EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer

Ke Gong; Gao Guo; Nishah Panchani; Matthew E Bender; David E Gerber; John D Minna; Farjana Fattah; Boning Gao; Michael Peyton; Kemp Kernstine; Bipasha Mukherjee; Sandeep Burma; Cheng-Ming Chiang; Shanrong Zhang; Adwait Amod Sathe; Chao Xing; Kathryn H Dao; Dawen Zhao; Esra A Akbay; Amyn A Habib

doi:10.1038/s43018-020-0048-0

. Author manuscript; available in PMC: 2020 Dec 1.

Published in final edited form as: Nat Cancer. 2020 Apr 6;1(4):394–409. doi: 10.1038/s43018-020-0048-0

EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer

Ke Gong ¹, Gao Guo ¹, Nishah Panchani ¹, Matthew E Bender ², David E Gerber ^3,^4,⁵, John D Minna ^3,^6,⁷, Farjana Fattah ⁴, Boning Gao ^6,⁷, Michael Peyton ^6,⁷, Kemp Kernstine ⁸, Bipasha Mukherjee ⁹, Sandeep Burma ⁹, Cheng-Ming Chiang ^4,^6,¹⁰, Shanrong Zhang ¹¹, Adwait Amod Sathe ⁵, Chao Xing ^5,^12,¹³, Kathryn H Dao ¹⁴, Dawen Zhao ¹⁵, Esra A Akbay ^2,⁴, Amyn A Habib ^1,^4,^16,^*

¹Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas TX 75390

²Department of Pathology, University of Texas Southwestern Medical Center, Dallas TX 75390

³Department of Internal Medicine, Division of Hematology-Oncology, University of Texas Southwestern Medical Center, Dallas TX 75390

⁴Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas TX 75390

⁵Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas TX 75390

⁶Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas TX 75390

⁷Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas TX 75390

⁸Department of Cardiovascular and Thoracic Surgery, University of Texas Southwestern Medical Center, Dallas TX 75390

⁹Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas TX 75390

¹⁰Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas TX 75390

¹¹Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas TX 75390

¹²Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas TX 75390

¹³Department of Bioinformatics at the University of Texas Southwestern Medical Center, Dallas TX 75390

¹⁴Baylor Research Institute, Dallas TX 75204

¹⁵Departments of Biomedical Engineering and Cancer Biology, Wake Forest School of Medicine, Winston Salem, NC 27157, USA

¹⁶VA North Texas Health Care System, Dallas TX 75216

Author Contributions

KG, GG, and AAH designed experiments. KG, GG, NP, MEB, SZ, and EAA performed or assisted with experiments. DEG, JDM, FF, BG, MP, KK, and KHD provided cell lines, PDX or human tissue specimens. KG, GG, DEG, BM, SB, C-MC, AAS, CX, KHD, DZ and AAH analyzed data. KG and AAH wrote the manuscript with contributions from GG, JDM, and C-MC. Study conception and supervision: AAH.

To whom correspondence should be addressed at: University of Texas Southwestern Medical Center, North Texas VA Healthcare System, Mail Code 151, 4500 South Lancaster Road, Dallas TX 75216, Phone: (214) 857-3610, Amyn.Habib@UTSouthwestern.edu

PMCID: PMC7706867 NIHMSID: NIHMS1637068 PMID: 33269343

Abstract

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. Here, we demonstrate that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. We propose that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients.

Introduction

Interferon regulatory factor 3 (IRF3) plays a central role in innate immunity. IRF3 is a transcription factor that is expressed constitutively and in response to viral infection, and induces the transcription of type I interferons¹. IRF3 is activated in response to cytosolic recognition of nucleic acids or tissue damage by a number of pattern recognition receptors (PRRs)². In response to viral infection, IRF3 becomes phosphorylated leading to its dimerization and nuclear translocation, leading to induction of Type I interferons and orchestration of the antiviral response³. IRF3 is activated by the TANK-binding kinase TBK1 and by IKKε⁴. TBK1 is ubiquitously expressed and activated in response to activation of pattern recognition receptors (PRRs) and associated adaptor signaling proteins such as RIG-I/MAVS, cGAS-STING, and TLR3/4-TRIF⁵.

