And CTL-Mediated Cytotoxicity Stephane� Terry1, Abderemane Abdou1, Agnete S.T

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 2019 American Association for Cancer Research. overexpressed in various human cancers. Evidence suggests AXL is

www.aacrjournals.org 1789

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 previously 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) was from Santa Cruz Biotechnology, and anti- hypoxic conditions (1% pO2 with 5% CO2) in a hypoxia chamber b-Actin (AC-74) was from Sigma-Aldrich. (InVivo2 400 Hypoxia Workstation; Ruskinn Technology), cancer For electrophoretic mobility shift assay (EMSA) analysis of clones were obtained by limiting dilution and clonal expansion. NF-kB DNA binding, nuclear extracts were prepared and analyzed For this study, low-passage cells were used (<13 passages) and for DNA binding activity using the HIV-LTR tandem kB oligonu- cultured in DMEM/F12 medium containing 10% heat-inactivated cleotide as kB probe (26). FCS, 1% penicillin–streptomycin, (Thermo Fisher Scientific), 1% Ultroser G (Pall Corporation) at 37C in a humidified atmo- Antibodies and flow cytometry sphere with 5% CO2. Cells were authenticated through cell Phenotypic analyses of carcinoma cells, NK, and CTL cells were morphology, and expression of TP53 and ACTN4 mutants in performed by direct immunostaining. Briefly, 0.2 106 cells were parental cells and derivatives. Mycoplasma testing was carried out collected and resuspended in FACS buffer (PBS with 2% FBS) and using the Venor GeM OneStep kit (Minerva Biolabs). The H33 stained for 30 minutes in the dark performed at 4C with the CTL clone originated from the same patient tumor specimen as following Abs for extracellular staining: APC-anti-ICAM1, PE- NSCLC IGR-Heu cells and EPI or MES derivative clones. The CTL anti-KLRG1, PE-anti-DNAM1 (CD226), APC-anti-CD96, PE- clone was cultured and stimulated in complete RPMI media anti-CD16, PE-anti-NKp30, PE-anti-NKp44, PE-anti-NKp46, supplemented with 10% human AB serum (Jacques Boy Institute) PE-anti-NKG2C, PE-anti-MICA/MICB, APC-anti-CD137 (4-1BB), and rIL2 in the presence of irradiated autologous NSCLC IGR-Heu PE-anti-human HLA-A,B,C, APC-HLA-A2, APC-anti-TCR a/b, (3 103/well) and lymphoblastoid (4 104/well) cell lines. Cells APC-anti-human CD262 (DR5, TRAIL-R2), PE-anti-human were fed every 3 days with complete media (23). The NSCLC cell CD261 (DR4, TRAIL-R1) and mouse isotopic controls were from lines ADC-Coco, IGR-Pub, ADC-Tor, ADC-Let as well as primary BioLegend; APC-anti-CD244 (2B4) APC-anti–LFA-1 (cd11a), PE- NSCLC cells were derived from tumor biopsies as described anti–NKG2D, PE-anti-NKG2A, PE-anti-KIR2DL1, PE-anti- previously and used at low passage (24, 25). Human NK cell KIR3DL1, PE-anti-KIR3DL2, and PE-anti-KIR2DL1 from Miltenyi clones (NK92 and NKL) were maintained in RPMI-1640 medium Biotec; PE-anti-TIGIT (FAB7898P), APC-anti-ULBP1 (MAB1380) supplemented with 10% FBS, and 200 or 300 U/mL IL2, respec- from R&D Systems; APC-Anti-CD94, PE-anti-CD3, and APC-anti- tively. Unless indicated, cells were maintained at 37Cina5% CD45 were from BD Biosciences. Acquisitions were performed CO2 and 95% air (21% O2) incubator. Isolation of peripheral using a BD Accuri C6 flow cytometer (BD Biosciences), and data blood NK cells from healthy donors (NKd) was from fresh were processed using the FlowJo program.

1790 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

Targeting AXL to Overcome Immune Resistance of NSCLC Cells

Fluorescence microscopy and conjugate formation was performed by preincubating effector or target carcinoma cells Conjugate formation between carcinoma target cells (T) and for 1 hour with antibodies (low endotoxin-free azide) against immune effector cells (E) was assessed after coculturing the cells CD54 (ICAM1), CD314 (NKG2D, 1D11), and control isotypes on poly-L-lysine–coated slides in a humidified incubator at 37C obtained from BioLegend, and used at 5 mg/mL concentration, for 30 minutes at 3:1 E/T ratio, respectively. Cells were then fixed which was maintained during cell lysis. with freshly prepared 3% paraformaldehyde for 10 minutes, incubated in a 50-mmol/L NH4Cl quenching solution (in PBS), Microarray analysis permeabilized with 0.25% Triton X-100 in PBS for 10 minutes. Total RNA was extracted using TRIzol reagent as above except Fixed and permeabilized cells were blocked with 10% FCS (v/v) in that nucleic acids were resolved in ethanol instead of isopropyl PBS for 20 minutes and stained using anti-LFA-1 (1:200, CD11a, alcohol and then transferred onto an RNeasy columns (Qiagen) clone EP1285Y, Abcam) diluted in PBS containing 1 mg/mL BSA for the rest of the procedure. Expression profiles of carcinoma cell for 2 hours or overnight at 4C followed by 1-hour incubation clones treated or not with bemcentinib were then generated using with secondary anti-rabbit Alexa Fluor 488 (Thermo Fisher Sci- an Agilent Technologies SurePrint G3 human GE 8 60K micro- entific) to identify effector cells. Cell nuclei were counterstained array (039494) with a single color technology. Data were pro- and mounted using ProLong Gold Antifade Mountant with DAPI cessed and analyzed as previously described (27). The microarray (Thermo Fisher Scientific). Conjugates were imaged on an IX83 data and protocols are available at the European Molecular microscope using the CellSense Dimension software (Olympus). Biology Laboratory European Bioinformatics Institute database The efficiency of conjugate formation between the effector and (https://www.ebi.ac.uk/arrayexpress/) under accession no. cancer cells was quantified as (the number of effector cells able to E-MTAB-8294. Gene set enrichment analyses (GSEA) were per- form mature conjugates with target cells/target cells 100). formed with the GSEA platform of the Broad Institute (http:// Conjugate formation was evaluated in 10 different visual fields www.broadinstitute.org/gsea/index.jsp) using the Molecular Sig- per experimental condition. For dual staining of ICAM1 and natures Database (MSigDB). F-actin, similar conditions were applied. Immunostaining was performed using anti-ICAM-1 (1:150, SC107, Santa Cruz) in Analysis of gene-expression data sets derived from NSCLC incubation buffer (0.1% BSA/PBS) at 4C for 16 hours followed patients by a 1-hour RT incubation with anti-mouse secondary antibody The Cancer Genome Atlas (TCGA) RNA-sequencing (RNA-seq) (A11029, Molecular Probes, Molecular Probes). After washing, expression data and sample information from human lung ade- cells were incubated with 165 nmol/L AF 647-conjugated phal- nocarcinoma from TCGA (http://cancergenome.nih.gov/) were loidin in 1% BSA/PBS (A22287, Molecular Probes). Images were accessed on April 28, 2018, from cBioPortal (28) and the TCGA obtained and processed on a Leica TCS SP8 STED 3X confocal public access data (http://tcga-data.nci.nih.gov/). Cases of the microscope (Leica Microsystems) equipped with a 100 objective LUAD TCGA with available expression data and follow-up infor- (HC PL Apo STED white, oil, NA ¼ 1.4, WD ¼ 0.13 mm). mation (n ¼ 504) were considered and Kaplan–Meier plots Phalloidin (F-actin) staining was also performed on carcinoma generated to assess the prognostic value of ICAM-1, ULBP1, cells cultured in poly-L lysine–coated chamber slides and pro- NKG2D, LFA-1, and AXL mRNA expression, as in RSEM normalized cessed as above to assess morphologic changes of the cells counts. Using http://kmplot.com, a similar analysis was performed following treatment. on a group of surgically managed NSCLC patients who did not receive radiotherapy or chemotherapy (n ¼ 227). Gene-expression Gene silencing by RNA interference data were automatically computed to generate Kaplan–Meier plots The functionally validated and predesigned siRNAs used in this as described (29). The Prat and colleagues data set (30) consisting study were from Thermo Fisher Scientific (Silencer select) and of 35 NSCLC patients treated with anti–PD-1 was investigated to Qiagen (FlexiTube) and directed against human ULBP1 compare mRNA mean expression of ULBP1, PD-1, LFA-1, and (SI02779847, SI02779868), ICAM1 (SI00004347, SI03093923), ICAM-1 in progression versus non-progression diseases. SLAMF7 (SI00130004, SI02644649), TNFRSF10B (SI03038665, SI03094063), HIF1A (SI02664053), MAPK1 (1027321), ZEB1 Statistical analysis (s229970, s229971), ZEB2 (SI02664277), TWIST1 (s14523), Data analyses were carried out using GraphPad (GraphPad SNAI2 (s13127, s13128), and SNAI1 (s13185, s13186). siRNAs Prism) and Excel (Microsoft Corp). Statistical tests were per- were transfected using Lipofectamine RNAiMAX Transfection formed using a two-tailed a ¼ 0.05 level of significance; , P reagent (Thermo Fisher Scientific), with appropriate controls. 0.05; , P 0.01; , P 0.001. Comparisons of the means between groups were performed using the Student t test, Mann– Cytotoxicity assay Whitney, or two-way ANOVA as appropriate. Overall survival The cytotoxic activity of NK and CTL clones was measured by a curves were generated by the Kaplan–Meier method and com- conventional 4-h 51Cr release assay as described (21) using 51Cr pared using the Mantel–Cox log-rank test. purchased from Perkin-Elmer. Briefly, after coculturing effector (immune cell):carcinoma target (E:T) cells at various ratios for 4 hours in round-bottomed 96-well plates, the percentage of Results specific lysis was calculated using the formula [(experimental Increase in AXL expression associated with resistance to lysis cpm spontaneous cpm)/total cpm incorporated] 100, and It has previously been shown that AXL is highly expressed in the results are expressed as the mean of triplicate samples. For lung cancers that have undergone EMT (15). We thus investigated some cytotoxic assays, carcinoma cells were incubated with com- the expression of AXL in a panel of EPI and MES NSCLC carci- pounds versus DMSO or siRNAs for 48 to 72 hours before their noma clones derived from IGR-Heu cell cultured under hypoxic use as targets. Cytotoxicity inhibition with blocking antibodies stress pressure (Fig. 1A). All the EPI clones, including EPI C2 cells,

