Oncogene (2014) 33, 5405–5414 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE Preclinical validation of AXL receptor as a target for antibody-based pancreatic cancer immunotherapy W Leconet1, C Larbouret1, T Charde`s1, G Thomas1, M Neiveyans1, M Busson1, M Jarlier2, N Radosevic-Robin3, M Pugnie`re1, F Bernex1, F Penault-Llorca3, J-M Pasquet4,APe`legrin1 and B Robert1 AXL receptor tyrosine kinase (RTK) is implicated in proliferation and invasion of many cancers, particularly in pancreatic ductal adenocarcinoma (PDAC), for which new therapeutic options are urgently required. We investigated whether inhibition of AXL activity by specific monoclonal antibodies (mAbs) is efficient in limiting proliferation and migration of pancreatic cancer cells. Expression of AXL was evaluated by immunohistochemistry in 42 PDAC. The AXL role in oncogenesis was studied using the short hairpin RNA approach in a pancreatic carcinoma cell line. We further generated antihuman AXL mAbs and evaluated their inhibitory effects and the AXL downstream signaling pathways first in vitro, in a panel of pancreatic cancer cell lines and then in vivo, using subcutaneous or orthotopic pancreatic tumor xenografts. AXL receptor was found expressed in 76% (32/42) of PDAC and was predominantly present in invasive cells. The AXL-knockdown Panc-1 cells decreased in vitro cell migration, survival and proliferation, and reduced in vivo tumor growth. Two selected anti-AXL mAbs (D9 and E8), which inhibited phosphorylation of AXL and of its downstream target AKT without affecting growth arrest-specific factor 6 (GAS6) binding, induced downexpression of AXL by internalization, leading to an inhibition of proliferation and migration in the four pancreatic cancer cell lines studied. In vivo, treatment by anti-AXL mAbs significantly reduced growth of both subcutaneous and orthotopic pancreatic tumor xenografts independently of their KRAS mutation status. Our in vitro and preclinical in vivo data demonstrate that anti-human AXL mAbs could represent a new approach to the pancreatic cancer immunotherapy. Oncogene (2014) 33, 5405–5414; doi:10.1038/onc.2013.487; published online 18 November 2013 Keywords: RTK; monoclonal antibodies; pancreatic cancer; targeted therapy INTRODUCTION migration, invasion, pro-inflammatory cytokine production, anti- Pancreatic cancer is the fourth leading cause of cancer death in apoptosis, proliferation and survival of cancer cells.8 both men and women, with an overall 5-year survival rate of about AXL was first identified as a transforming gene in chronic 5%.1 At diagnosis, 80% of patients already have metastases, myeloid leukemia9 and is expressed or upregulated in several resulting in a median overall survival of 5–6 months. Conventional cancers.10 AXL overexpression in breast and pancreatic cancers is treatments, which associate surgery and radiotherapy, often in significantly associated with higher metastasis frequency and combination with chemotherapy, show a marginal clinical benefit therefore with poor overall survival.11–13 with no real progress in patients’ survival. Targeted therapies, such Recently, AXL has been demonstrated to be required for the as antibodies or small molecule inhibitors of receptor tyrosine epithelial-mesenchymal transition (EMT) of malignant cells, kinases (RTKs) or their downstream signaling molecules have been induced by various stimuli, like H-RASV12 and overexpression of clinically successful in various cancers;2,3 however, the results in SLUG.14 The cancers which have undergone EMT display increased pancreatic cancer are still limited due to the complexity of the invasiveness, metastatic capacity and multidrug resistance. disease. Currently, gemcitabine remains the standard pancreatic Interestingly, AXL overexpression is associated with resistance cancer treatment but with modest therapeutic gains.4 either to standard chemotherapy or to tyrosine kinase inhibitors Therefore, it is now crucial to develop new therapeutic agents (TKI) in acute and chronic myeloid leukemia,15,16 gastrointestinal based on the recent findings on molecular mechanisms involved stromal tumors,17 breast,18 ovarian19 and lung cancers.20 in pancreatic cancerogenesis.5,6 In this study, we first investigated the expression of AXL in a AXL belongs, with TYRO3 and MER, to the TAM family of RTKs.7 series of pancreatic ductal adenocarcinoma (PDAC) and found it in Unlike other RTKs that are stimulated by growth factors, AXL is the great majority of cases, similarly to the previous reports. As activated by growth arrest-specific factor 6 (GAS6). GAS6 binding that indicated AXL could be a target for pancreatic cancer to AXL leads to AXL dimerization, autophosphorylation and immunotherapy,12 we generated monoclonal