Activation of IRF3 results in production of Type I interferons, cytokines essential for generating antiviral responses and activating innate immunity⁶. Type I interferons include interferon-α and interferon-β and bind to the IFNAR, composed of IFNAR1 and IFNAR2 chains. Type I interferons play a tumor suppressive role. Indeed, IFNs have been used for treating certain types of cancer (kidney cancer, melanoma, chronic myeloid leukemia) and are thought to function through multiple mechanisms including promotion of anti-tumor immunity, anti-angiogenesis, promoting inflammation in the tumor microenvironment and a direct role in suppression of proliferation and apoptosis in tumor cell ^7–9. Importantly, homozygous deletion of genes is common in 9p21.3, the locus for the type I interferon gene cluster. Homozygous deletion of the type I interferon genes is widespread in cancer, including about 10% of NSCLC¹⁰. Importantly, Type I IFN loss confers a worse prognosis in multiple cancer types¹⁰.

The EGFR is widely expressed in non-small cell lung cancer (NSCLC) and is an important target in NSCLC^11–13. EGFR inhibition using tyrosine kinase inhibitors (TKIs) is highly effective initially in the subset of patients with EGFR-activating mutations, who comprise about 10–15% of NSCLC patients in Western populations¹⁴. However, the majority of NSCLC tumors express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. Nevertheless, EGFR ligands are commonly expressed in lung cancer^15,16. Furthermore, a constitutive overexpression-induced EGFRwt signaling has also been reported ^17–19. Thus, it is possible that EGFRwt could play an oncogenic role in lung cancer¹⁵. Moreover, secondary resistance inevitably develops in initially responsive EGFR mutant tumors¹¹. The major mechanisms of resistance to EGFR inhibition in NSCLC include EGFR mutations such as the T790M mutation ²⁰ and activation of other RTKs such as Met²¹. In addition, EGFR inhibition triggers a rapid adaptive response in NSCLC that likely contributes to secondary resistance, but may also induce primary resistance to treatment ^11,22. This adaptive response is broad and may involve bypass signaling pathways such as STAT3 or NF-κB ^23,24. We recently reported that a rapid TNF-driven adaptive response plays a key role in resistance to EGFR inhibition in NSCLC²⁵ and in glioma^26–28. In this study we examine an adaptive response to EGFR inhibition in NSCLC mediated by upregulation of Type I IFNs.

Results

EGFR inhibition leads to activation of Type I interferon signaling in NSCLC

To better understand the early adaptive response to EGFR inhibition we undertook RNA sequencing in the EGFRwt/KRas mutant A549 cells following exposure to erlotinib. The transcriptional response to EGFR inhibition is quite broad and affects a large number of genes (Fig. 1A). Pathway analysis revealed that a Type I IFN gene signature was prominent among the signaling changes triggered by EGFR inhibition in these cells (Fig. 1B, and Table 1). EGFR inhibition in multiple NSCLC lines harboring EGFR mutant or EGFRwt with KRas mutation or other genetic alterations resulted in an upregulation of Type I interferons as determined by qPCR (Fig. 1C–F and Extended Data Fig. 1A–X) and by ELISA (Fig. 1G–H, and Extended Data Fig. 2A–R). We also found upregulation of Type I IFNs in HCC827 and A549 xenografts and in PDX models of EGFRwt/KRas mutant and EGFR mutant NSCLC when erlotinib was administered to tumor bearing mice (Extended Data Fig. 2S–Z). The level of type I IFN receptor was unchanged in response to erlotinib (Extended Data Fig. 1Q). We also detected an upregulation of interferon gamma in response to erlotinib, but IFN gamma siRNA knockdown did not result in a synergy with EGFR inhibition in cell survival assays (Extended Data Fig. 2AA–MM).

