Published OnlineFirst September 5, 2019; DOI: 10.1158/2326-6066.CIR-18-0903 Research Article Cancer Immunology Research AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity Stephane Terry1, Abderemane Abdou1, Agnete S.T. Engelsen1,2,Stephanie Buart1, Philippe Dessen3,Stephanie Corgnac1, Davi Collares4, Guillaume Meurice3, Gro Gausdal5, Veronique Baud4, Pierre Saintigny6,7, James B. Lorens2, Jean-Paul Thiery1,2,8,9, Fathia Mami-Chouaib1, and Salem Chouaib1,10 Abstract Immune resistance may arise from both genetic instability killer (NK)– and cytotoxic T lymphocyte (CTL)–mediated and tumor heterogeneity. Microenvironmental stresses such as killing. A small-molecule targeting AXL sensitized mesenchy- hypoxia and various resistance mechanisms promote carcino- mal lung cancer cells to cytotoxic lymphocyte–mediated ma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and killing. Mechanistically, we showed that attenuation of AXL- Mer) receptor tyrosine kinase family, is widely expressed in dependent immune resistance involved a molecular network human cancers and increasingly recognized for its role in cell comprising NF-kB activation, increased ICAM1 expression, and plasticity and drug resistance. To investigate mechanisms of upregulation of ULBP1 expression coupled with MAPK inhi- immune resistance, we studied multiple human lung cancer bition. Higher ICAM1 and ULBP1 tumor expression correlated clones derived from a model of hypoxia-induced tumor plas- with improved patient survival in two non–small cell lung ticity that exhibited mesenchymal or epithelial features. We cancer (NSCLC) cohorts. These results reveal an AXL-mediated demonstrate that AXL expression is increased in mesenchymal immune-escape regulatory pathway, suggest AXL as a candidate lung cancer clones. Expression of AXL in the cells correlated biomarker for tumor resistance to NK and CTL immunity, and with increased cancer cell–intrinsic resistance to both natural support AXL targeting to optimize immune response in NSCLC. Introduction mous cell carcinoma are the most prevalent histotypes. Like most malignancies, lung carcinoma is heterogeneous and composed of Lung cancer is the third most frequent cancer and the first cause subpopulations of cancer cells, or clones, with distinct molecular of cancer-related death worldwide (1, 2). Approximately 85% of and phenotypic features. High intratumoral heterogeneity can patients present with a disease defined as non–small cell lung pose challenges to the effectiveness of therapies for NSCLC and cancer (NSCLC), of which lung adenocarcinoma and lung squa- contributes to the emergence of therapy resistance (3). The underlying biology of tumor heterogeneity and the environmen- tal factors that shape tumor heterogeneity remain unclear. The 1INSERM UMR1186, Integrative Tumor Immunology and Genetic Oncology, immune system plays a role during tumor development, with the Gustave Roussy, Equipe Labellisee par la Ligue Contre le Cancer, EPHE, Faculte interplay between cancer cells and their tumor microenviron- de Medecine, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France. ment. The latter contributes to the development of refractoriness 2Department of Biomedicine, Centre for Cancer Biomarkers, Norwegian Centre of Excellence, University of Bergen, Bergen, Norway. 3Plateforme de Bioinfor- through mechanisms preventing cytotoxic immune effector T matique, UMS AMMICA, Gustave Roussy, Villejuif, France. 4NF-kB, Differentia- cells and natural killer (NK) cells from reaching and destroying tion and Cancer, Universite Paris Descartes, Sorbonne Paris Cite, Paris, France. their targets (4). Under microenvironmental stresses, carcinoma 5BerGenBio ASA, Bergen, Norway. 6Univ Lyon, Universite Claude Bernard Lyon 1, cells can undergo molecular and phenotypic changes referred to as INSERM, CNRS, Centre Leon Berard, Centre de Recherche en Cancerologie de carcinoma cell plasticity, which contributes to heterogeneity in 7 Lyon, Lyon, France. Department of Medical Oncology, Centre Leon Berard, tumors. Previously, we provided evidence that morphologic 8 Lyon, France. Department of Biochemistry, National University of Singapore, changes of carcinoma cells could affect their recognition and Singapore, Singapore. 9Institute of Molecular and Cell Biology, A-STAR, Singapore, Singapore. 