, MET- xenograft tumor tissue sections and confirmed the combination IHC diagnostic positivity, which is defined as a score of 2þ and effect (Supplementary Fig. S3D). Thus, we concluded that both 3þ, was estimated to be around 50% in patients with NSCLC (34).

Figure 4. Overexpression of HGF decreases MET protein levels. A, MET protein levels were decreased in HGF-overexpressing clones compared with the parent cells. Cell lysates of PC-9, PC-9/hHGF, HCC827, and HCC827/hHGF cells were prepared and total MET expression levels then detected by Western blotting. B, MET protein levels at the cell surface were increased by onartuzumab treatment. PC-9 and PC-9/hHGF cells were treated with 30 mg/mL of onartuzumab for 6 and 24 hours. After cells were collected, 30 mg/mL of anti-MET mouse monoclonal antibody, recognizing a different epitope to onartuzumab, was added. An FITC-conjugated anti-mouse IgG was used as a secondary antibody. Cell surface MET levels were analyzed by flow cytometry. Red line, mock control; green line, untreated cells; pink line, onartuzumab-treated cells. C, MET protein levels in PC-9/hHGF tumors were increased by onartuzumab treatment. Six mice bearing PC-9/hHGF xenograft tumors were administered onartuzumab (30 mg/kg body weight) for 24 hours and 50 mg/kg of erlotinib for 6 hours. Mice were sacrificed, and tumor lysates were used for Western blotting of MET. MET protein level was quantified by densitometry analysis of Western blotting bands. , P < 0.05. D, onartuzumab inhibits MET ubiquitination. Cells were treated with 50 mmol/L of MG-132, a proteasome inhibitor, in the presence or absence of 30 mg/mL of onartuzumab. Cell lysates were prepared and then immunoprecipitated with 1.2 mg/mL of anti-MET antibody. Ubiquitinated MET was detected by Western blotting using an anti-ubiquitin antibody. E, onartuzumab restored total MET protein levels in U-87MG and KP4 cells, which are HGF autocrine cells. U-87MG and KP4 cells were treated with 30 mg/mL of onartuzumab for 6, 12, or 24 hours; cell lysates were prepared and the total MET protein levels were examined by Western blotting. F, MET mRNA levels were analyzed by qRT-PCR in U-87MG and KP4 cells that had been treated with onartuzumab. U-87MG and KP4 cells were treated with 30 mg/mL of onartuzumab for 24 hours, and MET mRNA levels determined by qRT-PCR and normalized to GAPDH mRNA levels (n ¼ 3 wells; error bars, SD). G, autocrine HGF activated both cell proliferation and MET-degradation signals.

