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.

Translational Relevance

Alternate targeting of EGFR-mutant non–small cell lung cancer (NSCLC) tumors, a majority of which develop EGFR-TKI (tyrosine kinase inhibitor) resistance, is an unmet clinical need. Tumors which acquire resistance through epithelial-to-mesenchymal transition–associated EGFR-independent mechanisms are especially difficult to target. EGFR-mutant NSCLC tumors are poorly responsive to immune checkpoint blockade (ICB) therapy further highlighting the need for new therapeutic approaches for TKI-resistant tumors. This study identified that EGFR-mutant tumors which acquire oncogene-independent EGFR-TKI resistance express high levels of IL6 and exhibit a more mesenchymal phenotype. Utilizing an immunocompetent murine model of EGFR-mutant NSCLC, we show that IL6 suppressed infiltration and cytotoxic potential of tumor-infiltrating natural killer and T cells. Blockade of IL6 abrogated these immunosuppressive effects including a reduction in infiltrating regulatory T and Th17 cell populations. Inhibition of IL6 increased antitumor activity of ICB through increased infiltration of IFNγ+CD8+ T cells. These results support clinical testing of drugs targeting the IL6 pathway in combination with ICB.

Non–small cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. Around 10%–15% of patients with NSCLC in the United States and up to 40% in the Asian population bear tumors harboring activating mutations in the EGFR (1). For patients with advanced disease, EGFR tyrosine kinase inhibitors (TKI) such as the third-generation drug osimertinib are part of standard first-line therapy. While the vast majority of patients initially benefit from EGFR-TKIs, therapeutic resistance can emerge via either EGFR-dependent mechanisms, such as secondary EGFR mutations (e.g., C797S mutations), or via EGFR-independent mechanisms such as epithelial-to-mesenchymal transition (EMT) or small cell transformation (2–4). For patients with TKI resistance, standard treatment approaches are limited and include chemotherapy and immunotherapy with anti-PD-1/PD-L1 immune checkpoint blockade (ICB). Because immunotherapy is typically employed after the use of EGFR-TKI, there is a major unmet need for new treatment approaches for these patients.

Patients with EGFR-mutant NSCLC are typically resistant to ICB. In the TKI-naïve setting, an objective response rate (ORR) of 9% was observed for the PD-1 inhibitor pembrolizumab, and enrollment to this phase II trial (NCT02879994) was ceased early due to lack of efficacy (5). In the TKI-resistant setting, response rates were even lower, with ORRs to ICB typically less than 5% (6–12). The mechanisms driving immunotherapy resistance in EGFR-mutant NSCLC are not well understood and are likely multi-factorial. In comparison with EGFR-wildtype tumors, EGFR-mutant tumors have been reported to have a lower tumor mutational burden and expression of PD-L1, factors implicated in ICB resistance (13). However, patients with EGFR-mutant NSCLC appear to still have significantly lower response rates (12) than patients bearing EGFR-wildtype tumors, even in tumors expressing high levels of PD-L1.

Tumor-derived immunomodulators such as cytokines are known to impact the tumor immune microenvironment (TIME) and immunotherapy response. We and others reported that IL6 is a potential mediator of resistance to first-generation TKIs (14, 15) that occurs independent of secondary EGFR mutations or MET amplification. Given the known immunomodulatory role of IL6, in the current report we sought to investigate whether IL6 promotes an immunologically inert phenotype in EGFR-mutant TKI-refractory tumors and whether blockade of IL6 might enhance the activity of ICB therapy in EGFR-mutant NSCLC tumors.

Here we report that IL6 inhibited the activation of T and natural killer (NK) cells in the EGFR-mutant tumor microenvironment. In genetically engineered mouse models (GEMM) of EGFR-mutant NSCLC, knockout (KO) of IL6 extended overall survival (OS) and enhanced immune cell infiltration. Moreover, IL6 null tumors had reduced regulatory T and Th17 T-cell populations, which are implicated as contributing to tumor immunosuppression. In addition, depletion of IL6 resulted in increased infiltration of activated NK cells in vivo, and acute blockade of IL6 further sensitized EGFR-mutant cell lines to NK-mediated cytotoxic killing. Blockade of IL6 in combination with anti-PD-1 treatment increased tumor infiltration by activated CD8 T cells and extended OS of tumor-bearing animals. Our findings indicate that blockade of IL6 signaling reduces tumor immunosuppression and enhances both antitumor immunity and responsiveness to anti-PD-1 immunotherapy within the ICB-resistant, EGFR-mutant subset of NSCLC.

Cell culture and reagents

NSCLC cell lines HCC4006, HCC827, and H1975 were obtained from ATCC and maintained as described previously (16). EGFR-TKI–resistant cell lines were generated as described previously (3). YUL-0019 (N771delinsFH; ref. 17) were obtained by Dr. Politi (Yale Medical School). MDA-L-011 (L858R; ref. 3), MDA-L-004K (EGFR exon 20 mutation), MDA-L-0024 (E746_A750del mutation), MDA-L-0046 (EGFR exon 19 del), and MDA-L-0065 (EGFR exon 19 del) were derived from patients at MD Anderson that progressed on EGFR-TKIs and were cell lines generated from surgical specimens collected after patients provided written informed consent through an Institutional Review Board–approved protocol at MD Anderson Cancer and conducted in accordance with the Declaration of Helsinki and Belmont report. All cell lines were cultured in RPMI medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% l-glutamine. IL6 neutralizing antibodies were obtained from R&D Systems. Erlotinib and siltuximab were obtained from the institutional pharmacy at the University of Texas MD Anderson Cancer Center (Houston, TX).

