Oncogene (2013) 32, 2873–2881 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Oncogenic RAS simultaneously protects against anti-EGFR antibody-dependent cellular cytotoxicity and EGFR signaling blockade S Kasper1, F Breitenbuecher1, H Reis2, S Brandau3, K Worm2,JKo¨ hler1, A Paul4, T Trarbach1, KW Schmid2 and M Schuler1 Monoclonal antibodies against the epidermal growth factor receptor (EGFR) are effective cancer therapeutics, but tumors harboring RAS mutations are resistant. To functionally dissect RAS-mediated resistance, we have studied clinically approved anti-EGFR antibodies, cetuximab and panitumumab, in cancer models. Both antibodies were equally cytotoxic in vitro. However, cetuximab, which also triggers antibody-dependent cellular cytotoxicity (ADCC), was more effective than panitumumab in vivo. Oncogenic RAS neutralized the activity of both antibodies in vivo. Mechanistically, RAS upregulated BCL-XL in cancer cell lines and in primary colorectal cancers. Suppression of BCL-XL by short hairpin RNA or treatment with a BH3 mimetic overcame RAS-mediated antibody resistance. In conclusion, RAS-mutant tumors escape anti-EGFR antibody-mediated receptor blockade as well as ADCC in vivo. Pharmacological targeting of RAS effectors can restore sensitivity to antibody therapy. Oncogene (2013) 32, 2873–2881; doi:10.1038/onc.2012.302; published online 16 July 2012 Keywords: cetuximab; colorectal cancer; RAS; ADCC; anti-EGFR antibodies; BCL-XL INTRODUCTION products are involved in activating the mitogen-activated protein Overexpression of the epidermal growth factor receptor (EGFR) is kinase pathway and additional pathways downstream of the EGFR. observed in several human cancers. It associates with adverse Hence, the current model of mutant RAS-mediated resistance to prognosis in patients with colorectal cancer (CRC) and head and antibody therapy focuses on the ability of oncogenic RAS to neck cancer (HNSCC), thus defining the EGFR as an attractive compensate for upstream signals lacking owing to EGFR blockade therapeutic target.1 Combining anti-EGFR antibodies cetuximab by the antibody. In analogy, receptor blockade is regarded as the or panitumumab with cytotoxic chemotherapy in patients with main effector mechanism of the two clinically approved anti-EGFR metastatic CRC increased remission rates, prolonged progression- antibodies, cetuximab and panitumumab. Cetuximab is a chimeric free survival and, in some clinical trials, increased overall survival. IgG1 antibody capable to mediate antibody-dependent cellular Importantly, more patients with isolated liver metastases were cytotoxicity (ADCC) in vitro and in vivo.12,13 In contrast, converted to a curatively resectable stage when chemotherapy panitumumab is unable to induce classical, natural killer (NK) 14 was complemented by an anti-EGFR antibody. In addition, cell-mediated ADCC owing to its IgG2 isotype. So far, no head-to- monotherapy with anti-EGFR antibodies has clinical activity in head comparison of these two antibodies in a clinical trial or in a metastasized CRC patients relapsing after prior chemo- preclinical in vivo model has been reported. Accordingly, it therapies.2–5 Adding the anti-EGFR antibody cetuximab to remains speculative whether the additional ADCC activity of standard chemotherapy increased response rates, progression- cetuximab results in enhanced therapeutic activity over receptor free survival and overall survival in patients with recurrent or blockade alone. Interestingly, Fc receptor-g polymorphisms, which metastatic HNSCC.6 Also, locoregional control of advanced HNSCC may determine the efficacy of ADCC, have been correlated with was improved when cetuximab was applied simultaneously with outcome following cetuximab-based treatment in a retrospective radiotherapy.7 Recently, several retrospective analyses of studies analysis of CRC patients. This correlation was maintained in RAS- conducted in CRC patients have suggested that the clinical benefit mutant and RAS-wild-type tumors.15 In vitro, RAS-mutant cancer from both clinically approved anti-EGFR antibodies, cetuximab cell lines were protected against direct antiproliferative actitivies and panitumumab, is restricted to patients suffering from tumors of cetuximab and panitumumab, but not against ADCC enabled devoid of somatic mutations of KRAS and additional RAS family by cetuximab.16 These lines of evidence argue that CRC oncogenes.4,8–11 Accordingly, more than 40% of all metastatic CRC susceptibility to cetuximab-mediated ADCC is not determined by patients are excluded from this therapeutic modality. the mutation