IL6 Mediates Suppression of T- and NK-cell Function in EMT-associated TKI-resistant EGFR-mutant NSCLC

Abstract

Purpose: Patients with advanced non-small cell lung cancer (NSCLC) harboring activating EGFR mutations are initially responsive to tyrosine kinase inhibitors (TKI). However, therapeutic resistance eventually emerges, often via secondary EGFR mutations or EGFR-independent mechanisms such as epithelial-to-mesenchymal transition. Treatment options after EGFR-TKI resistance are limited as anti-PD-1/PD-L1 inhibitors typically display minimal benefit. Given that IL6 is associated with worse outcomes in patients with NSCLC, we investigate whether IL6 in part contributes to this immunosuppressed phenotype.

Experimental design: We utilized a syngeneic genetically engineered mouse model (GEMM) of EGFR-mutant NSCLC to investigate the effects of IL6 on the tumor microenvironment and the combined efficacy of IL6 inhibition and anti-PD-1 therapy. Corresponding in vitro studies used EGFR-mutant human cell lines and clinical specimens.

Results: We identified that EGFR-mutant tumors which have oncogene-independent acquired resistance to EGFR-TKIs were more mesenchymal and had markedly enhanced IL6 secretion. In EGFR-mutant GEMMs, IL6 depletion enhanced activation of infiltrating natural killer (NK)- and T-cell subpopulations and decreased immunosuppressive regulatory T and Th17 cell populations. Inhibition of IL6 increased NK- and T cell-mediated killing of human osimertinib-resistant EGFR-mutant NSCLC tumor cells in cell culture. IL6 blockade sensitized EGFR-mutant GEMM tumors to PD-1 inhibitors through an increase in tumor-infiltrating IFNγ+ CD8+ T cells.

Conclusions: These data indicate that IL6 is upregulated in EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance and suppressed T- and NK-cell function. IL6 blockade enhanced antitumor immunity and efficacy of anti-PD-1 therapy warranting future clinical combinatorial investigations.

Figure 1 –

EGFR mutant NSCLC tumor cells with acquired resistance to an EGFR-TKI have increased levels of IL-6. (A&B) ELISA analysis of IL-6 secretion by human EGFR mutant HCC4006 (A) and H1975 (B) cells and their osimertinib-resistant (OR) variants (p <0.0063). (C) IL-6 production in EGFR mutant TKI naïve NSCLC cells (HCC827 and YUL-0019) and cell lines derived from EGFR mutant patients with acquired resistance to EGFR-TKIs (p < 0.1509). (D) Overall survival of EGFR mutation positive NSCLC patients from the KMplotter lung cancer dataset with high vs low IL-6 expression (n = 1926, p < 0.0001). (E) Circulating IL-6 levels were measured in patients from CROSSOVER and NORTHSTAR studies stratified by clinical outcome of prior to progression of disease or post progression of disease (n = 12 matched pairs run in duplicate, p =0.0517). (mean +/− SEM, * p<0.05, ** p<0.01, *** p<0.001)

Figure 2 –

Depletion of IL-6 increases the number of infiltrating lymphocytes and overall survival in EGFR mutant NSCLC tumors. (A) Expression of IL-6 in the serum and BALF of EGFR^L858R and EGFR^L858R/ IL6^tKO tumor-bearing mice. (B) Knockout of IL-6 extended the survival of EGFR^L858R tumor-bearing mice (p = 0.0212). (C-D) Immune cell deconvolution analysis from RNA-sequencing data collected from control and IL-6 knockout tumors from EGFR^L858R mice showed a minor decrease in T-regulatory cells (p = 0.1004) and a slight increase of CD8+ T cells (p = 0.0595), CD4+ T cells (p = 0.0086), NK cells (p = 0.0032) and T follicular helper cells (p < 0.0001). (E) Tumor immune cell populations in EGFR^L858R tumors with or without anti-IL-6 treatment as determined by flow cytometry. (F) Kaplan-meier analysis of EGFR mutant GEMM treated acutely with monoclonal blocking antibody to IL-6 (p = 0.0385). (G) Flow cytometry analysis to directly assess infiltrating immune cells in T and NK cell infiltration in EGFR mutant GEMM treated with monoclonal blocking antibody to IL-6. (mean +/− SEM, * p<0.05, ** p<0.01, *** p<0.001)