Flow cytometry

The following mouse flow cytometry–conjugated antibodies were used at the concentration suggested on the manufacturer's datasheet for surface or intracellular staining: Live/Dead Ghost UV450, anti-CD45 BUV805, anti-PD1 BV421, anti-CD8 BV570, anti-NKG2D FITC, anti-CD278 BV785, anti-IFNγ PerCP-Cy5.5, anti-PD-L1 PE-Dazzle 594, anti-NK1.1 Pe-CY5, anti-CD3 Pe-Fire700, anti-CD4 AlexaFluor700, anti-Ki-67 Pacific Blue, anti-FoxP3 Alexa532, anti-granzyme B PE, anti-IL17A APC all purchased from BioLegend or Thermo Fisher Scientific. For intracellular staining of FoxP3, granzyme B, IL17A, and Ki-67, the cells were fixed and permeabilized using cold 70% ethanol. Immunostained cell percentages were assessed by a Cytek Aurora flow cytometer and analyzed by Flow-Jo software.

Multiplex cytokine array

Mouse Luminex discovery assay was performed on mouse serum collected from blood of EGFRL858R GEMMs.

Detection of IL6 in preclinical samples

NSCLC cells (200,000 cells/well in 6-well plates) were plated in 10% FBS serum RPMI medium and after 24 hours were replaced with serum-free medium, and then conditioned medium was collected. IL6 ELISA (R&D Systems) was performed according to manufacturer's instructions. For IL6 staining, antibodies (1:50; Millipore) were used on formalin-fixed paraffin-embedded tumor sections. For inhibitor studies, cells were pretreated with human IL6 inhibitor as described previously (14).

Detection of IL6 in clinical samples

Biospecimens were obtained after patients provided written informed consent under an Institutional Review Board–approved protocol and conducted in accordance with the Declaration of Helsinki and Belmont report. The CROSSOVER and NORTHSTAR datasets include patients from MD Anderson who received osimertinib treatment and if applicable local consolidated therapy. We analyzed 12 matched pairs of samples collected prior to progression of disease (after randomization and end of cycle 2 of osimertinib treatment) and at progression of disease. Each sample was analyzed in duplicate and analysis of circulating IL6 concentration was performed. Groups were compared using a paired two-tailed Student t test.

RT-PCR

Total cellular RNA was isolated using TRIzol Reagent (Invitrogen) and RT-PCR was performed in triplicate biological samples.

RNA sequencing

Total RNA was collected from the EGFR-mutant NSCLC GEMMs listed above and extracted and purified using the RNeasy Plus Mini Kit (Qiagen). RNA sequencing (RNA-seq) libraries were prepared and analyzed in triplicate using the Illumina Mouse NovaSeq6000. Human EGFR-mutant NSCLC cell line gene expression data from the Gene Expression Omnibus (GEO) repository (GSE 121634) have also been utilized (3).

Functional immune cell assays

Human peripheral blood mononuclear cells (PBMC) from healthy donors purchased from the Gulf coast consortium were obtained from whole blood samples and isolated by Ficoll-Paque density gradient centrifugation. The ex vivo expansion and activation of NK cells used K562 feeder cells (1:2 ratio) and 200 U/mL IL2 and 5 ng/mL IL15, and 2 mmol/L GlutaMAX purchased from Gibco. Healthy donor-derived NK cells were cultured in RPMI medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% l-glutamine. Cells were cultured for 14 days and counted using trypan blue. To isolate T cells, PBMCs were activated with anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio in RPMI1640 complete media (10% FBS, 2 mmol/L GlutaMAX, 100 U/mL penicillin, and 100 μg/mL streptomycin) supplemented with 100 U/mL IL2 (PreproTech). To assess cytotoxicity in vitro, NK or T cells were cocultured with target cells stably transfected with pHIV-Luc-ZsGreen (Addgene plasmid # 39196) and plated at varying effector-to-target ratios (10:1, 5:1, 2.5:1, and 1:1). Cytotoxic killing was quantified by luciferase signal after 4 hours of incubation. This signal was quantified using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG Labtech). Specific lysis was calculated using [(Target cells only − Experimental Target)/(Target cells only − no target cells) × 100]. Similar assays were performed with T cells derived from healthy donors. Target cells for T-cell cytotoxicity assay were transduced with luciferase pHIV-Luc-ZsGreen (Addgene plasmid #39196) and custom OKT3 construct (pPSFG-OKT3-CD86).

In vivo studies

CCSP-rtTA EGFRL858R genetically engineered mice were obtained from Dr. Katherine Politi (Yale School of Medicine; ref. 18). Mice were crossed with IL6 knockout mice (IL6−/−) obtained from Dr. Seyed Moghaddam (MD Anderson Cancer Center) to generate CCSP-rtTA EGFRL858R/IL6−/− mice. After 6 weeks of doxycycline (DOX) diet treatment, animals were randomized into treatment groups and treated with a monoclonal antibody against IL6 (10 mg/kg) or anti-PD-1 (10 mg/kg), intraperitoneally, twice a week. Antibodies were purchased from Bioxcell. Tumors and serum from mice were harvested after 3 weeks.

IHC

Frozen tissue sections were used to evaluate EMT and immune cell markers. Specimens were sectioned (8–10 μmol/L thickness) and stained with antibodies against E-cadherin, vimentin, SP-C, and TTF-1 at 4°C overnight. Staining was visualized using an AxioCam MRC5 camera and Axio vision software 4.6.

Timer2.0 analysis

This immune deconvolution tool used RNA-seq data analysis to estimate immune infiltrates through comparison of multiple deconvolution analyses (19).

KMplotter analysis

A total of 1,926 patients with NSCLC from the KMplotter (Kaplan-Meier Plotter; https://kmplot.com/analysis/) lung cancer database were stratified across expression level of IL6 and evaluated for OS in months.

Statistical analysis

For all studies, statistical analysis was performed using the Student t test (two-tailed) or one-way ANOVA. A P value ≤ 0.05 was considered statistically significant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. For RNA-seq data, ANOVA was used on a gene by gene basis. The resulting P values, computed from F-statistic, were modeled using the beta-uniform mixture model and used to determine a FDR cutoff to identify significantly differentially expressed genes.