A. A549 cells were treated with 1 µM erlotinib for 0, 2, and 24 hours. Three biologically independent RNA samples per group were sequenced. R-package DESeq2 was used to calculate the fold-change and p-value (unadjusted). Up-regulated and down-regulated genes were identified by the cutoff of p<0.05. Volcano plot shows the distribution of differentially expressed genes. B. The heatmap shows the up-regulated genes at 24 hours in the Reactome pathway “Antiviral mechanism by IFN-stimulated genes” within Table 1 pathways, representing mean values of normalized log-ratio between untreated and treated group. **C-F.** HCC827 and A549 cells were treated by 0.1 or 1 µM erlotinib respectively, and then qPCR was performed to detect IFNα1 and IFNβ1 mRNA. ATCB (β-Actin) expression was the loading control. **G-H.** HCC827 and A549 were treated with 0.1 µM or 1 µM erlotinib for two days, the concentration of IFNα1 and IFNβ1 was measured in supernatants by ELISA. **I-M.** HCC827 and A549 cells were transfected with IFNAR1 or control siRNA for 48h and then exposed to 0.01 µM or 1 µM erlotinib for 72h, or concurrently treated with 0.01 µM or 1 µM erlotinib, together with 10 µg/mL anifrolumab for 72h, followed by AlamarBlue assay. Western blot confirming the silencing of IFNAR1. **N-Q.** Four EGFR mutant cell lines were treated by 0.1 µM erlotinib and exogenous IFNα1 or IFNβ1 at 50 ng/mL for 72h followed by AlamarBlue assay. R. HCC827 cells were plated in a 96-well plate with 0.1 µM erlotinib and/or 10 µg/mL anifrolumab and cultured for extended periods as indicated. When cells reach 100% confluence, they were considered resistant. For experiments (C-F, I-J, L-Q) and experiment (R), n=3 and 20 technical replicates, respectively, representative of 3 independent repeats with similar results. Data (G-H) represent mean ± S.E.M, n=3 independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001, by two-tailed two-sample Student’s t-test (G-H). Western blots are cropped and representative of three independent experiments with similar results. Uncropped images are in Source_Data_Fig.1. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.1.

Table 1. Erlotinib-induced pathways.

Following RNAseq described in Figure 1A, pathway analysis was performed via Gene Set Enrichment Analysis (GSEA), from http://software.broadinstitute.org/gsea/index.jsp. The top erlotinib upregulated pathways are presented, ranked by normalized enrichment scores (NES), with numbers of upregulated genes and p-values.

Erl-induced Pathway	UP	NES	p	FDR-q
Asparagine N-linked Glycosylation	35	5.82	0.001	0.001
Antiviral Mechanism by IFN Stimulated Genes	31	5.47	0.001	0.001
Unfolded Protein Response	32	5.39	0.001	0.001
Transport of Mature Transcript to Cytoplasm	28	5.35	0.001	0.001
Telomere Maintenance	28	4.97	0.001	0.001
Respiratory Electron Transport	23	4.93	0.001	0.001
Metabolism of Non-Coding-RNA	26	4.84	0.001	0.001
Cleavage of Growing Transcript in the Termination Region	19	4.68	0.001	0.001
Meiotic Synapsis	28	4.65	0.001	0.001
Activation of NF-kappa-B in B cells	36	4.53	0.001	0.001

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Biological significance of Type I IFN upregulation by EGFR inhibition in NSCLC

Next, we found that siRNA knockdown of IFNAR1, the receptor for Type I interferons, resulted in enhanced sensitivity to EGFR inhibition in EGFR mutant NSCLC (Fig. 1I, K, and Extended Data Fig. 3A–D). In addition, silencing of IFNAR1 conferred sensitivity to EGFR inhibition in resistant EGFRwt/KRas cell lines (Fig. 1J–K, Extended Data Fig. 3E–H). We also examined the effect of anifrolumab, a monoclonal antibody directed against IFNAR that inhibits the binding of Type I IFNs to its receptor and is in clinical trials for lupus²⁹. Anifrolumab enhanced sensitivity to erlotinib in EGFR mutant NSCLC lines (Fig. 1L, Extended Data Fig. 3I–K) and in multiple resistant EGFRwt/KRas cell lines (Fig. 1M, Extended data Fig. 3L–N). Additionally, IFN inhibition resulted in sensitivity to erlotinib in EGFRwt NSCLC cell lines with Ros1 mutation, EML4/ALK fusion, Met amplification. and Braf mutation (Extended Data Fig. 3O–W).