www.aacrjournals.org Cancer Immunol Res; 7(11) November 2019 1791

Terry et al.

LO LO/- A B MES C23 (AXL ) MES C29 (AXL )

EPI C2EPI C5EPI C9 EPI C13EPI C16MES C23MES C25MES C33MES C30MES C29MES C32

AXL

Vimentin

β-Actin

MES C30 (AXLHI) MES C33 (AXLMed)

MES C23 C AXL LO cells MES C29 40 30 MES C33 AXL Med cells MES C30 AXL HI cells ** * * * ** * ** * * * * * 30 * * * * * * * * * * * 20 * * * * * ** * 20 * * ** * 10 ** 10 % Lysis (NK92 clone) % Lysis (CTL H33 clone)

0 0 15:1 10:1 5:1 1:1 20:1 10:1 3:1 E:T ratio (CTL/MES cancer clones) E:T ratio (NK92/MES cancer clones)

IL15/IL2-activated NK cells IL15/IL2-starved NK cells 40 40 MES C23 AXL LO cells MES C29 30 30 Med ** ** * * * * MES C33 AXL cells *** * * MES C30 HI * * * * * * * * AXL cells * * * 20 * * * * MES C25 20 *

10 10 % Lysis (NK donor) % Lysis (NK donor)

0 0 10:1 3:1 1:1 10:1 3:1 1:1 E:T ratio (NKd/MES cancer clones) E:T ratio (NKd/MES cancer clones)

Figure 1. Selective expression of AXL in MES lung carcinoma cells is associated with resistance to cell-mediated cytotoxicity. A, Western blot analysis of AXL expression in extracts from various NSCLC carcinoma clones with more EPI or MES phenotypes. Vimentin-negative clones were considered to have an EPI phenotype, whereas vimentin-positive clones were considered to have a MES phenotype. B, Morphology of four representative MES lung carcinoma clones. Scale bar, 40 mm. C, Results from cytotoxicity assays using the four MES carcinoma clones depicting the percentage of "target" cancer cells killed by "effector" CTL-mediated lysis (left), NK92-mediated lysis (right), or NK cells (bottom) puriﬁed from blood of healthy donors (Nkd) unactivated or activated with an IL2/IL15 cocktail. Different E:T ratios were used, and data represent mean percentage of lysis SEM for three replicates. , P < 0.05; , P < 0.01; , P < 0.001.

lacked detectable expression of AXL, whereas a spectrum of AXL investigated the susceptibility to lymphocyte-mediated killing of expression from low/undetectable to high was observed in vimen- the MES carcinoma clones displaying high (AXLHI), medium tin-positive MES carcinoma clones. MES C30 carcinoma cells (AXLMed), or low (AXLLO) AXL expression. In cytotoxicity assays displayed the highest AXL expression. The pronounced expression where carcinoma cells were challenged with either human NK92, of AXL in this clone may be due to stabilization of hypoxia- NKL cell clones, puriﬁed NKd cells or the autologous CTL clone inducible factor-1-a (HIF-1a) in the cells concomitant with H33, the killing was signiﬁcantly lower in AXLHI and AXLMed MES higher expression of the EMT transcription factors (EMT-TF) cancer cells compared with AXLLO cells (Fig. 1C; Supplementary ZEB-1, SNAI1, and TWIST1 (Supplementary Fig. S1). There were Fig. S2). These results indicated that AXL expression in MES no substantial differences in the morphology of MES carcinoma carcinoma cells may mediate intrinsic resistance to both NK- and cells expressing different amounts of AXL (Fig. 1B). We then CTL-mediated killing.

1792 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

Targeting AXL to Overcome Immune Resistance of NSCLC Cells

AXL inhibition increased MES carcinoma cell susceptibility to expression profiles of AXLHI MES C30 cells treated for 72 hours lysis with the AXL inhibitor or vehicle (Fig. 3). Gene-expression anal- AXL is associated with resistance to different BRAF/MEK and ysis from bemcentinib- versus control-treated indicated an aver- EGFR inhibitors (14–16, 31) and AXL inhibitors are in trials for age of 700 genes that were significantly changed (499 upregulated overcoming such resistance. We thus thought to explore the and 199 downregulated; Fig. 3A). In contrast, bemcentinib treat- potential of AXL inhibition in sensitizing MES cancer cells to ment of the AXLNeg EPI C2 or AXLLO MES clones showed a much cell-mediated cytotoxicity. Treatment with bemcentinib as an AXL lower variation (EPI C2: 50 genes upregulated and 6 genes down- kinase inhibitor increased sensitivity to CTL and NK lysis in MES regulated; MES C29: 39 genes upregulated and 13 genes down- carcinoma clones expressing medium to high amounts of AXL regulated; Supplementary Table S2). These results highlight the (Fig. 2A–C). A 2- to 10-fold higher percentage of cancer cell killing AXL-targeting specificity of bemcentinib. was observed for the AXLHI MES and the AXLMed MES cancer cells GSEA revealed enrichment in the expression of gene sets related when assayed in the presence of autologous CTLs or NK clones. A to the NK pathway or activation, and antigen processing and net sensitization was also observed in the presence of purified presentation (Fig. 3B). An enrichment of regulatory pathways NKd cells. This contrasted with no significant changes observed involving NF-kB and MAPK inhibition was also observed. Apo- for AXLLO MES cells. ptosis and EMT signatures were not affected by the treatment To investigate the mechanisms that operate under AXL inhi- conditions (Fig. 3B). No obvious changes were observed in the bition in the observed sensitization to lymphocyte-mediated EMT-related gene-expression pattern or in the morphology of the cytotoxicity in the MES carcinoma cells, we examined gene- cells (Fig. 3C and D). Nonetheless, staining of cortical actin A 25 DMSO 25 30 DMSO ** DMSO 20 AXL inhib. 20 AXL inhib. AXL inhib. 20 15 ** 15 ** 10 ** 10 ** 10 ** ** 5 5 ** % Lysis (CTL H33 clone) % Lysis (CTL H33 clone) % Lysis (CTL H33 clone) 0 0 0 15:1 10:1 5:1 1:1 15:1 10:1 5:1 1:1 15:1 10:1 5:1 1:1 E:T ratio (CTL/AXLHI MES cancer clone) E:T ratio (CTL/AXLMed MES cancer clone) E:T ratio (CTL/AXLLO MES cancer clone)

B 30 ** DMSO 25 DMSO 25 DMSO AXL inhib. AXL inhib. 25 20 20 AXL inhib. 20 ** ** 15 15 15 ** 10 10 10 **

% Lysis (NK92) 5 5 % Lysis (NK92) 5 ** % Lysis (NK92)

0 0 0 20:1 10:1 3:1 20:1 10:1 3:1 20:1 10:1 3:1 E:T ratio (NK92/AXLHI MES cancer clone) E:T ratio (NK92/AXLMed MES cancer clone) E:T ratio (NK92/AXLLO MES cancer clone)

C * 70 70 70 DMSO DMSO DMSO 60 60 60 * AXL inhib AXL inhib. AXL inhib. 50 50 * 50 40 40 * 40 30 * 30 30 % Lysis (NKd) % Lysis (NKd) % Lysis (NKd) 20 * 20 20 10 10 10 0 0 0 10:1 3:1 1:1 10:1 3:1 1:1 10:1 3:1 1:1 E:T ratio (NKd/AXLHI MES cancer clone) E:T ratio (NKd/AXLMed MES cancer clone) E:T ratio (NKd/AXLLO MES cancer clone)

Figure 2. Treatment of AXLHI MES and AXLMed lung carcinoma clones with an AXL inhibitor results in increased susceptibility to NK- and CTL-mediated lysis. A, CTL H33 cell lysis susceptibility of AXLHI MES (left), AXLMed MES (center), and AXLLO MES (right) clones after 72-hour treatment with the AXL inhibitor bemcentinib (1 mmol/L). Similar experiment evaluating susceptibility to NK92 cell lysis (B) and NKd cells (C). Data are presented as mean SEM for three replicate experiments. , P < 0.05; , P < 0.01. inhib., inhibitor.

www.aacrjournals.org Cancer Immunol Res; 7(11) November 2019 1793

Terry et al.