Figure 3 –

Oncogene-independent EGFR-TKI resistant EGFR mutant NSCLC tumors display an immunologically cold, mesenchymal phenotype. (A) Timeline of induction or withdrawal of doxycycline used as an inducing agent for the generation of EGFR mutant lung tumors in GEMMs. (B) Levels of EGFR are measured by qPCR of RNA collected from tumors collected from the oncogene-dependent (dox on) and oncogene-independent (dox off) models displaying a significant decrease in EGFR expression in the dox off samples. (C) CT imaging and (D) associated quantification displaying the significant increase in tumor volume (p = 0.0475) in the dox off tumor during EGFR-TKI treatment. (E) Oncogene-independent tumors display an enrichment of an epithelial-to-mesenchymal phenotype. (F) Oncogene-independent tumors show an increased expression of vimentin and loss of E-cadherin. (G-H) Immune cell deconvolution analysis from RNA-sequencing data collected from oncogene-dependent and oncogene-independent tumors showed a significant reduction of CD8+ T cells (p = 0.0194), CD4+ T cells (p < 0.0001), total NK cells (p < 0.0001), activated NK cells (p = 0.0122), and T follicular helper cells (p = 0.0061). (I) Multiplex ELISA analysis of serum collected from oncogene-dependent compared to oncogene-independent tumor-bearing mice. (J) IL-6 ELISA confirms elevated levels of IL-6 in oncogene-independent model compared to oncogene-dependent. (mean +/− SEM, * p<0.05, ** p<0.01, *** p<0.001)

Figure 4 –

IL-6 suppresses the activation of NK cells in EGFR mutant NSCLC tumors. (A) IL-6 knockout and (B) acute blockade of IL-6 increases expression of Ki-67+ proliferating NK cells (p=0.3501, p=0.4698), (C-D) activated NKG2D+ NK cells) (p=0.1042, p=0.0418). (E-F) Acute blockade of IL-6 of EGFR mutant NSCLC cell lines co-cultured with human NK cells isolated from healthy donor PBMCs significantly increased expression of NKG2D and Granzyme NK cells (G) MICA expression on EGFR mutant TKI resistant is increased with IL-6 blockade. (H) IL-6 blockade sensitizes EGFR mutant EGFR-TKI resistant cells to NK cell-mediated cytotoxic killing. (mean +/− SEM, * p<0.05, ** p<0.01, *** p<0.001)

Figure 5 –

IL-6 inhibits T cell-mediated anti-tumor response in EGFR mutant NSCLC tumors. (A-D) IL-6 knockout increased CD8+IFNγ+ T cells (p=0.0375) while IL-6 blockade had slightly more modest effects p=0.0375. Th17 T cell populations were significantly reduced in IL-6 knockout tumors (p=0.048) and those treated with IL-6 blocking antibody (p=0.032). (E) Human T cells isolated from healthy donor PBMCs co-cultured in conditioned media collected after the acute blockade of IL-6 of EGFR mutant NSCLC cell lines significantly increased expression of Granzyme B expression. (F) Anti-IL-6 treatment increased T cell-mediated cytotoxicity of EGFR mutant NSCLC cell lines. (G) Anti-IL-6 blockade induces increased survival of EGFR mutant GEMMs treated with anti-PD-1 immunotherapy (p = 0.0161). (H) Anti-IL-6 and anti-PD-1 combination treatment increases infiltration of activated T cells (PD-1+ CD8+) in EGFR mutant GEMMs (p = 0.0468). (mean +/− SEM, * p<0.05, ** p<0.01, *** p<0.001)