Next, we found that exogenous Type I IFNs protect from erlotinib-induced cell death in oncogene addicted cells in cell survival assays (Fig. 1N–Q). We found that prolonged culture in the presence of erlotinib²⁵ alone resulted in the emergence of resistant cells. However, a combined exposure to erlotinib plus anifrolumab inhibited the development of secondary resistance (Fig. 1R).

Although, the Type I interferons have a known role in cytotoxicity, they also have a pro-survival role and mediate resistance to radiation and chemotherapy, primarily through STAT1 activation^7,8,30–35. We found that EGFR inhibition results in activation of STAT1 in multiple EGFR wild type and EGFR mutant NSCLC cell lines (Figure 2A–F). Furthermore, anifrolumab or siRNA knockdown of IFNAR1 blocks erlotinib induced activation of STAT1 confirming that erlotinib induced Type I IFN upregulation is required for STAT1 activation. (Fig. 2G–J and Extended Data Fig. 4A–H). We also found that siRNA knockdown of STAT1 synergizes with EGFR inhibition in cell survival assays (Fig. 2K–M and Extended data Fig. 4I–N) and also abrogates the ability of exogenous Type I IFNs to rescue cells from EGFR inhibition induced cell death in EGFR mutant NSCLCs (Fig. 2N–Q). Thus, STAT1 activation provides a mechanistic explanation for the pro-survival effect of Type I IFNs in the context of EGFR inhibition.

**A-F.** Three EGFR mutant (HCC827, H3255, and HCC2279), as well as three EGFRwt (A549, H441, H1573), were treated with 0.1 or 1 µM erlotinib for the indicated time points. Cell lysates were collected and subjected to Western blot for detection of total and phosphorylated STAT1 expression. β-Actin was the loading control. **G-H.** HCC827 and A549 cells were concurrently treated by 0.1 or 1 µM erlotinib for 24h or the indicated time points, with or without 10 µg/mL anifrolumab. **I-J.** HCC827 and A549 cells were transfected with IFNAR1 or control siRNA for 48h, followed by 0.1 or 1 µM erlotinib for 24h or the indicated time points. Western blot was performed to detect total and phosphorylated STAT1. **K-M.** HCC827 and A549 cells were transfected with STAT1 or control siRNA for 48h, followed by indicated doses of erlotinib for 72h, and then cell viability was measured by AlamarBlue assay. STAT1 siRNA was confirmed by Western blot. **N-Q.** Three EGFR mutant cells were transfected with STAT1 or control siRNA for 48h, and then cells were concurrently treated by 0.1 µM erlotinib and exogenous IFNα1 or IFNβ1 at 50 ng/mL as indicated for 72h followed by AlamarBlue assay. STAT1 siRNA was confirmed by Western blot. For experiments (K-L, N-P), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are cropped and representative of three independent experiments with similar results. Uncropped Western blots are shown in Source_Data_Fig.2. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.2.

EGFR inhibition results in activation of a TBK1/IRF3 signaling axis in NSCLCs with EGFR activating mutations