HI A AXL inhib. vs. DMSO treated B AXL inhib. vs. DMSO treated AXL MES C30 cells AXLHI MES clone GO_DNA_DAMAGE_RESPONSE_DETECTION_OF_DNA_DAMAGE GO_PRECATALYTIC_SPLICEOSOME downregulated genes: upregulated genes: Volcano plots GO_CATALYTIC_STEP_2_SPLICEOSOME P < 0.05 199 499 Threshold : GO_DEOXYRIBONUCLEOSIDE_TRIPHOSPHATE_METABOLIC_PROCESS

) FC: 2 GO_REGULATION_OF_CHROMOSOME_SEGREGATION P P value : 0.05 GO_TRANSCRIPTION_COUPLED_NUCLEOTIDE_EXCISION_REPAIR KEGG_NUCLEOTIDE_EXCISION_REPAIR GO_REGULATION_OF_SISTER_CHROMATID_SEGREGATION GO_ENDORIBONUCLEASE_COMPLEX GO_RECOMBINATIONAL_REPAIR -log10(Adj. HALLMARK_INTERFERON_GAMMA_RESPONSE HALLMARK_TNFA_SIGNALING_VIA_NFKB NF-κB activation BIOCARTA_UCALPAIN_PATHWAY log2(FC) HALLMARK_INTERFERON_ALPHA_RESPONSE KEGG_SPHINGOLIPID_METABOLISM MAPK & NF-κB REACTOME_NFKB MAP_KINASES AXL inhib. vs. DMSO treated _AND_ reactomes GO_POSITIVE_REGULATION_OF_SKELETAL_MUSCLE_TISSUE_DEVELOPMENT EPI C2 BIOCARTA_NKCELLS_PATHWAY NK pathway downregulated genes: upregulated genes: KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION antigen presentation 6 50 GO_NEGATIVE_REGULATION_OF_MAPK_ACTIVITY MAPK inhibition REACTOME_TAK1_ACTIVATES_NFKB_BY_PHOSPHORYLATION_AND_ACTIVATION_OF_IKKS_COMPLEX NF-κB activation GO_NATURAL_KILLER_CELL_ACTIVATION NK activation GO_INTRINSIC_APOPTOTIC_SIGNALING_PATHWAY_IN_RESPONSE_TO_ENDOPLASMIC_RETICULUM_STRESS GO_ANTIGEN_PROCESSING_AND_PRESENTATION_OF_PEPTIDE_ANTIGEN_VIA_MHC_CLASS_I antigen presentation KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY GO_POSITIVE_REGULATION_OF_MITOCHONDRIAL_MEMBRANE_PERMEABILITY REACTOME_ACTIVATED_TAK1_MEDIATES_P38_MAPK_ACTIVATION -log10(Adj. P ) GO_INACTIVATION_OF_MAPK_ACTIVITY MAPK inhibition

2 1 log2(FC) - - 0 1 2 Normalized enrichment score

HI C DEAXLHI MES clone AXL MES clone AXLHI MES clone DMSO AXL inhib. AXLHI MES clone Relative mRNA expression * ICAM1 in treated vs. untreated DMSO * ULBP1 0.0 0.5 1.0 1.5 2.0 SNAI1 SNAI2 max ZEB1

ZEB2 Vehicle (DMSO) F-acn TAP1 TWIST1 DAPI * VIM * TAPBP TGFB1 AXL AXL inhib. SERPINE1 1 mmol/L μ mol/L)

CD44 intensity) 2 (probe Log FN1 min CDH2 CDH1 KRT18 * SLAMF7 EPCAM F-acn DAPI * ERAP2 AXL inhibitor (1 * TNFRSF10B

F Relative mRNA expression in treated vs. untreated Relative mRNA expression in treated vs. untreated Relative mRNA expression in treated vs. untreated 02468101214 012345 012345 TGFB1 TGFB1 TGFB1 ICAM1 ICAM1 ICAM1 ULBP1 ULBP1 ULBP1 TAP1 TAP1 TAP1 TAP2 TAP2 TAP2 HI Med LO TAPBP AXL MES TAPBP AXL MES TAPBP AXL MES ERAP2 clone ERAP2 clone ERAP2 clone TNFRSF10B TNFRSF10B TNFRSF10B MICA MICA MICA SLAMF7 SLAMF7 SLAMF7 TNFRSF10A TNFRSF10A TNFRSF10A ERAP1 ERAP1 ERAP1

Figure 3. Treatment of the AXLHI/Med MES carcinoma clones with the AXL inhibitor is accompanied by changes in expression of antigen-processing genes and tumor- related factors that mediate NK cell functions. A, Volcano plots generated from expression data depicting the distribution of genes downregulated (green) or upregulated (red) in AXLHI MES C30 and EPI C2 cells treated with 1 mmol/L bemcentinib versus vehicle (DMSO) for 72-hour treatment. Adj., adjusted; inhib., inhibitor. B, Bar graphs generated from GSEA analysis showing the enrichment scores for the top 28 differentially enriched gene sets in AXLHI MES cancer cells. A positive score indicates enrichment in the bemcentinib-treated cells as opposed to negative scores, indicating enrichment in vehicle-treated cells. inhib., inhibitor. C, Bar graphs from qRT-PCR experiments showing relative mRNA expression of a panel of EMT-related markers to assess the effect of such treatment on EMT status of the cells. D, Phase contrast microscopy images of AXLHI MES cancer cells under treatment with the AXL inhibitor (inhib.) or DMSO (left). Scale bar, 40 mm. Right, immunoﬂuorescence staining of F-actin of the cells. Scale bar, 20 mm. E, Heat map displaying expression levels of a panel of immune-related genes in AXLHI MES cells treated with either bemcentinib or vehicle control for 72 hours from three replicates. inhib., inhibitor. F, Bar graphs from qRT-PCR experiments showing relative mRNA expression of the indicated immune-related genes in inhibitor treated versus vehicle treated in the different MES carcinoma clones. Data are presented as mean SEM for three replicate experiments.

microﬁlaments in the treatment condition was indicative of molecule 1), ULBP1, the ligand for NKG2D activating NK receptor changes concomitant with more cell–cell contacts (Fig. 3D; Sup- (UL16 binding protein 1), SLAMF7, also known as CD2-like plementary Fig. S3). Expression of tumor-related factors that receptor–activating cytotoxic cell (CRACC), TNFRSF10B mediate NK cell functions [i.e., ICAM1 (intercellular adhesion (TRAIL-R2)] and antigen processing and presentation (TAP1,

1794 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

Targeting AXL to Overcome Immune Resistance of NSCLC Cells

TAPBP, and ERAP2) was upregulated upon AXL inhibition expressed on both CTLs and NK cells consistent with an effect of (Fig. 3E). In contrast, no effect on the expression of these genes bemcentinib via ICAM1/LFA-1 and ULBP1/NKG2D interactions was observed when EPI C2 carcinoma cells were treated (Supple- (Supplementary Fig. S9). mentary Fig. S4). We further assessed by qRT-PCR the impact of To confirm the finding that ICAM1 and ULBP1 operate as AXL inhibition on these genes upon bemcentinib treatment of immune-modulatory factors in the increased cancer cell lysis by AXLHI, AXLMed as well as AXLLO MES carcinoma clones. AXL cytotoxic effector cells, cytotoxic experiments were carried out kinase inhibition upregulated ICAM1, ULBP1, SLAMF7, and with blocking of the ICAM-1/LFA-1 and ULBP1/NKG2D interac- TNFRSF10B in the AXLHI MES and the AXLMed MES cells, but tions. Treatment with blocking anti-ICAM1 suppressed the had no significant effect on the AXLLO MES clone (Fig. 3F). In increased susceptibility of the carcinoma cells to NK and CTL complementary experiments, bemcentinib treatment of AXL- clones when pretreated with bemcentinib (Fig. 4F). Similar var- expressing NSCLC cell lines (H23, HCC44, H820, and A549), in iations in lysis were observed using a blocking antibody directed house generated NSCLC cell lines (ADC-PuB, ADC-TOR, ADC- to NKG2D (Fig. 4G). However, this effect was more apparent in Coco26, and ADC-Let), or primary carcinoma cell cultures freshly the presence of CTL effector cells compared with those seen in isolated from 5 NSCLC patients, resulted in a net upregulation of assays using NK effector cells. ULBP1, SLAMF7, and ICAM1 expression in multiple instances Together, these data suggest that MES NSCLC clones, which are (Supplementary Fig. S5). These observations suggested upregulation intrinsically resistant to immune effector cells, can be sensitized of ULBP1, SLAMF7, and ICAM1 as primary mechanisms of sensi- by targeted inhibition of AXL through a mechanism involving tization to lymphocyte-mediated cytotoxicity upon AXL inhibition. ICAM1 and ULBP1.

