RESEARCH ARTICLE CANCER Combining three antibodies nullifies feedback-mediated resistance to erlotinib in lung cancer Maicol Mancini,1 Nadège Gaborit,1 Moshit Lindzen,1 Tomer Meir Salame,2 Massimiliano Dall’Ora,1 Michal Sevilla-Sharon,1 Ali Abdul-Hai,1,3 Julian Downward,4,5 Yosef Yarden1* Despite initial responses to targeted kinase inhibitors, lung cancer patients presenting with primary epidermal growth factor receptor (EGFR) mutations acquire resistance, often due to a second-site mutation (T790M). However, clinical trials found no survival benefits in patients treated with a monoclonal antibody (mAb) to EGFR that should block activation of the mutated receptor and thus bypass resistance to molecules that target the catalytic or ATP-binding site. Using cell lines with the T790M mutation, we discovered that pro- longed exposure to mAbs against only the EGFR triggered network rewiring by (i) stimulating the extra- Downloaded from cellular signal–regulated kinase (ERK) pathway; (ii) inducing the transcription of HER2 (human epidermal growth factor receptor 2) and HER3, which encode other members of the EGFR family, and the gene encoding HGF, which is the ligand for the receptor tyrosine kinase MET; and (iii) stimulating the interaction between MET and HER3, which promoted MET activity. Supplementing the EGFR-specific mAb with those targeting HER2 and HER3 suppressed these compensatory feedback loops in cultured lung cancer cells. The triple mAb com- bination targeting all three receptors prevented the activation of ERK, accelerated the degradation of the re- ceptors, inhibited the proliferation of tumor cells but not of normal cells, and markedly reduced the growth of http://stke.sciencemag.org/ tumors in mice xenografted with cells that were resistant to combined treatment with erlotinib and the single function-blocking EGFR mAb. These findings uncovered feedback loops that enable resistance to treatment paradigms that use a single antibody and indicate a new strategy for the treatment of lung cancer patients. INTRODUCTION To overcome EGFR TKI resistance, several second-generation TKIs Lung cancer is the worldwide leading cause of cancer-related death (1). have been developed [reviewed in (17, 18)]. For example, afatinib (Gilotrif), Patients often present with advanced-stage disease, and the prognosis is an orally administered drug, covalently binds with the kinase domains of generally poor. The discovery in 2004 of mutant forms of the epidermal EGFR and HER2, resulting in irreversible inhibition of autophosphoryla- growth factor receptor (EGFR) in non–small cell lung cancer (NSCLC) tion as well as transphosphorylation of HER3 (19, 20). Another approach, on June 4, 2015 identified groups of patients who are sensitive to tyrosine kinase inhibitors which combines afatinib and an antibody to EGFR, has recently been re- (TKIs), including gefitinib and erlotinib (2–4). EGFR (also called ERBB1) ported (21, 22). Although afatinib is approved asafirst-linetreatmentof forms active heterodimers with other drug targets, namely, HER2 (human advanced NSCLC harboring activating EGFR mutations, third-generation epidermal growth factor receptor 2; also called ERBB2), HER3 (ERBB3), inhibitors, such as AZD-9291, CO-1686, and HM-61713, which inhibit both and HER4 (ERBB4) (5–7). Despite the initial efficacy of EGFR-specific EGFR-activating and resistance mutations while sparing wild-type EGFR, TKIs, all patients acquire resistance within about 1 year (8, 9). The most com- are in early-phase studies (23). Nevertheless, the therapeutic potential of mon (>50%) mechanism of acquired (secondary) resistance involves a spe- EGFR-specific antibodies, such as cetuximab, remains unclear. A clinical trial cific second-site mutation in EGFR, denoted T790M, which places a bulky that combined cetuximab and chemotherapy (cisplatin and vinorelbine) dem- amino acid, methionine, instead of a threonine in position 790 of EGFR onstrated relatively small but significant prolongations of patient survival (10–12). Amplification of the gene encoding another receptor tyrosine ki- (24). However, another study in unselected NSCLC patients failed to nase, MET, occurs in 5 to 10% of cases of acquired resistance (13, 14). It demonstrate a survival benefit of the combination of cetuximab and appears that MET amplification causes resistance because it stimulates chemotherapy (carboplatin and taxane) (25). Specific antibodies can re- HER3-dependent activation of the phosphatidylinositol 3-kinase (PI3K) duce the surface abundance of EGFR, and EGFR abundance is pathway. Consistent with this model, activation of PI3K and a downstream considered a predictor of patient survival (26–28). For example, analysis kinase AKT by an ectopically expressed active mutant of PI3K conferred TKI of EGFR abundance using immunohistochemistry (29) or fluorescence resistance in a cellular model, and down-regulation of PTEN (phosphatase in situ hybridization (30) associated high EGFR abundance with patient and tensin homolog), which inhibits PI3K signals, was found to be suffi- response to cetuximab. cient for emergence of TKI resistance in another model (15, 16). Because mutants of EGFR are associated with a small increase in EGFR gene copy number (31), these observations hint that monoclonal antibodies 1Department of Biological Regulation, Weizmann Institute of Science, Rehovot (mAbs) against EGFR might be effective on a subset of NSCLC patients. 