status of RAS proto-oncogenes. However, no Anti-EGFR antibodies block the receptor to interfere with ligand apparent clinical benefit of cetuximab was derived in patients binding and possibly receptor dimerization, thus preventing signal suffering from RAS-mutant CRC despite the theoretical possibility transduction via the EGFR tyrosine kinase. The RAS family gene of preserved ADCC. 1Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany; 2Department of Pathology and Neuropathology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany; 3Department of Otorhinolaryngology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany and 4Department of General, Visceral and Transplant Surgery, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany. Correspondence: Professor Dr M Schuler, Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany. E-mail: [email protected] Received 12 July 2011; revised 1 May 2012; accepted 31 May 2012; published online 16 July 2012 RAS-mediated antibody resistance S Kasper et al 2874 A full molecular understanding of relevant effector mechanisms non-obese diabetic severe combined immunodeficiency (NOD/ of, as well as resistance mechanisms to, anti-EGFR antibody SCID) mice. Injection of either cell line reproducibly led to therapy will allow the development of strategies (i) for potentially outgrowth of flank tumors, which could be followed in vivo. Two synergistic combination treatments, and (ii) to overcome antibody models were established: (A) in a ‘therapeutic setting’, NOD/SCID resistance in RAS-mutant cancers. To this end, we have established mice bearing palpable flank tumors were treated twice weekly by preclinical models for the study of anti-EGFR therapy in vitro and intraperitoneal antibody injections (1 and 0.1 mg), and tumor in vivo. We find that ADCC indeed provides a therapeutic benefit growth was monitored. (B) In an ‘adjuvant setting’, intraperitoneal in addition to EGFR blockade in vivo. However, oncogenic RAS antibody injections (1 mg twice weekly) were initiated 1 day after completely abolishes both, receptor blockade and ADCC, by the subcutaneous implantation of A431 cells in NOD/SCID mice, cetuximab in vivo. Dissecting RAS-mediated signaling alterations in and tumor development and survival were monitored. Applying cancer models and primary CRC, we identify anti-apoptotic BCL-XL cetuximab, panitumumab and rituximab in therapeutic model A, as a clinically and functionally relevant target for therapeutic we observed a dose-dependent activity of both anti-EGFR modulation of anti-EGFR antibody resistance. antibodies (Figure 2 and data not shown). In the ‘adjuvant’ model B, cetuximab prevented tumor development more effectively than panitumumab, resulting in superior survival of cetuximab-treated RESULTS mice (Figures 3a–c). Diverging efficacies of cetuximab and panitumumab in cancer As both anti-EGFR antibodies were equally effective in vitro models in vitro and in vivo (Figure 1), where cytotoxic effects are solely mediated by EGFR Using a panel of EGFR-expressing cell lines, we confirmed that the blockade, we reasoned whether the superiority of cetuximab cytotoxic activity of the anti-EGFR antibodies, cetuximab and in vivo resulted from its ability to trigger ADCC. Canonical ADCC is panitumumab, in vitro strictly correlated with the extent of EGFR executed by Fc receptor-bearing NK cells, which kill antibody- expression and KRAS mutation status (Supplementary Table 1). For marked target cells by granule-mediated cytotoxicity.17 While further studies, we selected two cell lines, which exhibited a NOD/SCID mice are lacking B and T cells, NK cell activity is at least profound reduction in clonogenic survival upon antibody treat- partially preserved and thus can contribute to cetuximab activity ment in vitro (Figures 1a and b). In short-term assays, anti-EGFR in vivo.18 To formally prove this hypothesis, NOD/SCID mice were antibodies effectively induced apoptosis in Difi cells, whereas radiodepleted (1.5 Gy) of cellular ADCC effectors prior to anti-EGFR A431 cells primarily responded by reduced proliferation (Figures antibody treatment. Interestingly, the in vivo activity of cetuximab 1c and d). Both anti-EGFR antibodies, cetuximab and panitumu- was markedly reduced in irradiated NOD/SCID mice as compared mab, were equally effective in vitro showing significant cytotoxi- with untreated mice. In contrast, panitumumab was equally city as compared with the chimeric monoclonal anti-CD20 effective in irradiated and non-irradiated NOD/SCID mice antibody rituximab (IgG1), which served as negative control (not