Data availability

Gene expression data from human EGFR-mutant NSCLC cell lines have been uploaded to the GEO repository (GSE 121634) as reported previously (3). Additional raw data for this study were generated at MD Anderson Cancer Center, and data related to this study are available from the corresponding author upon request.

IL6 secretion is elevated in human EGFR-mutant NSCLC cells with acquired TKI resistance

We and others have shown that EGFR-mutant NSCLC cells with acquired resistance to the first-generation EGFR inhibitor, erlotinib, overexpress IL6 (14, 15). To determine whether IL6 is similarly elevated in human NSCLC cell lines with acquired resistance to the third-generation EGFR-TKI osimertinib, we utilized a panel of osimertinib refractory cell lines previously shown to have undergone EGFR-TKI resistance (3) through EMT (3). In our models of EGFR-TKI resistance mediated by MET amplification, IL6 was not upregulated (14). Osimertinib-resistant (OR) variants secreted significantly greater levels of IL6 compared with EGFR-mutant parental cells HCC4006 (Fig. 1A; P = 0.0063) and H1975 (Fig. 1B; P = 0.0040). Likewise, IL6 RNA levels were upregulated in EGFR-TKI–resistant cells as compared with parental (Supplementary Fig. S1A). We tested IL6 secretion by YUL-0019 cells which harbor an exon 20 insertion (N771del insFH) and are sensitive to poziotinib along with their poziotinib-resistant variants that we previously reported had undergone EMT (20). YUL-0019 parental cells expressed low levels of IL6, whereas, poziotinib-resistant cells expressed significantly higher levels of IL6 (Supplementary Fig. S1B).

Figure 1.

EGFR-mutant NSCLC tumor cells with acquired resistance to an EGFR-TKI have increased levels of IL6. ELISA analysis of IL6 secretion by human EGFR-mutant HCC4006 (A) and H1975 (B) cells and their OR variants (P < 0.0063). C, IL6 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, OS of patients with EGFR mutation–positive NSCLC from the KMplotter lung cancer dataset with high versus low IL6 expression (n = 1,926, P < 0.0001). E, Circulating IL6 levels were measured in patients from CROSSOVER and NORTHSTAR studies stratified by clinical outcome of prior to progression of disease or after 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 1.

EGFR-mutant NSCLC tumor cells with acquired resistance to an EGFR-TKI have increased levels of IL6. ELISA analysis of IL6 secretion by human EGFR-mutant HCC4006 (A) and H1975 (B) cells and their OR variants (P < 0.0063). C, IL6 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, OS of patients with EGFR mutation–positive NSCLC from the KMplotter lung cancer dataset with high versus low IL6 expression (n = 1,926, P < 0.0001). E, Circulating IL6 levels were measured in patients from CROSSOVER and NORTHSTAR studies stratified by clinical outcome of prior to progression of disease or after progression of disease (n = 12 matched pairs run in duplicate, P = 0.0517). (mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

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Next, we sought to determine whether IL6 production was also upregulated in patient-derived models of EGFR-TKI resistance. Therefore, we generated cell lines from EGFR-mutant patients who progressed on EGFR-TKIs and evaluated IL6 secretion by ELISA. Patient-derived models of acquired EGFR-TKI resistance (MDA-L-004K, MDA-L0011, MDA-L-0024, MDA-L-0046, and MDA-L-0065) secreted higher amounts of IL6 as compared with EGFR-mutant TKI-naïve cells (Fig. 1C). We also observed increases in IL6 mRNA expression in tumors from EGFR-mutant, TKI-refractory patients as compared with TKI-naïve EGFR-mutant tumors from the MD Anderson GEMINI dataset (Supplementary Fig. S1C; P = 0.050), and these TKI-refractory specimens also displayed a mesenchymal phenotype as indicated by increased expression of ZEB1 and decreased expression of CDH1 (Supplementary Fig. S1D and S1E). To investigate whether high IL6 was associated with a worse clinical outcome in patients with NSCLC, we utilized the lung cancer KMplotter and found that high expression of IL6 was associated with a worse OS (HR = 1.48, P < 0.0001) than those with low expression of IL6 (Fig. 1D). Moreover, after investigating serum of patients enrolled in the MD Anderson CROSSOVER and NORTHSTAR clinical trial studies, we identified that circulating IL6 levels were higher in patients with progressive disease compared with blood from these same patients collected prior to progression of disease (Fig. 1E; P = 0.0517).

Depletion of IL6 increases OS and number of activated infiltrating lymphocytes in murine immunocompetent EGFR-mutant NSCLC tumor models

To evaluate the impact of IL6 on EGFR-mutant NSCLC tumor growth and the TIME, we utilized syngeneic, immune competent murine models. To do this, we crossed the DOX-inducible EGFRL858R GEMM (18) with IL6 knockout mice (Supplementary Fig. S2A and S2B). As expected, concentrations of IL6 were essentially eliminated in the serum and bronchoalveolar lavage fluid (BALF) in EGFRL858R/IL6tKO mice as compared with EGFRL858R mice (Fig. 2A). IHC staining validated the absence of IL6 protein in tumors from EGFRL858R/IL6tKO mice (Supplementary Fig. S2C). Depletion of IL6 in treatment-naïve EGFRL858R mice had a modest yet significant effect on survival (Fig. 2B; HR = 1.31, P = 0.0212). Interestingly, treatment-naïve EGFRL858R/IL6tKO tumors displayed increased necrotic area which may be indicative of increased cell death in the absence of IL6 (Supplementary Fig. S2D). To investigate the effects of IL6 on the TIME of EGFR-mutant tumors, we next analyzed EGFRL858R and EGFRL858R/IL6tKO tumors by RNA-seq data and performed immune cell deconvolution analysis. IL6 depletion resulted in increased NK-cell populations within the tumor (Fig. 2C; P = 0.0032). Moreover, knockout of IL6 increased the CD8+ T cell (P < 0.0001) and T follicular helper cell populations (P < 0.0001) while populations of regulatory T cells (P = 0.1004) decreased within the tumor (Fig. 2D). Flow cytometry analysis was performed to further analyze the presence of tumor-infiltrating immune cells in EGFR-mutant tumors with or without IL6. Total immune infiltration in tumors from EGFRL858R/IL6tKO trended toward a higher density compared with EGFRL858R (P = 0.06; Fig. 2E). Moreover, NK-cell (P = 0.0471) and T-cell (P = 0.0408) infiltration was significantly increased in EGFRL858R/IL6tKO tumors as compared with EGFRL858R tumors (Fig. 2E). Among T-cell subtypes, the CD4+ T-cell population was more notably increased with IL6 depletion (P = 0.0086) as compared with CD8+ T cells (P = 0.0595), and the Foxp3+ regulatory T cell population was not significantly altered.