10Thumbay Research Institute for Precision Medicine, Gulf Medical killing by CTLs (5). We and others demonstrated that carcinoma University, Ajman, United Arab Emirates. cell plasticity results from the activation of epithelial– mesenchymal transition (EMT) programs (6, 7). EMT also confers Note: Supplementary data for this article are available at Cancer Immunology – Research Online (http://cancerimmunolres.aacrjournals.org/). an immune-resistant phenotype (8 13). Our understanding of the mechanistic links between carcinoma cell plasticity, hetero- Corresponding Author: Salem Chouaib, Institut Gustave Roussy, U1186 INSERM, geneity, and the emergence of tumor immune escape remains 114 rue Edouard, Vaillant 94805, Villejuif cedex, France. Phone: 33142114547; Fax: 33142115288; E-mail: [email protected] poor due to the limited number of preclinical models that recapitulate carcinoma cell phenotypic diversity in the tumor Cancer Immunol Res 2019;7:1789–802 mass. doi: 10.1158/2326-6066.CIR-18-0903 AXL, a member of the TAM receptor tyrosine kinase family, is Ó2019 American Association for Cancer Research. overexpressed in various human cancers. Evidence suggests AXL is www.aacrjournals.org 1789 Downloaded from cancerimmunolres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst September 5, 2019; DOI: 10.1158/2326-6066.CIR-18-0903 Terry et al. associated with carcinoma cell plasticity as well as chemo-, radio-, apheresis products after Ficoll-Paque Plus centrifugation (GE and drug resistance associated with the mesenchymal Healthcare). We used the NK cell isolation kit with MACS system phenotype (14–17). AXL may be a promising therapeutic target (Miltenyi Biotec). NKhd were subsequently cultured in RPMI- for overcoming such resistance (16, 18, 19). However, the bio- 1640 media with 10% pooled human serum (Jacques Boy), 5% logical role of AXL as a driver of cancer cell–intrinsic immune horse serum (Thermo Fisher Scientific), with or without the resistance has not been investigated. presence of IL2 (150 UI/mL), and IL15 (15 ng/mL). TPCA-1 was One hypothesis postulates that microenvironmental stresses purchased from Sigma-Aldrich was prepared in DMSO. Bemcen- such as hypoxia, a hallmark of most solid tumors, promote tinib (formerly known as BGB324, or R428, BerGenBio ASA) was carcinoma cell plasticity, tumor progression, and immune sup- dissolved in DMSO (10 mmol/L stock solution) and used at the pression (20). We used the primary NSCLC IGR-Heu carcinoma final concentration of 1 mmol/L. cell line subjected to hypoxic stress as a model to generate phenotypic diversity (21). This model recapitulates some of the RNA preparation, cDNA synthesis, and quantitative real-time key features of tumor heterogeneity resulting in the emergence of PCR carcinoma cell clones residing in the epithelial (EPI) or the Total RNA extraction was performed using TRIzol reagent. mesenchymal (MES) states of the EMT spectrum. Reverse transcription was performed using Maxima Reverse Tran- Here, we use these clones to explore the mechanisms by which scriptase followed by qPCR using real-time PCR Master SYBR carcinoma cells are positioned in the mesenchymal domain of the Green on a StepOnePlus Real-Time PCR system. All products were EMT spectrum and expressing AXL could affect lysis by CTLs and from Thermo Fisher Scientific. Most oligonucleotide sequences NK cells. EPI and MES carcinoma cells were shown to differentially used were designed using Beacon Designer Free Edition and express various immune-related genes, which may affect their Primer3Plus. The full list is available as Supplementary susceptibility to lysis by cytotoxic effector cells. The present study Table S1. Heat maps were generated using Excel software. also showed that MES carcinoma clones with increased expression of AXL were more resistant to cytotoxic immune cell attacks. Protein extracts, Western blot analysis, and electrophoretic Inhibition of AXL in MES carcinoma cells resulted in the sensiti- mobility shift assays zation of these cells to NK- and CTL-mediated killing through a Adherent cells were lysed on ice with lysis buffer (62.5 mmol/L molecular network involving MAPK and NF-kB activities as well as Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol) containing a cocktail increased expression of ICAM1 and the NKG2D ligand ULBP1. of protease (Thermo Fisher Scientific) and phosphatase inhibitors (Roche Life Science). Western blotting was performed as previ- ously described (21). Antibodies for Western blots were as fol- Materials and Methods lows: antibodies to AXL (C89E7), phospho-p44/42 MAPK (Erk1/ Cell culture and reagents 2; Thr202/Tyr204) D13.14.4E, p44/42 MAPK (Erk1/2; L34F12), Carcinoma clones with EPI or MES phenotypes were isolated as phospho-Akt (Ser473; D9E), Akt (pan; 40D4) were from Cell described (21) from primary NSCLC IGR-Heu cells deriving from Signaling Technology. Phospho-AXL-y779-antibody was from a resection of a nonmetastatic NSCLC patient (22). Briefly, after R&D Systems. Anti-vimentin (Clone V9) was from Dako), anti- cultivating NSCLC IGR-Heu cells for one month and a half under ICAM1 (sc-107)