The transcription factor IRF3 plays a central role in transcription of Type I interferons. IRF3 is activated by TBK1. EGFR inhibition led to a rapid and robust activation of TBK1 and IRF3 in NSCLC cell lines harboring EGFR-activating mutations (Fig. 3A–B, Extended Data Fig. 5A–B) and in animal models (Fig. 3C–D). However, EGFR inhibition-induced TBK1 or IRF3 activation was not detected in EGFRwt cell lines, or in EGFRwt animal models (Fig. 3E, Extended Data Fig. 5C–E). Similarly, increased IRF3 transcriptional activity was detected upon EGFR inhibition in multiple EGFR mutant NSCLC cell lines but not in EGFRwt NSCLC cell lines (Fig. 3F–G, Extended Data Fig. 5F–K). We found that siRNA knockdown of TBK1 could not abrogate the ability of exogenous Type I IFNs to rescue EGFR mutant cells from EGFR inhibition induced cell death in EGFR presumably because TBK1 is upstream of IFN signaling (Extended Data Fig. 5L–O). We confirmed that pharmacological inhibition of TBK1 using BX-795 or siRNA knockdown of TBK1 results in a loss of EGFR inhibition-induced IRF3 phosphorylation (Extended Data. Fig. 5P–U). In addition, there is a loss of erlotinib-induced IRF3 transcriptional activity in multiple EGFR mutant cell lines in response to chemical or biological inhibition of TBK1 (Extended Data Fig. 5V–BB). IKKε is another kinase involved in the activation of IRF3 but does not appear to be expressed in NSCLC cell lines (Extended Data Fig. 5CC).

**A-B.** Two EGFR mutant lines were treated with 0.1 µM erlotinib for the indicated time points followed by collection of lysates and Western blot. C. Nude mice bearing HCC827 xenografts were treated with erlotinib 50 mg/kg for 1–14 days followed by removal of tumors and Western blot. D. A similar experiment was performed with HCC4190 PDX in NOD-SCID mice. Mice treated with erlotinib 50 mg/kg for 1–14 days. E. Erlotinib (1 µM) was used to treat A549 cells for different time points followed by Western blot. **F-G**. HCC827 and A549 cells were transfected with ISRE-Luc or IFI27-Luc reporters for 48 hours, then treated with erlotinib at 0.1 or 1 µM for 24h followed by a luciferase assay. **H-I.** HCC827 cells were transfected with the indicated TBK1, IRF or control siRNA for 48 hours, and then treated with 0.01 µM erlotinib for 72 hours followed by AlamarBlue assay. J. HCC827 cells were transfected with IRF3 expressing plasmid or empty vector for 48 hours, and then incubated with 0.1 µM erlotinib for 72 hours. Cell viability was detected by AlamarBlue assay and overexpression of IRF3 was confirmed by Western blot. **K-L.** AlamarBlue cell survival assays were conducted with multiple stably silenced HCC827 TBK1 and HCC827 IRF3 clones, with or without erlotinib 0.1 µM. Gene silencing was confirmed by Western blot in K-L. M. Animal experiments with stably silenced TBK1 or IRF3. Eight nude mice per group were injected with HCC827 shTBK1 (clone #37), shIRF3 (clone #35), or control lentivirus-infected stable cells per group and the rate of tumor formation was 5–7 as shown in Source_Data_Fig.3. Mice received 6.25 mg/kg/d erlotinib or Vehicle by oral gavage. Tumor sizes were monitored as described in the methods section and representative tumor images are shown. For experiments (F-L), n=3 technical replicates, representative of 3 independent repeats with similar results. Data (M) represent mean ± S.E.M. of animal tumor sizes. The rate of tumor formation was 5–7 per group as shown in the Source_Data_Fig.3 (n=5–7). #: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001, by two-way ANOVA adjusted by Bonferroni’s correction (M). Western blots are cropped and representative of three independent experiments with similar results. Uncropped Western blots are shown in Source_Data_Fig.3. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.3.