Figure 2.

Depletion of IL6 increases the number of infiltrating lymphocytes and OS in EGFR-mutant NSCLC tumors. A, Expression of IL6 in the serum and BALF of EGFRL858R and EGFRL858R/IL6tKO tumor-bearing mice. B, Knockout of IL6 extended the survival of EGFRL858R tumor-bearing mice (P = 0.0212). C and D, Immune cell deconvolution analysis from RNA-seq data collected from control and IL6 knockout tumors from EGFRL858R mice showed a minor decrease in regulatory T 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 EGFRL858R tumors with or without anti-IL6 treatment as determined by flow cytometry. F, Kaplan–Meier analysis of EGFR-mutant GEMM treated acutely with monoclonal blocking antibody to IL6 (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 IL6. (mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 2.

Depletion of IL6 increases the number of infiltrating lymphocytes and OS in EGFR-mutant NSCLC tumors. A, Expression of IL6 in the serum and BALF of EGFRL858R and EGFRL858R/IL6tKO tumor-bearing mice. B, Knockout of IL6 extended the survival of EGFRL858R tumor-bearing mice (P = 0.0212). C and D, Immune cell deconvolution analysis from RNA-seq data collected from control and IL6 knockout tumors from EGFRL858R mice showed a minor decrease in regulatory T 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 EGFRL858R tumors with or without anti-IL6 treatment as determined by flow cytometry. F, Kaplan–Meier analysis of EGFR-mutant GEMM treated acutely with monoclonal blocking antibody to IL6 (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 IL6. (mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

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Given our finding that tumor immune infiltration was enhanced in EGFR-mutant tumors with IL6 knockout, we next assessed whether blockade of IL6 signaling could similarly alter the TIME. Tumors were initiated in EGFRL858R mice, and animals were randomized to receive control antibodies or anti-IL6 blocking antibodies. IL6 blockade significantly extended the OS of tumor-bearing animals (HR = 2.301, P = 0.0385; Fig. 2F). Moreover, consistent with the knockout experiment, we observed an increase in infiltration of NK and CD4+ T cells in the tumor microenvironment as determined by flow cytometry (Fig. 2G) in mice treated with IL6 blockade. Collectively, these data indicated that IL6 in TKI-naïve EGFR-mutant NSCLC tumors causes a reduction in NK and T lymphocytic infiltration, particularly in CD4+ T cells, which can be restored with IL6 blockade.

EGFR-mutant NSCLC tumors with acquired EGFR-TKI resistance associated with EMT display an immunologically cold phenotype and secrete IL6

As there are currently no established syngeneic murine models of EGFR-independent TKI resistance to facilitate the study of the TIME, we used an approach previously described for KRAS (21, 22) and other oncogenes of “extinguishing” the driver oncogene using an inducible system after tumors were established. We chose to investigate TKI resistance in this setting because due to the reduced tumor burden in syngeneic EGFR-mutant models, it is not likely that osimertinib resistance emerges with continuous dosing at therapeutic levels, although some investigators have used dosing of osimertinib for a limited period of time (e.g., 2 weeks) followed by drug discontinuation to evaluate posttumor regrowth (23). To establish this driver extinguishing model, we used the DOX-inducible EGFRL858R GEMM (18) with chow containing DOX to induce tumor growth (oncogene-dependent tumors; Fig. 3A). To develop oncogene-independent resistance in the EGFRL858R GEMM, we likewise induced expression using DOX-containing chow, and then after 6 weeks when lung tumors could be visualized by CT imaging, DOX chow was withdrawn in an effort to mimic the development of EGFR-TKI resistance that occurs independently of EGFR secondary mutations (oncogene-independent tumors; Fig. 3A). The withdrawal of DOX was initially associated with loss of mutant EGFR expression (Fig. 3B) and initial tumor shrinkage followed by resumed tumor growth (Supplementary Fig. S3A). To confirm that oncogene-independent tumors were EGFR-TKI–resistant, animals were treated with or without osimertinib for 2 weeks. As observed by CT imaging (Fig. 3C), osimertinib treatment led to a marked reduction in the volume of oncogene-dependent tumors (P = 0.0002), while oncogene-independent tumors increased in size (P = 0.0475; Fig. 3D).

Figure 3.