Increase in ICAM1 and ULBP1 upon AXL inhibition restored AXL inhibition regulates ICAM-1 and ULBP1 expression cancer cell lysis through NF-kB and MAPK signaling Assessment of the MES carcinoma clones for ICAM1 and As ICAM1 is an NF-kB target gene, we examined the status of ULBP1 expression indicated lower amounts in AXLHI and AXLMed NF-kBactivationbyEMSA.AlthoughbasalNF-kB DNA bind- MES compared with AXLLO MES clones (Supplementary Fig. S6A ing activity was observed in the vehicle-treated AXLHI MES and S6B), which could explain immune resistance of these cells cancer cells, treatment with bemcentinib led to an increase in (Fig. 1C). Certain AXLHI also appeared to have less MHC class I NF-kB activity (Fig. 5A). In order to substantiate this finding molecules at the cell surface (Supplementary Fig. S6C). Ligands of and examine the contribution of the IKK/NF-kB pathway in the NKG2D such as ULBP1 play a role in the recognition of stressed upregulation of ICAM-1, we performed experiments wherein target cells by the immune system, whereas cancer–lymphocyte cells were treated with the AXL inhibitor in the presence or conjugation and cytotoxic function rely in part on heterophilic absence of the IKK/NF-kB inhibitor TPCA-1. Exposure to TPCA-1 interactions between ICAM1 expressed on the target cell and the abrogated ICAM1 induction following bemcentinib treatment integrin LFA-1 expressed on immune effector cells. We thus asked (Fig. 5B; Supplementary Fig. S10). Similar results were obtained whether bemcentinib treatment could affect these interactions. after evaluating three other NF-kB target genes, IL6, IL8, and We first validated the increased expression of ICAM-1 protein in CCL20 (Fig. 5B). bemcentinib-treated AXLHI MES and AXLMed MES cancer cells As AXL inhibition can affect both MAPK/ERK and AKT signal- (Fig. 4A). Upregulation of ICAM1 expression was observed at the ing (14, 18), we examined the phosphorylation status of these cell surface of AXLHI MES clones and in various NSCLC cell lines proteins. Although a moderate reduction of phospho-AKT was such as NCI-H460 (Fig. 4B and C; Supplementary Fig. S7A–S7C). perceived at 24 hours, bemcentinib treatment had no significant We then assessed whether bemcentinib treatment could affect effect on phospho-AKT at 48 or 72 hours (Fig. 5C), nor did target–effector conjugate formation. For this purpose, bemcenti- this treatment significantly change phospho-GSK3-b amounts). nib-treated cancer "target" cells were coincubated for 30 minutes As expected, phospho-AXL but not total AXL amounts were with NK "effector" cells, and conjugate formation was evaluated reduced upon exposure to bemcentinib and phospho-ERK/MAPK by imaging. AXL inhibition resulted in an increased number of amounts decreased (Fig. 5C). This result was consistent with the mature conjugates established between effector and target cells GSEA analysis showing an enrichment of gene sets associated with (Fig. 4D). In this respect, effector cells were more distant and had MAPK inhibition. To further define the importance of ERK/MAPK more protrusions toward the vehicle-treated target cells, which in these molecular events, we examined whether MAPK knock- may reflect their relative inability to establish stable cell–cell down using siRNAs could directly affect gene expression of the contacts (Fig. 4D; Supplementary Fig. S8). We then examined abovementioned deregulated genes in the absence of bemcenti- whether the increased expression of ULBP1 and ICAM1 observed nib (Fig. 5D). We observed an increase in ULBP1 and TNFRSF10B upon AXL inhibition could explain the increased susceptibility to expression following MAPK knockdown. Collectively, these data lymphocyte-mediated lysis. To this end, we performed cytolytic suggested that the AXL kinase inhibition increases the expression assays following AXL inhibition and knockdown of ICAM1, immune-modulatory genes, including ICAM-1 and ULBP1 ULBP1, SLAMF7, and TNFRSF10B in AXLHI MES cells. Compared expression, through NF-kB activation and concurrent inhibition with siRNA control-treated cells, siRNA-ICAM1– and siRNA- of the MAPK signaling, respectively. ULBP1–treated AXLHI MES cells exhibited lower sensitivity to NK cells (Fig. 4E). siRNAs directed to SLAMF7 showed modest effects, Different amounts of ICAM1, ULBP1, and MHC class I found in whereas siRNAs to TNFRSF10B had no significant effect on cancer MES carcinoma cells compared with their EPI counterparts susceptibility to NK-mediated lysis. We verified the expression of We have previously demonstrated that hypoxia-induced various molecules that regulate cell–cell interactions on the MES and EPI NSCLC cells have different susceptibility to NK- surface of NK and CTL clones. Accordingly, the cognate ICAM1 and CTL-mediated killing (21). However, the molecular basis receptor, b2 integrin LFA-1, and the ULBP1 receptor NKG2D were associated with this differential susceptibility has not been

www.aacrjournals.org Cancer Immunol Res; 7(11) November 2019 1795

Terry et al.

A B AXLHI MES clone C ctrl IgG AXL inhib. CTL MES clone DMSO AXL inhib. MES clone CTL MES clone DMSO HI LO Med AXL HI CTL 100 ICAM1 CTL CTL MES CTL AXL AXL AXL HI AXL inhib. 80 CTL AXL (1 μmol/L) − + − + − + MES CTL 60 AXL HI ICAM1 40 MES CTL CTL ICAM1 ICAM1 CTL ICAM1 20 Fold change 1115.7 1.2 2.7 F-acn F-acn Events (% of Max) CTL F-acn Actin 0 Dapi Dapi Dapi Fluorescence intensity

siRNA_control + DMSO D AXLHI MES cells + NK cells E siRNA_control 40 * * siRNA_ICAM1 NK + AXL inhib. * siRNA_ULBP1 DMSO Target 30 * siRNA_SLAMF7 40 * siRNA_TNFRSF10B NK Target NK 30 20 LFA-1 * 20 *

% Lysis (NK92) 10 NK 10 AXL 0 inhib. 0 Target 20:1 10:1 3:1 Conjugate formation efficiency DMSO LFA-1 AXL inhib. E:T ratio (NK92/AXLHI MES cancer clone)

F ICAM1 blocking ICAM1 blocking

DMSO/IgG 40 ** DMSO/IgG DMSO/anti-ICAM1 40 ** DMSO/anti-ICAM1 ** AXL inhib./IgG AXL inhib./IgG 30 ** AXL inhib./anti-ICAM1 30 AXL inhib./anti-ICAM1 ** 20 20 **

10 % Lysis (NK92) 10 % Lysis (CTLH33)

0 0 10:1 3:1 1:1 20:1 10:1 3:1 E:T ratio (CTL/AXLHI MES cancer clone) E:T ratio (NK92/AXLHI MES cancer clone)

G NKG2D blocking NKG2D blocking DMSO/IgG DMSO/IgG 40 40 DMSO/anti-NKG2D DMSO/anti-NKG2D AXL inhib./IgG * AXL inhib./IgG * 30 30 AXL inhib./anti-NKG2D ** AXL inhib./anti-NKG2D * 20 20 **

10 % Lysis (NK92) 10 % Lysis (CTLH33)

0 0 10:1 3:1 1:1 20:1 10:1 3:1 E:T ratio (CTL/AXLHI MES cancer clone) E:T ratio (NK92/AXLHI MES cancer clone)

