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Heymach, John

Le, Xiuning

Nilsson, Monique B

Patel, Sonia A

Poteete, Alissa

Ren, Xiaoyang

Shen, Li

Wang, Jing

Yang, Yan

Yu, XIaoxing

Zhang, Fahao

English

OpenWorks @ MD Anderson

IntroductionIL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC•Sonia A. Patel1, Monique B. Nilsson1, Yan Yang1, Xiaoxing Yu1, Fahao Zhang1, Alissa Poteete1, Xiaoyang Ren1, Xiuning Le1, Li Shen2, Jing Wang2, John V. Heymach11Department of Thoracic and Head and Neck Medical Oncology, 2Department of Cancer Biology,  3Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USAAcknowledgementsConclusionHypothesisFigure 1. Osimertinib-resistant (OR)variants secreted significantly greaterlevels of IL-6 compared to EGFR-mutant parental cells HCC4006 (A)and H1975 (B). Patient-derivedmodels (MDA-L-004K, MDA-L0011,MDA-L-0024, MDA-L-0046, andMDA-L-0065) of EGFR-TKIresistance also secreted higheramounts of IL-6 as compared toEGFR-mutant TKI-naïve cells (C).From TCGA analysis, patients withEGFR-mutant NSCLC, altered levelsof IL-6 were associated with a worsedisease-free survival (D).• Patients with NSCLC with activating mutations in epidermal growthfactor receptor (EGFR) receive clinical benefit from treatment withEGFR tyrosine kinase inhibitors (TKIs)• The majority of these patients will acquire resistance which can bemediated by various mechanisms including secondary EGFRmutations such as T790M, MET amplification, or EMT.• Anti-PD-1/PD-L1 immune checkpoint-blockade demonstratedclinical benefit in NSCLC patients. However, among patients withEGFR-mutant NSCLC, response rates to immunotherapy areminimal.• Previous studies show that IL-6 is a critical mediator of EGFR-TKIresistance. Thus, we sought to investigate the impact of IL-6 onanti-tumor immunity in EGFR-mutant NSCLC.Emerson Collective, Lung SPORE grant 5 P50 CA070907, 1R01 CA190628, 1R01 CA234183-01A1, Stading Fund for EGFR inhibitor resistance, CPRIT Training Award (RP210028).IL-6 is upregulated in EGFR-mutant NSCLC tumors with acquired resistance to EGFR-TKIs and impairs anti-tumor immunity through suppression of T and NK cell function.ResultsFigure 2. Survival analysis of EGFRL858R GEMMs and EGFRL858R GEMMs crossed with an IL-6knockout mouse showed that knockout of IL-6 significantly increased overall survival (A). EGFR-mutant NSCLC tumors treated with mouse anti-IL-6 blocking antibody significantly increased overallsurvival of mice (B). Knockout (KO) of IL-6 resulted in increased NK cell populations and the CD8 Tcell populations but a decreased T-regulatory and T follicular helper cell populations (C).Figure 3. Schematic of the development EGFR-independent EGFR-mutant model using doxycycline-inducible EGFRL858R GEMMs (A). CT imaging after 2-week treatment with osimertinib indicates strongsensitivity to EGFR-TKI in the EGFR-dependent butnot EGFR-independent model (B). EGFR-independenttumors displayed an enrichment in an EMT geneexpression signature (C). EGFR-independent tumorsshowed evidence of a colder immunemicroenvironment (decreased CD8 T cells, CD4 Tcells, total NK cells, and decreased activated NK cells)(D). IL-6 was differentially overexpressed in EGFR-independent tumors as assessed by multiplex cytokineanalysis of serum (E).Given the immunosuppressive role of IL-6, we hypothesized that IL-6 in part mediates the immunosuppressive phenotype responsible for EGFR-TKI resistant NSCLC’s marginal response to anti-PD-1/PD-L1 therapy through altering the tumor infiltrating immune cell populations and modulating their cytotoxic potential.ADACAAcquired EGFR-TKI resistance is associated with increased levels of IL-6Depletion of IL-6 increases overall survival and number of infiltrating lymphocytes in EGFR