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However, in our study, 23.2% of Japanese patients were found to By screening lung cancer cell lines with HGF qRT-PCR, we be MET-positive (Table 1), a discrepancy that may be associated found that HCC2108 expressed high levels of endogenous HGF with ethnic differences. Interestingly, 35.4% of patients with lung (Supplementary Fig. S6A), and the proliferation of HCC2108 was cancer with EGFR mutations were found to be MET-positive, as inhibited by HGF siRNA, suggesting that the proliferation compared with 15.5% of patients without EGFR mutations. This depends on HGF (Supplementary Fig. S6B). Next, we treated result implied that the activation of the EGFR pathway might be HCC2108 cells with onartuzumab or crizotinib to observe the involved in inducing MET transcription and/or stabilizing the changes in MET protein levels. As expected, treatment with onar- MET protein. tuzumab or crizotinib resulted in increased MET protein levels (Supplementary Fig. S6C) without significant changes in the mRNA levels (Supplementary Fig. S6D). We therefore concluded MET levels in PC-9/hHGF that the MET degradation pathway is activated, in addition to We were surprised to observe that MET protein levels in the activation of a proliferation signal, in most HGF-autocrine cells HGF-transfected cells were less than in the parent cells for both (Fig. 4G). PC-9 and HCC827 cell lines (Fig. 4A). Moreover, the decreased MET levels recovered upon treatment of PC-9/hHGF with onar- HGF tuzumab or MET-TKI, even though phospho-MET levels were mRNA levels in NSCLC specimens HGF reduced (Fig. 2E, Supplementary Fig. S5A). Taken together, this We then performed qRT-PCR using NSCLC clinical sam- EGFR indicated that HGF overexpression stimulates MET downregula- ples. Of NSCLC patients with activating mutations, 34.4% tion as well as cell proliferation. (21 of 61) expressed HGF levels higher than that seen in U-87MG We also examined the change in MET levels at the cell surface by cells, in which tumor growth was regressed by treatment with flow cytometric analysis. In PC-9 cells, onartuzumab treatment more than 7.5 mg/kg onartuzumab (28), suggesting that HGF fi did not shift the MET peak, but in the PC-9/hHGF cells, onartu- levels in U-87MG cells are suf cient for onartuzumab to be highly zumab treatment resulted in a shift of the MET peak (Fig. 4B). effective. These 34.4% patients with NSCLC with high levels of EGFR Similar results were obtained with MET flow cytometric analysis HGF and activating mutations may not respond well to when PC-9 parent cells were treated with recombinant HGF in the erlotinib monotherapy, but a combination of onartuzumab and fi presence of onartuzumab (Supplementary Fig. S5B). We con- erlotinib may be more ef cacious in these patients. firmed that onartuzumab administration resulted in increased MET protein levels as quantified by densitometric analysis of Discussion Western blots of proteins obtained from a PC-9/hHGF xenograft Several studies have confirmed the efficacy of erlotinib and model (Fig. 4C). On the basis of these results, we speculated that gefitinib in patients with NSCLC with activating EGFR muta- when onartuzumab reduces the phospho-MET level, MET is no tions (39–41); therefore, these drugs have been approved for longer recognized by Cbl, a ubiquitin ligase of MET, and escapes use as first-line treatment. However, not all patients with from ubiquitin-dependent degradation. activating EGFR mutations respond to erlotinib, indicating that To prove this hypothesis, we examined the effect of onartuzu- activating EGFR mutation is not the sole factor affecting erlo- mab on MET ubiquitination. We treated cells with MG-132, a tinib sensitivity. proteasome inhibitor, with or without onartuzumab. Cell lysates Yano and colleagues observed that about 29% (13 of 45) of were immunoprecipitated using an anti-MET antibody, and ubi- patients who did not respond to EGFR-TKI despite having EGFR quitinated MET was then detected with an anti-ubiquitin anti- mutations showed high HGF expression in their tumors (15). body on Western blots. As shown in Fig. 4D, MET was ubiqui- Several reports have also shown that high HGF expression is tinated in the presence of MG132 in PC-9/hHGF cells; however, its associated with acquired and intrinsic resistance to EGFR-TKI level was decreased by onartuzumab treatment, suggesting that (13, 15, 16). We here confirmed that activation of MET signaling inhibition of MET-ubiquitination by onartuzumab treatment by HGF overexpression causes EGFR-TKI resistance in PC-9/hHGF causes recovery of the total MET level. cells. Because both MET–HGF and EGFR pathways are simulta- neously stimulated in PC-9/hHGF cells, neither a MET inhibitor fi MET levels in other HGF-autocrine cell lines nor an EGFR inhibitor alone was suf cient to completely inhibit Next, we also investigated whether this event was observed in native cancer cell lines. We selected U-87MG and KP4 cells, both of which are well known for overexpressing HGF; onartuzumab Table 2. High positive ratio of HGF in Japanese lung cancer patients has also been shown to have strong growth-inhibition activity in HGF xenograft tumors derived from these cell lines (26, 27). Similar to mRNA Positive EGFR status High Low Total ratio (%) our findings in PC-9/hHGF cells, MET protein levels were Mutation (exon19 del þ L858R) 21 40 61 34.4 increased by onartuzumab treatment in U87-MG and KP4, Wild-type 10 42 52 19.2 although phospho-MET levels were decreased (Fig. 4E). The Other mutation 1 10 11 9.1 difference in MET protein levels did not derive from the tran- Total 32 92 124 25.8 scriptional level, as no significant changes in mRNA levels were NOTE: Statistical significance between mutation (exon 19 deletion þ L858R) and observed between the control and the onartuzumab-treated sam- wild-type, P ¼ 0.0913. Statistical significance between mutation (exon 19 ples (Fig. 4F). We also obtained similar results when we used a deletion þ L858R) and wild-type þ other mutations, P ¼ 0.0402. RNA was MET-TKI or HGF siRNA instead of onartuzumab to inhibit MET– extracted from frozen tumor samples, and cDNA was synthesized. HGF qRT-PCR assay was performed using SYBR green. Normalization was done using GAPDH HGF activation (Supplementary Fig. S5C and S5D). We also mRNA level. KP4, U87-MG, and PC-9/hHGF were used as positive control confirmed that HGF siRNA significantly inhibited proliferation samples. HGF-high was defined as higher HGF expression levels higher than of U-87MG and KP4 cells (Supplementary Fig. S5E). that in U-87MG.