Oncogene-independent EGFR-TKI–resistant EGFR-mutant NSCLC tumors display an immunologically cold, mesenchymal phenotype. A, Timeline of induction or withdrawal of DOX 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. CT imaging (C) and associated quantification (D) 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 EMT phenotype. F, Oncogene-independent tumors show an increased expression of vimentin and loss of E-cadherin. G and H, Immune cell deconvolution analysis from RNA-seq 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 with oncogene-independent tumor-bearing mice. J, IL6 ELISA confirms elevated levels of IL6 in oncogene-independent model compared with oncogene dependent. (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 DOX 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. CT imaging (C) and associated quantification (D) 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 EMT phenotype. F, Oncogene-independent tumors show an increased expression of vimentin and loss of E-cadherin. G and H, Immune cell deconvolution analysis from RNA-seq 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 with oncogene-independent tumor-bearing mice. J, IL6 ELISA confirms elevated levels of IL6 in oncogene-independent model compared with oncogene dependent. (mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

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Next, we evaluated the molecular changes associated with acquired EGFR independence. After verification of the tumor by pathology (Supplementary Fig. S3B and S3C), tumor tissue from oncogene-independent and oncogene-dependent tumors was evaluated by RNA-seq and gene set enrichment analysis which revealed an enrichment in EMT-associated gene expression (Fig. 3E). The shift toward a mesenchymal phenotype in the oncogene-independent tumors was confirmed by IHC showing increased expression of vimentin and decreased expression of E-cadherin in oncogene-independent tumors compared with oncogene-dependent tumors (Fig. 3F). Previous studies have shown an association between a mesenchymal phenotype and a cold immune microenvironment in lung adenocarcinomas (24). Therefore, we hypothesized that oncogene-independent (mesenchymal) tumors may display a colder immune microenvironment compared with the oncogene-dependent (epithelial) tumors. Immune cell deconvolution analysis revealed significantly decreased CD8+ T cells (P = 0.0194), CD4+ T cells (P < 0.0001), regulatory T cells (P = 0.0061), total NK cells (P < 0.0001), and decreased activated NK cells (P = 0.0122) in oncogene-independent tumors as compared with oncogene-dependent tumors (Fig. 3G and H).

We next evaluated expression of 16 immunomodulatory-related cytokines in the serum from mice bearing oncogene-dependent and oncogene-independent tumors by multiplex ELISA and found that IL6 and IL9 were the most highly upregulated cytokines in oncogene-independent tumor-bearing animals as compared with oncogene-dependent tumor-bearing animals (Fig. 3I). Increased circulating levels of IL6 and tumor levels of IL6 in oncogene-independent mice was confirmed by ELISA assay and IHC, respectively (Fig. 3J; Supplementary Fig. S3D). Collectively, these data show that IL6 is upregulated in EGFR-mutant NSCLC tumors which have developed oncogene-independent, EMT-associated TKI resistance.

IL6 suppresses NK-cell activation in EGFR-mutant NSCLC tumors

IL6 signaling has been shown to impair NK activity in other cancer types (25). Thus, we next assessed whether IL6 modulates NK-cell proliferation in EGFR-mutant tumors. As determined by IHC, NK-cell proliferation was similar in EGFRL858R tumors with or without IL6 knockout (Fig. 4A). Likewise, treatment of EGFRL858R tumor-bearing mice with anti-IL6 antibodies did not affect NK-cell proliferation (Fig. 4B; Supplementary Fig. S4A). However, the number of activated NKG2D+ NK cells was increased by IL6 depletion or antibody-mediated IL6 blockade (Fig. 4C and D; Supplementary Fig. S4B). To further investigate the impact of IL6 on NK activation markers, NK cells isolated from healthy donor PBMCs were expanded ex vivo (Supplementary Fig. S4C) and then incubated in conditioned media collected from human HCC4006 EGFR-mutant cell lines and their OR variants alone or in combination with the IL6 antibody siltuximab. After 48 hours, NK cells were analyzed by flow cytometry. IL6 blockade enhanced expression of the NK activation marker NKG2D in NK cells cultured in conditioned media from HCC4006 OR cells (Fig. 4E), but not parental HCC4006 cells. Similarly, IL6 blocking antibodies increased granzyme B expression in NK cells incubated in HCC4006 OR4 and OR7 (Fig. 4F). However, perforin expression, which is an indicator of cytotoxic potential, was not altered in NK cells treated with OR cell conditioned media or anti-IL6 (Supplementary Fig. S4D). NK activation and inhibitory receptors can bind to corresponding ligands expressed on tumor cells. Thus, we investigated the effect of IL6 on tumor cell expression of NK receptor ligands. Treatment of HCC4006 OR cells with IL6 neutralizing antibodies increased expression of MICA, a NK activation ligand which typically binds to NKG2D+ NK cells (Fig. 4G), and enhanced expression of ULBP1, another NK activation ligand (Supplementary Fig. S4E), although expression of HLA-E, a NK inhibitory ligand which typically binds inhibitory NK cells, was not significantly different between parental and osimertinib-resistant cells with or without IL6 antibodies (Supplementary Fig. S4F).

Figure 4.

IL6 suppresses the activation of NK cells in EGFR-mutant NSCLC tumors. IL6 knockout (A) and acute blockade (B) of IL6 increases expression of Ki-67+ proliferating NK cells (P = 0.3501, P = 0.4698). C and D, Activated NKG2D+ NK cells (P = 0.1042, P = 0.0418). E and F, Acute blockade of IL6 of EGFR-mutant NSCLC cell lines cocultured 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 cells is increased with IL6 blockade. H, IL6 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 4.

IL6 suppresses the activation of NK cells in EGFR-mutant NSCLC tumors. IL6 knockout (A) and acute blockade (B) of IL6 increases expression of Ki-67+ proliferating NK cells (P = 0.3501, P = 0.4698). C and D, Activated NKG2D+ NK cells (P = 0.1042, P = 0.0418). E and F, Acute blockade of IL6 of EGFR-mutant NSCLC cell lines cocultured 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 cells is increased with IL6 blockade. H, IL6 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).

Close modal

We next assessed whether human EGFR-TKI–resistant cells that have acquired EGFR-TKI resistance through EMT-mediated mechanisms are sensitive to NK-mediated killing and whether inhibition of IL6 can enhance this effect. We hypothesized that IL6 blockade would enhance NK-mediated killing of TKI-resistant EGFR-mutant cells to a greater extent than TKI-naïve cells. Consistent with this hypothesis, we observed that while IL6 neutralizing antibodies did not impact NK-mediated killing of HCC4006 parental cells (low IL6 expressing), blockade of IL6 did sensitize EGFR-mutant TKI-refractory cell lines to NK-mediated cytotoxic killing (Fig. 4H), indicating that in EGFR-TKI–refractory tumors, secretion of IL6 impairs NK cytotoxicity, and IL6 blockade may at least partially restore NK cytotoxic potential.