antibodies (mAbs) subsequent activation of signaling pathways, such as the PI3K/ against the extracellular domain of AXL and tested their efficacy AKT, mitogen-activated protein kinase (MAPK), STAT and NF-kB in vitro in several pancreatic cancer cell lines and in vivo, using cascades.7 GAS6-induced AXL signaling promotes adhesion, xenografted nude mice. Two selected anti-AXL mAbs induced AXL 1INSERM-U896, IRCM, Universite´ Montpellier1, CRLC Val d’Aurelle-Paul Lamarque, 208 rue des Apothicaires, Montpellier-Cedex 5, France; 2Unite´ de Biostatistiques, CRLC Val d’Aurelle-Paul Lamarque, 208 rue des Apothicaires, Montpellier-Cedex 5, France; 3De´partement de biopathologie Centre Jean-Perrin 63011 Clermont-Ferrand Cedex 1; ERTICa EA4677, Universite´ d’Auvergne, Clermont-Ferrand, France and 4INSERM-U876, He´matopoı¨e`se Leuce´mique et Cible The´rapeutique, Universite´ Victor Se´galen, Laboratoire d’he´matologie CHU de Bordeaux, Bordeaux Cedex, France. Correspondence: Dr B Robert, IRCM-INSERM-U896, CRLC Val d’Aurelle-Paul Lamarque, 208 rue des Apothicaires, Montpellier-Cedex 5, Languedoc-Roussillon 34298, France. E-mail: [email protected] Received 12 April 2013; revised 22 August 2013; accepted 20 September 2013; published online 18 November 2013 Anti-AXL mAb for pancreatic cancer immunotherapy W Leconet et al 5406 downregulation and inhibited its phosphorylation induced by features (Figure 1c). AXL-positive single invasive carcinoma cells GAS6, as well as the subsequent AKT activation and cell were observed in 2 cases (Figure 1d). In another 2 cases we proliferation. In vivo, these anti-AXL mAbs reduced the growth noted the presence of emboli containing AXL-expressing cancer of subcutaneous and orthotopic pancreatic tumor xenografts, cells (Figure 1e). In contrast to cancer cells, no or weak AXL independently of their KRAS mutation status. Finally, when staining is observed on normal pancreatic cells (Supplementary compared to the standard gemcitabine treatment, the Figures S1C and E). antibodies induced more complete responses and longer survival of the mice. To our best knowledge, this is the first study that evaluates antihuman AXL antibodies in both cell and AXL silencing reduces cell viability and migration in pancreatic animal models of pancreatic cancer. The results indicate that our cancer cell anti-AXL mAbs are effective candidates for the targeted therapy of To ascertain the role of AXL in pancreatic cancer growth, we this deadly disease. generated two stable Panc-1 cell lines in which AXL was silenced by lentiviral transfection of two different anti-AXL shRNA (Panc-1- sh-AXL1 and -AXL2 cells) and a stable Panc-1 cell line transfected RESULTS with empty vector (Panc-1-sh-CONTROL; Supplementary. Fig. S2A). AXL expression is predominantly observed in invasive pancreatic Cell viability was reduced by 26–35% after 5 days of culture cancer cells (Figure 2a) and colony formation was decreased by 30% AXL was detected by IHC in 32 out of 42 PDAC (76%). In 13 out of (Figure 2b) in Panc-1-sh-AXL1 and -AXL2 cells in comparison to 32 positive cases (41%) the AXL protein expression was control cells. AXL silencing also inhibited cell migration, as moderate to strong and in 19 remaining cases, weak. Almost indicated by the reduced wound healing in Panc-1-sh-AXL1 and all high-expressors (12 out of 13) and 9 out of 19 low-expressors, -AXL2 cells in comparison to control cells after 24 h (Figure 2c). or 66% of all AXL-positive cases demonstrated presence of Finally, the Panc-1-sh-AXL1 xenografted tumors grew smaller than invasive cells (Figure 1a) in which AXL expression was stronger those obtained with Panc-1 sh-CONTROL (Figure 2d). These results than in the remaining tumor cells. These cells were localized at demonstrate that AXL is involved in pancreatic cancer cell the periphery of the tumor and often displayed anaplastic proliferation and migration. AXL EXPRESSION NEGATIVE POSITIVE 24% (10/42) 76% (32/42) WEAK MEDIUM/STRONG 59% (19/32) 41% (13/32) INVASIVE CELLS 47% (9/19) 92% (12/13) Figure 1. AXL expression in pancreatic cancer cells. AXL protein was detected by IHC in 32 out of 42 PDAC (b–AXL-negative case; c, d and e–AXL-positive cases). Its strongest expression was observed in the invasive cells, localized at the periphery of the tumors and frequently of anaplastic morphology (c). In 2 cases AXL-expressing single invasive cells were observed (d; arrows). Two more cases had AXL-positive cells in the emboli (e; arrow). Oncogene (2014) 5405 – 5414 & 2014 Macmillan Publishers Limited Anti-AXL mAb for pancreatic cancer immunotherapy W Leconet et al 5407 Figure 2. AXL knockdown in the Panc-1 pancreatic cancer cell line affects cell viability, colony formation, migration and impairs tumor growth. Analysis