Figure 4. AXL inhibition led to increased expression of ICAM-1 and ULBP1 in conjunction with increased NF-kB activity and inhibition of MAPK. A, Immunoblots showing induction of ICAM1 after treatment with 1 mmol/L bemcentinib. Increased ICAM1 is mostly visible in the AXLHI and AXLMed MES cancer cells. B, Increased cell- surface expression of ICAM1 on AXLHI MES cells following 72 hours of treatment as assessed by flow cytometry. C, ICAM-1 expression assessed by immunofluorescence staining after coculturing CTLs and AXLHI MES cells pretreated or not with 1 mmol/L bemcentinib for 48 hours. F-actin and counterstaining with DAPI are also shown. Scale bar, 10 mm. Single channel images are provided as Supplementary Fig. S7A. D, Efficiency of conjugate formation between NK92 cells and target cells, AXLHI MES cells, pretreated or not with 1 mmol/L bemcentinib for 72 hours. Immunofluorescence staining for LFA-1 was used to discriminate NK effector cells from target cells. Representative images showing mature conjugates in the bemcentinib-treated condition, which contrasted with vehicle condition, wherein effector cells appeared more distant to the target cells illustrating deficient immunologic synapses. Scale bar, 10 mm. E, Cytotoxicity assay for NK-mediated lysis of the AXLHI MES carcinoma cells after siRNA targeting of ICAM1, ULBP1, SLAMF7, or TNFRSF10B in cells treated with 1 mmol/L bemcentinib for 72 hours. Bars represent the means of three independent experiments SEM performed in triplicate. F and G, Cytotoxicity assays for NK-mediated lysis (F)or CTL-mediated lysis (G) of the AXLHI MES cancer cells after antibody neutralization of ICAM1 or the ULBP1 receptor NKG2D. Bars represent the means of three independent experiments SEM performed in triplicate. For D–G, , P < 0.05; , P < 0.01. inhib., inhibitor.

1796 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

Targeting AXL to Overcome Immune Resistance of NSCLC Cells

CCL20 A B 1.2 10-04 ***

1.0 10-04

7.5 10-05

IL6 5.0 10-05

ICAM1 relative mRNA levels 5 10-05 ** DMSO AXL inhib. 6 10-04 *** 2.5 10-05 -05 5 10-04 4 10 CCL20 0.0 -04 μ NF-kappaB 4 10 3 10-05 AXL inhib. ( mol/L) − 1 1 1 TPCA-1 (μmol/L) − − complexes 3 10-04 1 2.5 2 10-05 2 10-04 IL8 6 10-03 ** HI -05 AXL MES clone -04 1 10

1 10 IL6 relative mRNA levels 5 10-03 ICAM1 relative mRNA levels 0 0 4 10-03 AXL inhib. (μmol/L) − 1 1 1 AXL inhib. (μmol/L) − 1 1 1 -03 TPCA-1 (μmol/L) − − 1 2.5 TPCA-1 (μmol/L) − − 1 2.5 3 10 2 10-03 relative mRNA levels mRNA relative -03 IL8 1 10

0 AXL inhib. (μmol/L) − 1 1 1 TPCA-1 (μmol/L) − − 1 2.5

C AXL inhib. (1 μmol/L) AXL inhib. (1 μmol/L) D siRNA_MAPK1 24 h 48 h 72 h 24 h 48 h 72 h siRNA_ctrl ctrl IgG − + − + − + − + − + − + 100 ULBP1

80 p-AXL p-AKT siRNA_ctrlsiRNA_MAPK1

Tyr779 exposure 60 ERK/MAPK long AXL AKT 40

ERK/MAPK short 20 Events (% of Max)

p-ERK/MAPK Actin 0 Actin Fluorescence intensity

HI Relative mRNA expression p-GSK3β 100 ERK/MAPK AXL MES clone 024681012 TRAILR2 (Ser9) 80 in MAPK1 siRNA treated vs. control cells

Actin GSK3β TAP1 60 ICAM1 ULBP1 TAPBP ERAP2 TGFB1 MAPK1 SLAMF7 HI 40 AXL MES clone GAPDH

TNFRSF10B 20 Events (% of Max) HI AXL MES clone 0 Fluorescence intensity

Figure 5. Targeting the ICAM1/LFA-1 and ULBP1/NKG2D axes impairs bemcentinib's effect on cancer cell susceptibility to killing. A, Nuclear extracts from the AXLHI MES carcinoma clone treated with 1 mmol/L bemcentinib for 72 hours were analyzed by EMSA to evaluate NF-kB DNA binding activity. B, qRT-PCR data showing that in the AXLHI MES cancer cells, ICAM1 induction under bemcentinib treatment is blocked when combined with 48-hour treatment with the IKK/NF-kB inhibitor TPCA-1 at the indicated doses. Results obtained for known NF-kB targets IL6, IL8, and CCL20 are also presented. Bars represent the means of three independent experiments SEM performed in triplicate. , P < 0.01; , P < 0.001. C, Immunoblots comparing expression of phosphorylated-AKT, phosphorylated-ERK, and phosphorylated-AXL in AXLHI MES cancer cells treated for 24, 48, and 72 hours (h) with 1 mmol/L bemcentinib. D, AXLHI MES cancer cells were pretreated for 72 hours with MAPK1 siRNAs, and effects on the indicated immune-related genes were evaluated by quantitative RT-PCR and ﬂow cytometry. inhib., inhibitor.

elucidated. We thus asked which mechanisms would also either downregulated or upregulated, respectively (Fig. 6B). mediate different susceptibility between MES and EPI NSCLC Flow cytometry analysis further indicated that MES carcinoma cells. We investigated gene-expression proﬁles from two (EPI cells donwnregulated ICAM1, ULBP1, and MHC class I mole- C2 vs. MES C30) representative clones with EPI versus MES cules compared with EPI carcinoma cells (Fig. 6C). This obser- phenotypes arising from hypoxic stress. As expected, GSEA vation indicates that carcinoma cells with EPI (AXL null) or revealed enrichment in expression of EMT signature in the MES (AXL HI/Med) features differ in their expression of ICAM1 MES compared with EPI carcinoma cells (Fig. 6A). Gene sets and ULBP1, which likely regulates their susceptibility to NK- previously known to be associated with immune-suppressive and CTL-mediated killing. properties and resistance to cell-mediated killing, including TGFb,NF-kB, hypoxia, and STAT3/STAT5 signatures were also ULBP1 and ICAM1 tumor expression is associated with better found to be enriched. We noted expression differences only in survival in NSCLC patients a small number of immune-related gene transcripts. MES To test whether the results obtained are relevant in the clinical C30 was found to have downregulated expression of CDH1 setting of immune surveillance of human lung tumors, we exam- (E-cadherin) and upregulated expression of TGFb1 and TGFb2, ined the gene expression of ICAM1 and ULBP1 in public data sets known to be associated with EMT program. Genes encoding for and investigated their prognostic value in terms of overall survival the immune-modulatory molecules such as ICAM1, ULBP1, in NSCLC patients (29, 32). NSCLC patients were stratiﬁed into and MICA (MHC class I polypeptide-related sequence A) were two groups, with high or low expression of these genes, and

www.aacrjournals.org Cancer Immunol Res; 7(11) November 2019 1797

Terry et al.

A MES C30 vs. EPI C2 B cancer clones Hallmark gene sets EPI C2 MES C30 PANCREAS_BETA_CELLS HEDGEHOG_SIGNALING SPERMATOGENESIS PEROXISOME PROTEIN_SECRETION ESTROGEN_RESPONSE_EARLY BILE_ACID_METABOLISM ESTROGEN_RESPONSE_LATE APICAL_SURFACE COMPLEMENT IL2_STAT5_SIGNALING TGF_BETA_SIGNALING TNFA_SIGNALING_VIA_NFKB APOPTOSIS HYPOXIA UV_RESPONSE_DN WNT_BETA_CATENIN_SIGNALING P < 0.05 IL6_JAK_STAT3_SIGNALING ANGIOGENESIS EMT

5 0.00 0.25 0.50 0.75 1.00 −1.00 −0.75 −0.50 −0.2 Min Max Enrichment score Log 2 (probe intensity)

C MES lung carcinoma cells EPI lung carcinoma cells

ICAM1 ULBP1 MICA/B HLA-A,B,C

EPI C2 EPI C2 EPI C2

EPI C9 EPI C9 EPI C9

MES C23 MES C23 MES C23 AXLLO AXLLO AXLLO

MES C30 MES C30 MES C30 AXLHI AXLHI AXLHI MES and EPI carcinoma clones MES and EPI carcinoma

ctrl IgG ctrl IgG ctrl IgG ctrl IgG

2 3 4 5 6 2 3 4 5 6 2 3 4 5 6 2 3 4 5 6 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Fluorescence intensity Fluorescence intensity Fluorescence intensity Fluorescence intensity

Figure 6. Hypoxia-induced EMT is associated with differences in the expression of immune-modulatory genes, including ULBP1 and ICAM1. A, Top 20 differentially enriched gene sets in AXLHI MES C30 versus EPI C2 from GSEA analysis, as classiﬁed by enrichment scores. A positive score indicates enrichment in the MES C30

cells, whereas a negative score indicates enrichment in the EPI C2 cells. B, Heat map displaying log2-transformed expression levels of a panel of immune-related genes in the respective cancer clones from three replicates. C, Fluorescence intensity histograms displaying the surface expression of ICAM1, ULBP1, MICA/B, and HLA-A,B,C in two MES and two EPI carcinoma clones. Isotype control (Ctrl IgG) is shown in gray.