IL6 inhibits T-cell activity in EGFR-mutant NSCLC tumors

T cells are prominent effector cells in antitumor responses, and IL6 has been shown to impair T-cell antitumor activity (26, 27). We evaluated T cells in tumors from murine EGFRL858R tumors with and without IL6. We observed an increase in activated CD8+IFNγ+ T-cell population in EGFRL858R/IL6tKO tumors as compared with EGFRL858R tumors (Fig. 5A; P = 0.038). Similarly, EGFRL858R tumor-bearing animals treated with IL6 antibodies also displayed a slight increase in the activated CD8+IFNγ+ T-cell population compared with control mice (Fig. 5B; Supplementary Fig. S5A; P = 0.060). IL6 has been shown to promote development of immunosuppressive Th17 T-cell subsets (28). We observed that the Th17 T-cell subpopulation was reduced in EGFRL858R/IL6tKO tumors as compared with EGFRL858 tumors (Fig. 5C; P = 0.048), and likewise Th17 T cells were reduced in EGFRL858R tumors treated with IL6 blocking antibodies (P = 0.032; Fig. 5D; Supplementary Fig. S5B). Next, we incubated T cells derived from healthy donor PBMCs (Supplementary Fig. S5C) with conditioned media from EGFR-mutant HCC4006 cells, or their OR variants, with or without the IL6 blocking antibody siltuximab. For T cells incubated with conditioned media from HCC4006 OR4 and OR7 cells, IL6 blockade enhanced expression of granzyme B, an indicator of T-cell activation and killing potential (Fig. 5E), but not perforin or Ki-67, which stimulate membrane disruption prior to cytotoxic killing and proliferation, respectively (Supplementary Fig. S5D and S5E).

Figure 5.

IL6 inhibits T cell–mediated antitumor response in EGFR-mutant NSCLC tumors. A–D, IL6 knockout increased CD8+IFNγ+ T cells (P = 0.0375) while IL6 blockade had slightly more modest effects (P = 0.0375). Th17 T-cell populations were significantly reduced in IL6 knockout tumors (P = 0.048) and those treated with IL6 blocking antibody (P = 0.032). E, Human T cells isolated from healthy donor PBMCs cocultured in conditioned media collected after the acute blockade of IL6 of EGFR-mutant NSCLC cell lines significantly increased expression of granzyme B expression. F, Anti-IL6 treatment increased T cell–mediated cytotoxicity of EGFR-mutant NSCLC cell lines. G, Anti-IL6 blockade induced increased survival of EGFR-mutant GEMMs treated with anti-PD-1 immunotherapy (P = 0.0161). H, Anti-IL6 and anti-PD-1 combination treatment increased 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).

Figure 5.

IL6 inhibits T cell–mediated antitumor response in EGFR-mutant NSCLC tumors. A–D, IL6 knockout increased CD8+IFNγ+ T cells (P = 0.0375) while IL6 blockade had slightly more modest effects (P = 0.0375). Th17 T-cell populations were significantly reduced in IL6 knockout tumors (P = 0.048) and those treated with IL6 blocking antibody (P = 0.032). E, Human T cells isolated from healthy donor PBMCs cocultured in conditioned media collected after the acute blockade of IL6 of EGFR-mutant NSCLC cell lines significantly increased expression of granzyme B expression. F, Anti-IL6 treatment increased T cell–mediated cytotoxicity of EGFR-mutant NSCLC cell lines. G, Anti-IL6 blockade induced increased survival of EGFR-mutant GEMMs treated with anti-PD-1 immunotherapy (P = 0.0161). H, Anti-IL6 and anti-PD-1 combination treatment increased 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).

Close modal

To assess the impact of IL6 on T cell–mediated cytotoxicity, we cocultured T cells with EGFR-mutant tumor cell lines transfected to express membrane-bound anti-CD3 (OKT3), which binds CD3 on T cells and facilitates T-cell killing independent of antigen-specific recognition. In EGFR-TKI–refractory cells, IL6 blockade significantly increased T cell–mediated killing (Fig. 5F); IL6 blockade did not, however, increase T cell–mediated killing against HCC4006 parental cells which secrete markedly lower levels of IL6.

Next, given that ICB therapy is driven by a T cell–mediated antitumor response, we assessed whether IL6 blockade could enhance the activity of ICB against EGFR-mutant tumors. Consistent with clinical observations, single-agent anti-PD-1 did not significantly improve OS in EGFRL858R syngeneic tumor-bearing mice (HR = 1.53, P = 0.310). Anti-IL6 monotherapy, on the other hand, significantly extended OS in EGFRL858R tumor-bearing mice (HR = 2.30, P = 0.039). Combination treatment with anti-IL6 with anti-PD-1 treatment even further increased OS (Fig. 5G; HR = 3.15, P = 0.016). Moreover, we observed an increase in the number of activated T cells (CD8+ PD-1+) in EGFRL858R tumors treated with the combination of anti-IL6 and anti-PD-1 (Fig. 5H; P = 0.047), suggesting that IL6 blockade may improve the efficacy of anti-PD-1 treatment, even in TKI-naïve models, in part by enhancing CD8 activation and IFNγ production (Fig. 5A, B, and H).

The cytokine IL6 is known to associated with TKI resistance and have multiple potentially immunomodulatory effects. Given these observations, we hypothesized that IL6 plays a role in the immunosuppressive, ICB-resistant immune microenvironment of EGFR-mutant NSCLC. Furthermore, we hypothesized that this effect would be more pronounced in the setting of EGFR-TKI resistance, which is the relevant clinical setting in which these patients typically receive ICB.