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Sano et al.

cell growth. Thus, blocking both EGFR and MET–HGF pathways is number confounded the correlation between blood HGF levels required for complete inhibition of cell growth (Supplementary and survival rate in the phase II trial of onartuzumab and erlotinib Fig. S3E). Because mouse HGF does not activate human MET, we (38), it is likely that high HGF levels, not in the blood, but in the used the HGF-autocrine model in vivo. Similar results in a para- tumor region, can be a novel biomarker for onartuzumab use in crine model in PC-9 coinoculated with HGF-producing human future. fibroblasts have been reported (32). In conclusion, we showed that the combined use of onartu- We analyzed the HGF expression levels by qRT-PCR in frozen zumab and erlotinib has an additive effect in a model that NSCLC surgical specimens. Consistent with the report of Yano expresses high levels of human HGF in cells with an activating and colleagues (15), high expression of HGF was observed in EGFR mutation. This suggests that the use of a combination of 34% (21 of 61) of patients with exon 19 deletion and the L858R onartuzumab and erlotinib may achieve higher efficacy in the mutation in EGFR. Interestingly, the HGF-high ratio (HGF-high first-line treatment of patients with NSCLC with activating EGFR was defined as higher HGF expressionthanthatinU-87MG mutations and high levels of HGF expression. cells) in EGFR-mutated patients was higher than that seen in EGFR WT patients (19%; 10 of 52), although this difference was Disclosure of Potential Conflicts of Interest fi P ¼ not statistically signi cant ( 0.0913; Table 2). As well as HGF, Y. Yatabe has speakers' bureau honoraria from Chugai Pharmaceuticals the MET-diagnostic positive ratio is higher in EGFR-mutated Co., Ltd. and Ventana Roche and is a consultant/advisory board member for patients than in EGFR WT patients by IHC (P ¼ 0.00135; Table Ventana Roche. No potential conflicts of interest were disclosed by the other 1). Although the precise mechanisms have not been elucidated, authors. EGFR signaling, activated by a mutation, may enhance MET– HGF transcription or protein stability. We also found that Authors' Contributions erlotinib decreased not only phospho-EGFR, but also phos- Conception and design: Y. Sano, E. Hashimoto, N. Nakatani, M. Abe, Y. Yatabe pho-MET in PC-9 and PC-9/hHGF cells (Fig. 2E), suggesting Development of methodology: Y. Sano, N. Nakatani, Y. Satoh, Y. Yatabe that mutated EGFR trans-activates MET signaling. These results Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Sano, E. Hashimoto, N. Nakatani, Y. Yatabe imply a rationale for MET and EGFR combination therapy in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, NSCLC. computational analysis): Y. Sano, E. Hashimoto, N. Nakatani, K. Sakata, In the ongoing MetDriver study, which is a phase III clinical trial T. Fujii, M. Sugimoto, H. Motegi, E. Sasaki, Y. Yatabe using a combination of erlotinib and onartuzumab, or erlotinib Writing, review, and/or revision of the manuscript: Y. Sano, E. Hashimoto, alone, as first-line treatment in patients with NSCLC with acti- N. Nakatani, K. Fujimoto-Ouchi, M. Sugimoto, H. Motegi, Y. Yatabe vating EGFR mutations, MET expression status was tested by IHC, Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Sano, E. Hashimoto, Y. Yatabe and only MET-diagnostic positive patients (MET-IHC scores of 2þ þ Study supervision: Y. Sano, N. Nakatani, K. Fujimoto-Ouchi, M. Sugimoto, and 3 ) were included; however, HGF-high patients were not S. Nagahashi, M. Aoki, Y. Yatabe prospectively selected (ClinicalTrial.gov, NCT01887886). In our PC-9/hHGF model, the combined use of onartuzumab and Acknowledgments fi erlotinib showed high ef cacy, despite the low MET expression The authors thank Robin G. Taylor, Ellen Filvaroff, Mark Merchant, Dale Dei levels. In addition, onartuzumab showed high efficacy in U- Rossi, and See Phan from Genentech, Inc., for their support, advice on this study, 87MG and KP4 cells, in which elevated HGF levels resulted in and in preparing this article. The authors also thank Ikuko Matsuo, Asuka decreased MET protein levels. We also observed that MET protein Motoda, and Yumiko Hashimoto from Chugai Pharmaceutical Co., Ltd., for in vitro levels in HGF-autocrine cells, including PC-9/hHGF, U-87MG, assistance with experiments. and KP4 cells, were less than in A549 or HOP-92 cells, representative cell lines with MET-IHC scores of þ2 (Supplementary Fig. Grant Support S7). We demonstrated that autocrine HGF activates not only This study was funded by Chugai Pharmaceutical Co., Ltd., Kanagawa, Japan. proliferation but also MET downregulation via the ubiquitin- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked proteasome pathway, in which Cbl triggers ubiquitination and advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate degradation of MET by binding to phospho-MET (42, 43). These this fact. data suggested that measuring HGF expression levels in addition to MET-IHC may be important for selecting patients who are likely Received June 5, 2014; revised November 17, 2014; accepted December 2, to benefit from combination therapy. Although the small sample 2014; published OnlineFirst December 18, 2014.

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www.aacrjournals.org Mol Cancer Ther; 14(2) February 2015 541

Combining Onartuzumab with Erlotinib Inhibits Growth of Non− Small Cell Lung Cancer with Activating EGFR Mutations and HGF Overexpression

Yuji Sano, Eri Hashimoto, Noriaki Nakatani, et al.

Mol Cancer Ther 2015;14:533-541. Published OnlineFirst December 18, 2014.

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