Kaplan–Meier plots were generated to compare survival of NKG2D, respectively. Akin to the impact observed for ICAM1 and patients (Fig. 7). ULBP1, LFA-1 and NKG2D were consistently associated with In these NSCLC patients, high expression of ICAM1 and ULBP1 better survival in these patients (Fig. 7A and B; Supplementary was associated with better survival in the Gyorffy€ and colleagues Fig. S11A and S11B). ULBP1 also positively correlated with and TCGA LUAD data sets (Fig. 7A and B; Supplementary survival in the TCGA LUSQ data set (Supplementary Fig. S12). Fig. S11A and S11B). AXL mRNA expression was not prognostic Moreover, in a cohort of NSCLC patients treated with anti–PD-1, in the NSCLC data sets analyzed, despite a more unfavorable LFA-1 and NKG2D were found to be more highly expressed in the trend in the AXL-high group (Supplementary Fig. S11C). We next nonprogressor patients (Fig. 7C). Thus, the ICAM1/LFA-1 and queried the impact on patient survival of cognate ICAM1 and ULBP1/NKG2D axes regulate the balance between cancer cell ULBP1 receptors that are expressed by lymphocytes, LFA-1 and susceptibility and tumor progression.

1798 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

Targeting AXL to Overcome Immune Resistance of NSCLC Cells

A Györffy et al. dataset

100 100 100 100 HR = 0.46 (0.28–0.76) HR = 0.49 (0.32–0.76) HR = 0.54 (0.35−0.82) HR = 0.52 (0.35−0.78) logrank P = 0.0021 logrank P = 0.00097 logrank P = 0.0034 logrank P = 0.0015 80 80 80 80

60 60 60 60

40 40 40 40

20 20 20 20 ULBP1 high ICAM1 high LFA-1 (ITGAL) high NKG2D (KLRK1) high

Proportion of survival (%) 0 ULBP1 low 0 ICAM1 low 0 LFA-1 (ITGAL) low 0 NKG2D (KLRK1) low 050100 150 0 50 100 150 0 50 100 150 050100 150

Overall survival time (months) B TCGA LUAD dataset

100 100 100 100 HR = 0.66 (0.46−0.94) HR = 0.6 (0.44−0.85) HR = 0.61 (0.45−0.84) HR = 0.62 (0.44−0.88) logrank P = 0.021 logrank P = 0.0026 logrank P = 0.002 logrank P = 0.007 80 80 80 80

60 60 60 60

40 40 40 40

20 20 LFA-1 (ITGAL) high 20 20 ULBP1 high ICAM1 high NKG2D (KLRK1) high ULBP1 low ICAM1 low LFA-1 (ITGAL) low NKG2D (KLRK1) low 0 0 0 Proportion of survival (%) 0 024 48 72 96 120 144 024 48 72 96 120 144 240 48 72 96 120 144 240 48 72 96 120 144

Overall survival time (months)

C PD-1 (PDCD1) NKG2D (KLRK1) ICAM1 LFA-1 (ITGAL) 10 10 15 12 ** * NS * 9 9 14 11 13 10 8 8 12 9 11 7 7 8 10 6 7 6 9 6 5 5 8 7 5 4 4 6 4 3 3 Relative transcript abundance 3 5 D D PD PD PD PD NPD NP NP NPD

Figure 7. Both ICAM1/LFA-1 and ULBP1/NKG2D axes are associated with better survival in NSCLC. Kaplan–Meier curves were generated to analyze the association between ULBP1, ICAM1, LFA-1, and NKG2D mRNA expression in the whole tumors of primary lung adenocarcinomas and overall survival of patients from the Gyorffy€ et al. data set (ref. 29; A), and from TCGA lung adenocarcinoma data set (B; ref. 32). Patients were stratiﬁed into two groups (low and high expression) based on the best-calculated cutoff. C, Box and whisker plots generated from the Prat et al. data set for expression of PD-1, NKG2D, ICAM1, and LFA-1 mRNA in 35 NSCLC patients treated with anti–PD-1. , P < 0.05; , P < 0.01. PD; progression disease; ns, not signiﬁcant; NPD; nonprogression disease (stable þ objective response) under anti–PD-1 therapy.

AXL mRNA expression was not prognostic in the NSCLC data sets Discussion analyzed. Studies suggest AXL activity in both tumor and stromal Although immunotherapy has improved the survival rates for cells, which complicates interpretation (35). AXL targeting with patients with advanced malignancies, the prevalence of nonre- bemcentinib in transgenic pancreatic cancer mouse models both sponders prompts further investigation of the events underlying reversed tumor EMT and repolarized M2 macrophages (35). AXL the immune resistance of tumors to allow them to be prevented or inhibition has been reported to enhance the sensitivity of cancer overcome. Genetic and epigenetic intratumoral heterogeneity cells to chemotherapeutic agents through means including DNA contributes to the emergence of resistant clones and immune damage response, perturbations of cell death receptors, and escape (33). EMT of adenocarcinoma cells promotes the emer- increased expression of nucleoside transporters (18, 34, 35). gence of MES cancer cell populations that exhibit chemo-, radio-, However, how AXL contributes to immune evasion is unknown. and drug resistance. AXL expression is linked with such resistance In this study, we found that carcinoma cells positioned in the and EMT in many cancer types (14, 15, 18, 19, 34). Our survey mesenchymal states of the EMT spectrum, and expressing AXL, further showed heterogeneous AXL expression in the different can contribute to tumor resistance to NK and CTL immunity. We MES cancer clones. How combinatorial expression of various show that AXL-expressing MES NSCLC clones resist CTL- and NK EMT-TF and trans-acting microenvironmental factors such as cell–mediated lysis and that AXL kinase inhibition using bem- HIF-1a regulates this heterogeneity remains to be investigated. centinib, a pharmacologic inhibitor in several clinical trials

www.aacrjournals.org Cancer Immunol Res; 7(11) November 2019 1799

Terry et al.