IRF3 and TBK1 activation protect EGFR mutant cells from a loss of EGFR signaling

We found that pharmacological inhibition of TBK1 with BX-795 or siRNA knockdown of TBK1 synergized with EGFR inhibition in multiple NSCLC cell lines with mutant EGFR in cell viability assays (Fig. 3H, and Extended Data Fig. 6A–H). Also, siRNA knockdown of IRF3 synergizes with EGFR inhibition in multiple NSCLC cell lines with mutant EGFR in cell viability assays (Fig. 3I and Extended Data Fig. 6I–L). Conversely, overexpression of IRF3 in EGFR mutant cells results in resistance to EGFR inhibition-induced cell death in EGFR mutant NSCLC cell lines (Fig. 3J and Extended Data Fig. 6M–O). Next, we used shRNA to stably silence TBK1 in EGFR mutant cell lines (Fig. 3K). We confirmed that cells with stable silencing of TBK1 or IRF3 were sensitized to EGFR inhibition (Fig. 3K–L). Next, HCC827 cells with shTBK1, shIRF3, or with control shRNA were injected into the flanks of mice to form subcutaneous tumors. Once tumors became visible, treatment was started with control vehicle, or erlotinib. Stable silencing of TBK1 or IRF3 resulted in enhanced sensitivity of xenografted HCC827 and PC9 tumors to erlotinib (Fig. 3M and Extended Data Fig. 6P–R).

TBK1 undergoes a TRIM32 dependent K63-linked ubiquitination in response to EGFR inhibition

To understand the mechanism of EGFR inhibition dependent TBK1 activation, we analyzed proteins that associate with TBK1 in response to EGFR inhibition using mass spectrometry (Fig. 4A). We chose Tripartite motif-containing protein 32 (TRIM32) for further investigation as a potential candidate in the activation of TBK1. TRIM32 has an E3-ubiquitin ligase activity, and has previously been reported to have a role in Type I interferon induction in response to viral infection ^36,37 TRIM32 forms a complex with TBK1 in response to erlotinib in EGFR mutant NSCLC cell lines (Fig. 4B–D). TRIM32 can mediate K63-linked ubiquitination resulting in specific pathway activation. Importantly, several studies have reported a key role for K63-linked ubiquitination in TBK1 phosphorylation and activation^38–40. We found that EGFR inhibition leads to K63-linked ubiquitination of TBK1 in NSCLC cells (Fig. 4B–D). Importantly, siRNA knockdown of TRIM32 inhibits EGFR inhibition-induced K63 ubiquitination of TBK1 (Fig. 4E–G) and phosphorylation (Fig. 4H–J). These data indicate that TRIM32 is required for TBK1 and IRF3 activation in response to EGFR inhibition.

A. HCC827 cells were treated with 0.1 µM erlotinib for 0, 2, 6, and 24 hours. Cell lysates were immunoprecipitated by TBK1 antibody and Mass spectrometry was performed. The heatmap indicates the proteins that binds to TBK1 after 24h of erlotinib treatment with an affinity increase of over two folds. **B-D.** Three EGFR mutant cell lines were treated with 0.1 µM erlotinib for 0, 2, 6, and 24 hours followed by preparation of cellular lysates. This was followed by immunoprecipitation with a TBK1 antibody and Western blot with TRIM32, K63-Ubiquitin, and TBK1 antibodies. TRIM32 and β-Actin from the input samples were also tested. **E-G.** Cells were transfected with TRIM32 siRNA or control siRNA for 48 hours. This was followed by treatment with 0.1 µM erlotinib for 24 hours, followed by immunoprecipitation with TBK1 antibodies and Western blot with K-63 ubiquitin or TBK1 antibody as described. **H-J**. EGFR mutant cell lines with silenced TRIM32 were treated with erlotinib (24h) followed by Western blot with pTBK1 or pIRF3 antibodies. Western blots are cropped and representative of three independent experiments with similar results. Uncropped Western blots are shown in Source_Data_Fig.4.

Upregulation of RIG-I but no activation of STING or Ahr in response to EGFR inhibition

Retinoic acid inducible gene I (RIG-I) is a pattern sensing receptor that plays a key role in sensing RNA viruses⁴¹. We found that RIG-I is strongly induced by EGFR inhibition in multiple EGFR mutant NSCLC cell lines (Fig. 5A–D). Also, siRNA knockdown of RIG-I blocked TBK1 and IRF3 activation in response to EGFR inhibition (Fig. 5E–H). Furthermore, a loss of RIG-I enhances the sensitivity of EGFR mutant cell lines to erlotinib in cell survival assays (Fig. 5I–N). These data suggest that RIG-I is upregulated in response to EGFR inhibition and leads to activation of IRF3 culminating in resistance to EGFR inhibition.