mutant GEMMsBlockade of IL-6 induces T cell activity in EGFR-mutant NSCLC microenvironmentEGFR-independent TKI resistant tumors display mesenchymal, immunologically cold phenotype and secrete IL-6ABFigure 4. Blockade of IL-6 increased NKG2D (A) and Granzyme B (B) expression in the NK cells co-cultured with EGFR-mutant NSCLC tumor cell lines. Blockade of IL-6 in the EGFR-TKI refractory cellsco-cultured with NK cells increased NK cell-mediated killing (C).IL-6 suppresses the activation of NK cells in the EGFR-mutant microenvironmentAFigure 5. Blockade of IL-6 in the EGFR-TKI refractory cells transduced withOKT3 co-cultured with T cellsmoderately increased T cell-mediatedkilling (A). Anti-PD-1 therapy did notsignificantly improve overall survival inthe EGFRL858R GEMM model. However,combination treatment of anti-IL-6 withanti-PD-1 treatment significantlyincreased overall survival (B).HCC40064006 OR14006 OR24006 OR44006 OR70100020003000** *********Concentration (pg/mL)H1975 1975 OR21975 OR51975 OR131975 OR180100020003000** *********Concentration (pg/mL)HCC827YUL-0019MDA-L-004KMDA-L-0011MDA-L-0024MDA-L-0046MDA-L-0065050010001500Concentration (pg/mL)*******B CDisease Free (Months)Disease Free (%)0 5 10 15 20050100Weeks L858RL858R-IL6tKOWildtypep=0.0212Percent Survival (%)0 20 40 60020406080100Days of Treatment Vehicleanti-IL-6p=0.0385BT cell  F0.1100.1150.1200.1250.1300.135CD4 T cells****  0.0000.0050.0100.0150.020T-regulatory cells**  0.000.010.020.030.040.05T follicularhelper cells***  0.0000.0020.0040.0060.008CD8 T cell*CIL-6IL-9GM-CSFMIP-1aIL-15IL-12 (p70)IL-12 (p40)IL-7IL-1bIFNyIL-13IL-10IL-1aTNFaIL-2G-CSFKCIP-10MCP-1IL-4RANTESMIP-2IL-5IL-17MIP-1b-202468Fold change****10 5 2.5 1.25020406080100Effector: Target RatioSpecific Lysis (%)anti-IL-6Vehicle10 5 2.5 1.25020406080100Effector: Target RatioSpecific Lysis (%)Vehicleanti-IL-6*10 5 2.5 1.25020406080100Effector: Target RatioSpecific Lysis (%)anti-IL-6Vehicle**0 20 40 60020406080100Days of TreatmentPercent survivalVehicleanti-IL-6anti-PD-1anti-IL-6/ anti-PD-110 5 2.5 1.25020406080100Effector: Target RatioSpecific Lysis (%)anti-IL-6Vehicle10 5 2.5 1.25020406080100Effector: Target RatioSpecific Lysis (%)anti-IL-6Vehicle*10 5 2.5 1.25020406080100Effector: Target RatioSpecific Lysis (%)anti-IL-6Vehicle*NK cell  0.00.10.20.30.4NK cells****  0.000.010.020.030.040.05Activated NK cells*  0.000.020.040.060.080.10NK resting cells*EDCHCC4006 OR2 OR4 OR7 HCC4006 OR2 OR4 OR70 0Anti-IL-6          - + - +         - +        - + Anti-IL-6          - +        - +        - +         - +HCC4006 OR2 OR7HCC4006 OR2 OR7Granzyme B   MFINKG2D+ NK cellsReferences1. Engelman, et al. Clin Cancer Res 14, 2895-2899 (2008).2. Nilsson, et al. Sci Transl Med 9 (2017).3. Lee et al., JAMA Oncology (2018). BNK cells   0.0000.0050.0100.0150.0200.025NK cell**   0.000.050.100.15NK resting cellp=0.0546   O0.000.010.020.030.04Activated NK cellsp=0.201T cells   0.000.010.020.030.040.05CD8 T cell**** N  O0.000.010.020.030.04T follicularhelper cell****   O0.0000.0050.0100.0150.020T cell CD4+*   0.0000.0050.0100.0150.020T-regulatory cellsp=0.1004EGFR-dependentEGFR-independentBeeks Days of TreatmentIL-6 KO      - +                  - +                - +                       - +                        - +                 - +                  - +              Gene signature analysis (EGFR-dependent vs EGFR-independent)EGFR dependence (dox)      - +               - +                - +               - +              - +       - +            - +              EGFR-dependent EGFR-independentPre-TKIPost-TKI

IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1032&context=leading-edge

IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC

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