including NSCLC, led to sensitization to NK and CTL-mediated MHC class I molecules such as HLA-A. Nevertheless, AXLHI MES killing. carcinoma cells showed lower amounts of MHC class I molecules, The mechanism of AXL-mediated intrinsic mesenchymal cell and in line with previous studies (8, 10), MES cancer clones resistance to effector cell cytotoxicity involved decreased ICAM1 frequently exhibited reduced expression of MHC class I molecules and ULBP1 expression and reduced conjugate formation. ICAM-1 compared with more EPI cancer clones. As observed in EMT-like on target cells binds to its cognate receptor LFA-1 on effector triple-negative breast carcinoma cells, it will be interesting to see if lymphocytes, strengthening the interaction between the cytotoxic prominent accumulation of actin microfilaments at the immu- killer cells (CTLs and NK cells) and carcinoma target cells. Ligands nologic synapse affects cancer cell lysis in this setting (45). In of NKG2D such as ULBP1 play a role in the recognition of stressed contrast, other reports have raised the possibility that under some target cells by the immune system. Previous studies implied circumstances, such as forced expression of SNAIL or following ICAM1 was useful for prognosis in patients with colorectal cancer TGFb exposure, EMT may lead to increased susceptibility to NK or breast cancer (36–38). Higher expression of ICAM1, ULBP1, killing due to upregulation of MHC class I chain–related mole- and their receptors in NSCLC tumors correlated with better cules A and B (MICA/B; ref. 46), or increased expression of cell patient survival in two data sets of NSCLC patients, suggesting adhesion molecule (CADM1; ref. 47), respectively. Our current a contribution of the ICAM-1/LFA-1 and ULBP1/NKG2D axes study has focused on MES lung cancer cell populations exhibiting during immune surveillance. high resistance to both NK- and CTL-mediated killing. We Antibody-based therapies targeting T-cell inhibitory receptors, observed heterogeneity in target cell susceptibility in the MES mainly PD-1, to reactivate immune cells have led to objective cancer cell population. However, in contrast to these previous long-term responses for a subset of NSCLC patients (20% to 30%) studies, we did not observe variability of MICA/B, and could not with a very unfavorable prognosis (2). Other promising find evidence for deregulation of CADM1 in the EPI versus MES approaches are being developed that may widen the spectrum carcinoma clones even under bemcentinib treatment. However, it of immunotherapeutic options to include autologous cell ther- would be interesting to test whether AXL signaling or related apy, engineered T cells (such CAR-T cell therapy), or NK pathways could regulate MICA/B or CADM1 via various para- cells (39, 40). Nevertheless, some cancer patients fail to respond meters regulating the microenvironmental niche, such as hypoxia. to these therapeutic interventions and others show disease recur- In this regard, Huergo and colleagues showed that NK cells could rence after an initial response (2, 41). Future studies should confer an EMT-like/NK-protective phenotype to neighboring address the role of AXL targeting in these settings. melanoma cells (48). Studies in mouse models have provided Although our knowledge of regulation of the NKG2D ligand evidence linking AXL to immune-suppressive activities involving (ULBP1) expression remains fragmentary, our studies support the various stromal cellular components, including polarization of notion that AXL and EMT-associated cellular mechanisms may be dendritic cells, myeloid cells, T-cell exclusion, and tumor- exploited by carcinoma cells to reduce the expression of stress- associated macrophages (35, 44, 49). induced ligands and escape immune recognition. Among the AXL has received much attention for its contribution to inva- NSCLC cell lines tested, not all were responsive to the bemcenti- sion, metastasis, and targeted therapy resistance. The current study nib treatment. The factors that influence this response are just suggests a link between AXL expression in NSCLC cells and beginning to be investigated. resistance to cytotoxic immune effector cells. Collectively, our Although the NF-kB pathway is best characterized for its results provide insights into how an AXL-based mechanism may protective activity in response to proapoptotic stimuli, it also control cancer cell susceptibility to immune killer cells and supports EMT and suppresses cell death in certain cell types. In the establish AXL as a driver of mesenchymal cells' resistance to course of these studies, we demonstrated that long-term AXL cell-mediated cytotoxicity. Our results provide a rationale to test inhibition with bemcentinib resulted in slow but persistent NF- the ability of AXL inhibitors to overcome EMT-mediated resis- kB activation coinciding with an increase in ICAM1 expression tance to immunotherapy. and the subsequent increase in target susceptibility to cell- mediated cytotoxicity. Conversely, AXL inhibition resulted in Disclosure of Potential Conflicts of Interest MAPK inhibition leading to ULPB1 upregulation. AXL targeting G. Gausdal has ownership interest (including patents) in BerGenBio reduced MAPK signaling and reversed drug resistance in melano- ASA. J.B. Lorens is a scientific advisor for, reports receiving a commercial ma cells (42). Various drug-related stresses could contribute to a research grant from, and has ownership interest (including patents) in k BerGenBio ASA. J.-P. Thiery reports receiving other commercial research wide range of NF- B responses including alterations in the DNA- support from Biocheetah and is a consultant/advisory board member for fi repair machinery identi ed in the GSEA analysis (43). Gene sets Biosyngen, Aim Biotech, and ACT Genomics. No potential conflicts of related to DNA repair appeared to be affected upon treatment with interest were disclosed by the other authors. bemcentinib as shown by negative enrichment scores in the GSEA (Fig. 4). Similar observations were reported by Balaji and collea- Authors' Contributions gues, who used another AXL inhibitor (34). Moreover, it will be Conception and design: S. Terry, A.S.T. Engelsen, F. Mami-Chouaib, S. Chouaib Development of methodology: S. Terry, A. Abdou, A.S.T. Engelsen, J.B. Lorens, essential to determine how AXL signaling interferes with factors S. Chouaib such as cytokines that modulate NF-kB and MAPK activation. Acquisition of data (provided animals, acquired and managed patients, In previous studies, we and others have reported that EMT of provided facilities, etc.): S. Terry, A. Abdou, A.S.T. Engelsen, S. Buart, breast carcinoma cells can confer resistance to CTL-mediated S. Corgnac, D. Collares, V. Baud, F. Mami-Chouaib, S. Chouaib killing through mechanisms such as increased TGFb signaling or Analysis and interpretation of data (e.g., statistical analysis, biostatistics, reduced antigen presentation (8, 10, 13). Although AXL was computational analysis): S. Terry, A.S.T. Engelsen, P. Dessen, G. Meurice, V. Baud, P. Saintigny, F. Mami-Chouaib, S. Chouaib previously shown to suppress antigen-presenting MHC class I Writing, review, and/or revision of the manuscript: S. Terry, A.S.T. Engelsen, molecules in the mouse PyMT model (44), here, AXL inhibition in G. Gausdal, V. Baud, P. Saintigny, J.B. Lorens, J.-P. Thiery, F. Mami-Chouaib, MES cancer cells had no effect on cell-surface expression of S. Chouaib

1800 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

Targeting AXL to Overcome Immune Resistance of NSCLC Cells

Administrative, technical, or material support (i.e., reporting or organizing and the Research Council of Norway [FRIPRO Mobility Grant to A.S.T. Engelsen, data, constructing databases): S. Terry, A. Abdou, G. Meurice co-funded by the EU's 7th Framework Programme's Marie Curie Actions (MCA Study supervision: S. Terry, J.-P. Thiery, S. Chouaib COFUND grant agreement no. 608695)].

Acknowledgments The costs of publication of this article were defrayed in part by the The authors thank Nathalie Droin and Betty Leite (Gustave Roussy, Genomics payment of page charges. This article must therefore be hereby marked Platform), Yann Lecluse (Gustave Roussy, Imaging and Cytometry Platform), as advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate well as Avsnes Dale and Endy Spriet (University of Bergen, Molecular Imaging this fact. Center),Aurelie Durgeau, ElodieVoilin, Pierre Savagner, Vincent Lavergne, Marine Leclerc, and Gwendoline Gros for technical help and advice. This work was supported by la Ligue Contre le Cancer (EL2015.LNCC/SaC), Institut National Received January 2, 2019; revised May 29, 2019; accepted August 30, 2019; du Cancer (PLBIO15-266; INCa-DGOS-PRT-K), the SIRIC-SOCRATE program, published ﬁrst September 5, 2019.