2 76100, Israel. Department of Biological Services, Weizmann Institute of Science, We addressed this possibility by using two models of TKI-resistant NSCLC. Rehovot 76100, Israel. 3Kaplan Medical Center, Rehovot 76100, Israel. 4Signal Transduction Laboratory, Francis Crick Institute, London WC2A 3LY, UK. 5Lung We found feedback regulatory loops that increase the abundance of HER2 Cancer Group, The Institute of Cancer Research, London SW3 6JB, UK. and HER3 and activate MET in response to antibody-induced blockade of *Corresponding author. E-mail: [email protected] EGFR. Preventing this complex compensatory response effectively inhibited www.SCIENCESIGNALING.org 2 June 2015 Vol 8 Issue 379 ra53 1 RESEARCH ARTICLE Fig. 1. Antibodies specific to EGFR increase the abundance A B of HER2 and HER3 in TKI-resistant NSCLC cells. (A) Survival 120 PC9ER H1975 assay of PC9, PC9ER, and H1975 human NSCLC cells treated 100 0 1 2 8 12 24 48 0 1 2 8 12 24 48 (h) for 72 hours with increasing concentration of erlotinib (top) or 80 pEGFR cetuximab (bottom). Data are means ± SD of three independent 60 EGFR experiments. (B) Immunoblotting of erlotinib-resistant PC9ER 40 pHER2 and H1975 cells exposed to mAbs specific to EGFR (mAb 565 20 HER2 m Cell survival (%) or cetuximab; 10 g/ml) for the indicated times. Tubulin served 0 mAb 565 pHER3 1 as a loading control. Blots are representative of three experi- 0.1 10 0.01 HER3 ments. (C) HER2 and HER3 mRNA levels of H1975 and PC9ER 0.001 Erlotinib (µ ) Tubulin cells treated for the indicated time intervals with antibodies spe- 120 m cifictoEGFR(10 g/ml). Data are means ± SD from three experi- 100 pEGFR ments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-way analysis of 80 EGFR variance (ANOVA) with Tukey’s comparison. (D) Relative lucif- 60 pHER2 erase activity in PC9ER cells stably cotransfected for 48 hours PC9 40 PC9ER with luciferase reporter plasmids for the HER2 (left) or HER3 HER2 20 H1975 Cetuximab Cell survival (%) pHER3 (right) promoter and treated with mAb 565 (top) or cetuximab 0 (bottom). Data are means ± SEM from two experiments, each 1 HER3 0.1 10 performed in technical triplicate. A.U., arbitrary unit. 100 Downloaded from Cetuximab (µg/ml) Tubulin C 4 5 *** H1975 PC9ER *** *** NSCLC tumor growth in an animal model, thus offering a novel *** 3 *** HER2 4 * ** * strategy to treat relatively aggressive lung tumors. * 3 2 HER3 (fold change) 2 http://stke.sciencemag.org/ RESULTS 1 1 mRNA mRNA 0 0 EGFR-specific antibodies increase the 0 1 12 24 48 0 1 12 24 48 abundance of HER2 and HER3 in Time (h) Time (h) TKI-resistant NSCLC cells D EGFR mAbs avidly bind both the wild-type and mutant forms of 80 250 EGFR; hence, antibody treatment is expected to overcome HER2 promoter HER3 promoter resistance to TKIs. To experimentally examine this possibility, 60 200 we selected two NSCLC cell lines: patient-derived H1975 cells 150 40 that express a double-mutant EGFR (L858R and T790M) and 100 on June 4, 2015 the PC9ER cell line, which is a derivative of PC9 cells that have 20 50 565 mAb – Luciferase activity (A.U.) a deletion in EGFR (del746 750 EGFR, also called LREA de- 0 0 letion) and have acquired in vitro the T790M secondary muta- 0281224 0281224 tion (32). To evaluate the effect of EGFR-specific treatments, 80 250 we monitored the survival of cells treated for 4 days with in- creasing doses of either erlotinib or cetuximab (Fig. 1A). As 60 200 150 expected, the survival of parental PC9 cells was strongly inhib- 40 ited by erlotinib and weakly inhibited by cetuximab. However, 100 20 in line with a previous study (33), PC9ER and H1975 cells ex- 50 Cetuximab Luciferase activity (A.U.) pressing EGFR T790M showed resistance to both treatments. 0 0 Concentrating on the resistant cell lines, we noted that long- 0281224 0281224 term exposure to cetuximab or to another EGFR-specific anti- Time (h) Time (h) body, mAb 565, which was generated in our laboratory (34, 35), caused a gradual disappearance in the abundance of EGFR (Fig. 1B). This was accompanied by increased abundance of HER2 and displayed moderate, time-dependent increases in the abundance of the re- HER3, as well as increased tyrosine phosphorylation of EGFR, HER2, and spective transcripts (Fig. 1C). Second, promoter-reporter plasmids verified HER3 (Fig. 1B), suggesting the emergence of positive feedback that com- that cetuximab treatment strongly or weakly increased promoter activity of pensates for EGFR down-regulation. Similar to mAb treatment, depletion HER2 or HER3, respectively (Fig. 1D and fig. S1B). of EGFR abundance with small interfering RNA (siRNA) oligonucleo- tides was followed by increased abundance of HER2 and HER3 proteins A combination of three antibodies against (fig.