To test this hypothesis, we characterized the immune landscape of murine models of treatment-naïve and oncogene-independent EGFR-TKI resistance. In TKI-naïve EGFR-mutant tumors, the presence of IL6 was associated with reduced immune infiltration, which could partially be reversed by IL6 blockade. Depletion of IL6 increased the infiltration and activation of both T and NK cells. This in vivo observation was supported by the in vitro finding that the tumor cell killing capacity of T and NK cells could be enhanced by IL6 blockade. Finally, we demonstrate that in EGFR-mutant tumor models, blockade of IL6 enhances the efficacy of anti-PD-1 therapy.

The management of EGFR-TKI–refractory NSCLC is a major clinical challenge, and unfortunately, immunotherapy approaches have been unsuccessful in treating patients in the first-line setting (ORR 9%; ref. 5) and appear to be even less effective in the TKI-refractory setting (ORR 5%). In an earlier retrospective cohort, monotherapy treatment with anti-PD-1/PD-L1 inhibitors managed a response rate of only 3.6% in EGFR-mutant or ALK-positive patients compared with 23.3% in EGFR-wildtype and ALK-negative/unknown patients (12). A meta-analysis showed that single-agent ICB immunotherapy yielded worse results for patients with EGFR-mutant lung cancers compared with docetaxel (8), and in retrospective analyses PD-1/PD-L1 inhibitors demonstrated a median progression-free survival of 1.8 to 2.1 months (10, 11). Attempts have been made to combine ICB immunotherapy with EGFR-TKIs; however, the prevalence of severe side effects prohibited the further development of these immune and targeted therapy combination approaches (29). Studies are needed to identify factors that promote ICB immunotherapy resistance in the context of EGFR-mutant NSCLC and identify biologically based combinatorial ICB immunotherapy strategies for these patients. In the current report, we investigated the role of IL6 in driving ICB resistance in EGFR-mutant NSCLC.

IL6 is an inflammatory cytokine that has been associated with resistance to first-, second-, and third-generation EGFR-TKIs through investigations by our group and others (14, 15). Our previous study illustrated that IL6 levels were not markedly elevated in EGFR-mutant cells where EGFR-TKI resistance was associated with MET amplification or secondary EGFR mutations (14). In the phase III ZEST clinical study, high plasma levels of IL6 were associated with worse OS in unselected patients with NSCLC treated with the EGFR-TKI erlotinib (14). In addition, a recent study also observed a positive association between high levels of circulating IL6 with osimertinib—a third-generation EGFR-TKI—resistance (30). While the increased IL6 secretion observed in cells with acquired EGFR-TKI resistance has been shown to promote tumor cell survival and therapeutic resistance (14, 15, 30), the impact of elevated IL6 in EGFR EMT-driven TKI-resistant tumors on the TIME has not been fully appreciated. Moreover, given the recent shift of osimertinib to first-line treatment of patients with EGFR-mutant NSCLC, and the activity of osimertinib against the most common genomic mechanism of resistance to first- and second-generation TKIs (EGFR T790M secondary mutations), it is anticipated that EMT-mediated resistance will increase in frequency.

IL6 has pleiotropic effects on effector cells in the antitumor response, promoting CD4+ T-cell differentiation into regulatory T cells and inhibiting NK-cell cytotoxicity. IL6 has been implicated in promoting other immunosuppressive signaling pathways which may mediate resistance to ICB including the CD73-adenosine axis (31, 32). We previously reported that CD73 expression is upregulated in EGFR-mutant tumors compared with EGFR wildtype tumors (13). Therefore, it is feasible that in EGFR-TKI–resistant tumors IL6 upregulation may also drive an immunosuppressive microenvironment through CD73 signaling. Likewise, in EGFR-mutant murine models of lung cancer, combination treatment of durvalumab (anti-PD-L1) and oleclumab (anti-CD73) exhibited synergistic antitumor effects (33). This treatment combination is under investigation in patients with NSCLC including: oleclumab with osimertinib in NCT03381274 and durvalumab with oleclumab in the COAST (NCT03822351; ref. 34) and NEOCOAST (NCT03794544; ref. 35) trials. Therefore, we sought to investigate the role of IL6 in the immunosuppressive EGFR-mutant microenvironment, which is highly refractory to ICB immunotherapy. Because responses to ICB response are heavily dependent on immune effector cells, we investigated the effects of IL6 on T- and NK-cell function in EGFR-mutant NSCLC.

In the current report, we utilized an immune-competent model of EGFR-mutant NSCLC in which expression of the mutant EGFR transgene could be extinguished to characterize oncogene-independent, EGFR-TKI resistance. We find that unlike models studying tumor regrowth after discontinuing osimertinib (36, 37), this model is resistant to osimertinib dosing causing regression in the oncogene-dependent models, with tumors demonstrating EMT and increased IL6 levels consistent with clinical resistance.

We find that the elevated expression of IL6 associated with TKI resistance reduces the cytotoxic potential of T and NK cells in the EGFR-mutant NSCLC microenvironment. Moreover, IL6 increased the protumorigenic Th17 T-cell population and reduced infiltration of cytotoxic CD8+ T cells. In addition, IL6 reduced expression of activation markers like granzyme B and NKG2D on tumor-infiltrating NK cells and by in vitro assays, IL6 reduced the sensitivity of EGFR-mutant TKI-resistant cells to NK-mediated cytotoxicity. Consistent with our findings, studies in other malignancies have observed a similar role of IL6 on impairing the function of infiltrating NK cells in the tumor microenvironment (25). In addition, other work has highlighted that mesenchymal tumors may be more sensitive to NK-mediated cytotoxic killing than epithelial tumors (38). These findings support our preclinical observation that NK-mediated cytotoxic killing occurs in the setting of EGFR-TKI–resistant tumors treated with IL6 blockade.