References 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. 19. Wilson C, Ye X, Pham T, Lin E, Chan S, McNamara E, et al. AXL inhibition Cancer incidence and mortality worldwide: sources, methods and major sensitizes mesenchymal cancer cells to antimitotic drugs. Cancer Res 2014; patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359–86. 74:5878–90. 2. Herbst RS, Morgensztern D, Boshoff C. The biology and management of 20. Terry S, Faouzi Zaarour R, Hassan Venkatesh G, Francis A, El-Sayed W, non-small cell lung cancer. Nature 2018;553:446–54. Buart S, et al. Role of hypoxic stress in regulating tumor immunogenicity, 3. McGranahan N, Swanton C. Biological and therapeutic impact of intra- resistance and plasticity. Int J Mol Sci 2018;19:3044. tumor heterogeneity in cancer evolution. Cancer Cell 2015;27:15–26. 21. Terry S, Buart S, Tan TZ, Gros G, Noman MZ, Lorens JB, et al. Acquisition of 4. Joyce JA, Fearon DT. T cell exclusion, immune privilege, and the tumor tumor cell phenotypic diversity along the EMT spectrum under hypoxic microenvironment. Science 2015;348:74–80. pressure: consequences on susceptibility to cell-mediated cytotoxicity. 5. Abouzahr S, Bismuth G, Gaudin C, Caroll O, Van Endert P, Jalil A, et al. Oncoimmunology 2017;6:e1271858. Identification of target actin content and polymerization status as a 22. Echchakir H, Vergnon I, Dorothee G, Grunenwald D, Chouaib S, Mami- mechanism of tumor resistance after cytolytic T lymphocyte pressure. Chouaib F. Evidence for in situ expansion of diverse antitumor-specific Proc Natl Acad Sci U S A 2006;103:1428–33. cytotoxic T lymphocyte clones in a human large cell carcinoma of the lung. 6. Ye X, Weinberg RA. Epithelial-mesenchymal plasticity: a central regulator Int Immunol 2000;12:537–46. of cancer progression. Trends Cell Biol 2015;25:675–86. 23. Dorothee G, Echchakir H, Le Maux Chansac B, Vergnon I, El Hage F, 7. Nieto MA, Huang RY, Jackson RA, Thiery JP. Emt: 2016. Cell 2016;166: Moretta A, et al. Functional and molecular characterization of a KIR3DL2/ 21–45. p140 expressing tumor-specific cytotoxic T lymphocyte clone infiltrating a 8. Akalay I, Janji B, Hasmim M, Noman MZ, Andre F, De Cremoux P, et al. human lung carcinoma. Oncogene 2003;22:7192–8. Epithelial-to-mesenchymal transition and autophagy induction in breast 24. Dorothee G, Vergnon I, El Hage F, Le Maux Chansac B, Ferrand V, Lecluse Y, carcinoma promote escape from T-cell-mediated lysis. Cancer Res 2013;73: et al. In situ sensory adaptation of tumor-infiltrating T lymphocytes to 2418–27. peptide-MHC levels elicits strong antitumor reactivity. J Immunol 2005; 9. Chen L, Gibbons DL, Goswami S, Cortez MA, Ahn YH, Byers LA, et al. 174:6888–97. Metastasis is regulated via microRNA-200/ZEB1 axis control of tumour cell 25. Le Floc'h A, Jalil A, Vergnon I, Le Maux Chansac B, Lazar V, Bismuth G, et al. PD-L1 expression and intratumoral immunosuppression. Nat Commun Alpha E beta 7 integrin interaction with E-cadherin promotes antitumor 2014;5:5241. CTL activity by triggering lytic granule polarization and exocytosis. J Exp 10. Dongre A, Rashidian M, Reinhardt F, Bagnato A, Keckesova Z, Ploegh HL, Med 2007;204:559–70. et al. Epithelial-to-mesenchymal transition contributes to immunosup- 26. Authier H, Billot K, Derudder E, Bordereaux D, Riviere P, Rodrigues- pression in breast carcinomas. Cancer Res 2017;77:3982–9. Ferreira S, et al. IKK phosphorylates RelB to modulate its promoter 11. Hamilton DH, Huang B, Fernando RI, Tsang KY, Palena C. WEE1 inhibi- specificity and promote fibroblast migration downstream of TNF recep- tion alleviates resistance to immune attack of tumor cells undergoing tors. Proc Natl Acad Sci U S A 2014;111:14794–9. epithelial-mesenchymal transition. Cancer Res 2014;74:2510–9. 27. Djenidi F, Adam J, Goubar A, Durgeau A, Meurice G, de Montpreville V, 12. Tripathi SC, Peters HL, Taguchi A, Katayama H, Wang H, Momin A, et al. et al. CD8þCD103þ tumor-infiltrating lymphocytes are tumor-specific Immunoproteasome deficiency is a feature of non-small cell lung cancer tissue-resident memory T cells and a prognostic factor for survival in lung with a mesenchymal phenotype and is associated with a poor outcome. cancer patients. J Immunol 2015;194:3475–86. Proc Natl Acad Sci U S A 2016;113:E1555–64. 28. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio 13. Terry S, Savagner P, Ortiz-Cuaran S, Mahjoubi L, Saintigny P, Thiery JP, cancer genomics portal: an open platform for exploring multidimensional et al. New insights into the role of EMT in tumor immune escape. cancer genomics data. Cancer Discov 2012;2:401–4. Mol Oncol 2017;11:824–46. 29. Gyorffy B, Surowiak P, Budczies J, Lanczky A. Online survival 14. Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activation of the analysis software to assess the prognostic value of biomarkers using AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. transcriptomic data in non-small-cell lung cancer. PLoS One 2013;8: Nat Genet 2012;44:852–60. e82241. 15. Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. An 30. Prat A, Navarro A, Pare L, Reguart N, Galvan P, Pascual T, et al. Immune- epithelial-mesenchymal transition gene signature predicts resistance related gene expression profiling after PD-1 blockade in non-small cell to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target lung carcinoma, head and neck squamous cell carcinoma, and melanoma. for overcoming EGFR inhibitor resistance. Clin Cancer Res 2013;19: Cancer Res 2017;77:3540–50. 279–90. 31. Boshuizen J, Koopman LA, Krijgsman O, Shahrabi A, van den Heuvel EG, 16. Muller J, Krijgsman O, Tsoi J, Robert L, Hugo W, Song C, et al. Low MITF/ Ligtenberg MA, et al. Cooperative targeting of melanoma heterogeneity AXL ratio predicts early resistance to multiple targeted drugs in melanoma. with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat Med Nat Commun 2014;5:5712. 2018;24:203–12. 17. Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Coan JP, et al. 32. Network TCGA. Comprehensive molecular profiling of lung adenocarci- AXL is a logical molecular target in head and neck squamous cell carcinoma. Nature 2014;511:543–50. noma. Clin Cancer Res 2015;21:2601–12. 33. Holzel M, Bovier A, Tuting T. Plasticity of tumour and immune cells: a 18. Gay CM, Balaji K, Byers LA. Giving AXL the axe: targeting AXL in human source of heterogeneity and a cause for therapy resistance? Nat Rev Cancer malignancy. Br J Cancer 2017;116:415–23. 2013;13:365–76.

www.aacrjournals.org Cancer Immunol Res; 7(11) November 2019 1801

Terry et al.

34. Balaji K, Vijayaraghavan S, Diao L, Tong P, Fan Y, Carey JP, et al. AXL 42. Miller MA, Oudin MJ, Sullivan RJ, Wang SJ, Meyer AS, Im H, et al. Reduced inhibition suppresses the DNA damage response and sensitizes cells to proteolytic shedding of receptor tyrosine kinases is a post-translational PARP inhibition in multiple cancers. Mol Cancer Res 2017;15:45–58. mechanism of kinase inhibitor resistance. Cancer Discov 2016;6:382–99. 35. Ludwig KF, Du W, Sorrelle NB, Wnuk-Lipinska K, Topalovski M, Toombs 43. Janssens S, Tschopp J. Signals from within: the DNA-damage-induced NF- JE, et al. Small-molecule inhibition of Axl targets tumor immune suppres- kappaB response. Cell Death Differ 2006;13:773–84. sion and enhances chemotherapy in pancreatic cancer. Cancer Res 2018; 44. Aguilera TA, Rafat M, Castellini L, Shehade H, Kariolis MS, Hui AB, et al. 78:246–55. Reprogramming the immunological microenvironment through radiation 36. Kabashima H, Nagata K, Maeda K, Iijima T. Involvement of substance P, and targeting Axl. Nat Commun 2016;7:13898. mast cells, TNF-alpha and ICAM-1 in the infiltration of inflammatory cells 45. Al Absi A, Wurzer H, Guerin C, Hoffmann C, Moreau F, Mao X, et al. Actin in human periapical granulomas. J Oral Pathol Med 2002;31:175–80. cytoskeleton remodeling drives breast cancer cell escape from natural 37. Ogawa Y, Hirakawa K, Nakata B, Fujihara T, Sawada T, Kato Y, et al. killer-mediated cytotoxicity. Cancer Res 2018;78:5631–43. Expression of intercellular adhesion molecule-1 in invasive breast cancer 46. Lopez-Soto A, Huergo-Zapico L, Galvan JA, Rodrigo L, de Herreros AG, reflects low growth potential, negative lymph node involvement, and good Astudillo A, et al. Epithelial-mesenchymal transition induces an antitumor prognosis. Clin Cancer Res 1998;4:31–6. immune response mediated by NKG2D receptor. J Immunol 2013;190: 38. Schroder C, Witzel I, Muller V, Krenkel S, Wirtz RM, Janicke F, et al. 4408–19. Prognostic value of intercellular adhesion molecule (ICAM)-1 expression 47. Chockley PJ, Chen J, Chen G, Beer DG, Standiford TJ, Keshamouni VG. in breast cancer. J Cancer Res Clin Oncol 2011;137:1193–201. Epithelial-mesenchymal transition leads to NK cell-mediated metastasis- 39. Galluzzi L, Chan TA, Kroemer G, Wolchok JD, Lopez-Soto A. The hallmarks specific immunosurveillance in lung cancer. J Clin Invest 2018;128: of successful anticancer immunotherapy. Sci Transl Med 2018;10. pii: 1384–96. eaat7807. 48. Huergo-Zapico L, Parodi M, Cantoni C, Lavarello C, Fernandez-Martinez 40. Wrangle JM, Velcheti V, Patel MR, Garrett-Mayer E, Hill EG, Ravenel JG, JL, Petretto A, et al. NK-cell editing mediates epithelial-to-mesenchymal et al. ALT-803, an IL-15 superagonist, in combination with nivolumab in transition via phenotypic and proteomic changes in melanoma cell lines. patients with metastatic non-small cell lung cancer: a non-randomised, Cancer Res 2018;78:3913–25. open-label, phase 1b trial. Lancet Oncol 2018;19:694–704. 49. Guo Z, Li Y, Zhang D, Ma J. Axl inhibition induces the antitumor immune 41. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and response which can be further potentiated by PD-1 blockade in the mouse acquired resistance to cancer immunotherapy. Cell 2017;168:707–23. cancer models. Oncotarget 2017;8:89761–74.

1802 Cancer Immunol Res; 7(11) November 2019 Cancer Immunology Research

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity

Stéphane Terry, Abderemane Abdou, Agnete S.T. Engelsen, et al.

Cancer Immunol Res 2019;7:1789-1802. Published OnlineFirst September 5, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-18-0903

Supplementary Access the most recent supplemental material at: Material http://cancerimmunolres.aacrjournals.org/content/suppl/2019/09/04/2326-6066.CIR-18-0903.DC1

Cited articles This article cites 48 articles, 22 of which you can access for free at: http://cancerimmunolres.aacrjournals.org/content/7/11/1789.full#ref-list-1

Citing articles This article has been cited by 1 HighWire-hosted articles. Access the articles at: http://cancerimmunolres.aacrjournals.org/content/7/11/1789.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department Subscriptions at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerimmunolres.aacrjournals.org/content/7/11/1789. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.