Previous studies in lung adenocarcinoma have highlighted a strong association between the mesenchymal status of tumors and a highly inflammatory tumor microenvironment (24). Currently, there are limited clinical approaches to overcome EMT-driven oncogene-independent TKI resistance. Here, by utilizing both cell line and GEMMs, we observed that IL6 expression is enhanced in EGFR-mutant tumor cells which acquired EMT-associated EGFR-TKI resistance. These data indicate that IL6 may be an actionable target to mitigate its role in promoting EMT-driven immunosuppression.

Using an EGFR-mutant model of NSCLC, we show that blockade of IL6 is more effective when used in combination with anti-PD-1 treatment, which resulted in an increase in OS. Together, these studies show that blockade of IL6 favorably modulates the TIME and enhances response to ICB predominantly through activation of T and NK cells. Future studies should investigate the effect of IL6 on macrophage infiltration given that myeloid cell immunosuppressive function and its subsequent effect on regulatory T cells and CD8 T cells.

Our previous studies identified a novel targetable mechanism of EGFR-TKI resistance by which stress hormones activate β2-adrenergic receptors and acutely upregulates IL6. The current report elucidates an alternative mechanism of IL6 upregulation which is EMT associated. Therefore, both stress hormone signaling and EMT may be independent drivers of IL6-mediated immunosuppression, and blockade of β-adrenergic receptors may also suppress an IL6-mediated immunosuppressive tumor microenvironment. Moreover, others have reported that metformin can block IL6 expression and EMT in EGFR-TKI–resistant cells (39). Given that beta blockers and metformin are inexpensive and well tolerated, the testing of these agents in combination with EGFR-TKIs and ICB should be considered. Agents targeting IL6 are clinically available as FDA-approved therapies. While in previous studies blocking IL6 did not reverse TKI resistance when tested on EGFR-mutant cells in vitro or in vivo using immunocompromised mouse models, the results reported here indicate that IL6 blockade may, in part, reduce immunosuppression in the tumor environment. These findings support further investigation of IL6 targeting in EGFR-TKI–refractory NSCLC as a therapeutic approach which may sensitize tumors to ICB therapy.

M.B. Nilsson reports personal fees from Spectrum Pharmaceuticals outside the submitted work. X. Le reports grants and personal fees from EMD, Janssen, and Eli, as well as personal fees from AstraZeneca, Spectrum, Blueprint, Daiichi, Novartis, Boehringer Ingelheim, Hengrui Therapeutics, and AbbVie outside the submitted work. Y.Y. Elamin reports personal fees from Blueprint, Spectrum Pharmaceuticals, BMS, Turning Point Therapeutics, AstraZeneca, Takeda, and Sanofi outside the submitted work. S.J. Moghaddam reports grants from Arrowhead Pharma and Boehringer Ingelheim outside the submitted work. T. Cascone reports personal fees from Society for Immunotherapy of Cancer, Roche, Medscape, IDEOlogy Health, PeerView, Merck & Co., Genentech, Arrowhead Pharmaceuticals, and Regeneron; grants, personal fees, and other support from Bristol Myer Squibb, MedImmune/AstraZeneca, and EMD Serono; and other support from Dava Oncology and Boehringer Ingelheim outside the submitted work. M. Curran reports grants and personal fees from Immunogenesis, Inc., as well as personal fees from Alligator Bioscience, Inc., ImmunOs, Inc., Oncoresponse, Inc., Nurix, Inc., Aptevo, Inc., Kineta, Inc., Xencor, Inc., and AstraZeneca, Inc. outside the submitted work. D.L. Gibbons reports grants and personal fees from AstraZeneca, Sanofi, and Astellas; personal fees from Menarini Recherche, Eli Lilly, 4D Pharma, and Onconova; and grants from Janssen, Takeda, Ribon Therapeutics, NGM Biopharmaceuticals, Boehringer Ingelheim, and Mirati Therapeutics outside the submitted work. J.V. Heymach reports personal fees and other support from AstraZeneca, Boehringer Ingelheim, Spectrum, and Takeda, as well as personal fees from EMD Serono, Catalyst, Genentech, GlaxoSmithKline, Hengrui Therapeutics, Eli Lilly, Sanofi, Mirati Therapeutics, Bristol Myers Squibb, BrightPath Biotherapeutics, Janssen Global Services, Nexus Health Systems, Pneuma Respiratory, RefleXion, and Chugai Pharmaceuticals outside the submitted work. No disclosures were reported by the other authors.

S.A. Patel: Conceptualization, resources, data curation, software, formal analysis, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. M.B. Nilsson: Conceptualization, supervision, funding acquisition, project administration, writing–review and editing. Y. Yang: Conceptualization, data curation, methodology. X. Le: Data curation, funding acquisition. H.T. Tran: Data curation, validation. Y.Y. Elamin: Data curation, validation. X. Yu: Data curation, validation, methodology. F. Zhang: Data curation, investigation. A. Poteete: Resources, investigation. X. Ren: Resources, investigation. L. Shen: Software, formal analysis. J. Wang: Software, formal analysis, supervision. S.J. Moghaddam: Conceptualization, resources, writing–review and editing. T. Cascone: Conceptualization, resources, formal analysis, supervision. M. Curran: Conceptualization, investigation, methodology, writing–review and editing. D.L. Gibbons: Conceptualization, resources, investigation, writing–review and editing. J.V. Heymach: Conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, project administration, writing–review and editing.

This research was supported by the Emerson Collective, Lung SPORE grant 5 P50 CA070907, 1R01 CA190628, 1R01 CA234183-01A1, Stading Fund for EGFR inhibitor resistance. S.A. Patel was supported by the CPRIT Training Award (RP210028), the Dr. John J. Kopchick Award, and the Schissler Foundation Award. The authors would like to thank the MD Anderson Flow Cytometry Core for supporting this project. The authors would like to also thank Dr. Katerina Politi for providing the YUL-0019 and EGFRL858R mouse model utilized in this article.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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