**A-D.** EGFR mutant cells were treated with 0.1 µM erlotinib for the time points indicated followed by Western blot with RIG-I and β-Actin antibodies. **E-H.** Cells were transfected with RIG-I siRNA or control siRNA for 48h and then exposed to 0.1 µM erlotinib for 24h followed by Western blots. **I-N** Cells were transfected with RIG-I siRNA or control siRNA for 48h and then exposed to 0.01 µM erlotinib for 72h and cell viability was tested using AlamarBlue assay. RIG-I siRNA interference was confirmed by Western blot. For experiments (I-J, L-M), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are cropped and representative of three independent experiments with similar results. Uncropped Western blots are shown in Source_Data_Fig.5. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.5.

cGAS/STING activates IRF3 leading to induction of Type I IFNs⁴². STING is upregulated in EGFR TKI persister cells⁴³. However, we were unable to detect increased phosphorylation of STING in response to erlotinib in either EGFR mutant or EGFRwt cell lines (Extended Data Fig. 7A–F). We found that siRNA knockdown of STING decreased the basal level of Type I IFNs in EGFR mutant and EGFRwt NSCLC cell lines. However, the erlotinib induced upregulation of IFNs does not require STING in any of the cell lines tested (Extended Data Fig. 7G–V). Finally, siRNA knockdown of STING does not synergize with EGFR inhibition in cell survival assays (Extended Data Fig. 7W–BB).

The aryl hydrocarbon receptor (Ahr) has been implicated as a mechanism of resistance to EGFR TKIs in EGFR mutant NSCLC ⁴⁴. We found that Ahr levels are not altered in response to erlotinib (Extended Data Fig. 8A–D). Ahr is localized to the nucleus when activated⁴⁵. We did not detect erlotinib-induced nuclear localization of Ahr (Extended Data Fig. 8E–J), suggesting that Ahr activation is not a component of the adaptive response to EGFR inhibition. It certainly remains possible that Ahr may modulate the sensitivity to EGFR inhibition⁴⁴.

Regulation of PD-L1 by EGFR inhibition.

Although EGFR inhibition leads to decreased levels of PD-L1 in EGFR mutant lung cancer cell lines⁴⁶, immunotherapy is not effective in EGFR mutant NSCLC^47,48. Since type I IFNs were reported to regulate PD-L1 expression⁴⁹, we examined the PD-L1 expression upon erlotinib treatment. Interestingly, while a high concentration of erlotinib leads to decreased PD-L1 levels (Extended Data Fig. 8K–M), low concentrations of erlotinib leads to upregulation of PD-L1 in EGFR mutant NSCLC cell lines and in animal tumors (Extended Data Fig. 8N–Q). In EGFRwt NSCLC lines we do not detect an increase in PD-L1 levels in response to erlotinib (Extended Data Fig. 8R–Y). Importantly, this erlotinib-induced upregulation of PD-L1 in EGFR mutant NSCLCs can be abrogated by anifrolumab or siRNA knockdown of IFNAR1 (Extended Data Fig. 8Z–CC), suggesting a possible explanation for why immunotherapy with PD1 inhibitors is not effective in EGFR mutant NSCLC⁴⁸. We also examined on some other PD-1 pathway members and found only PD-L1 level was affected by EGFR inhibition (Extended Data Fig. 8DD–EE). Thus, prior treatment with erlotinib may result in elevated PD-L1 levels in EGFR mutant tumors and render them resistant to immunotherapy. Such resistance could potentially be averted by the use of anifrolumab.

The mechanism of EGFR inhibition-induced IFN induction is distinct in EGFR mutant and EGFRwt NSCLC

EGFR inhibition in EGFR mutant NSCLCs results in activation of a TBK1-IRF3 signaling axis Also, pharmacological inhibition of TBK1 using BX-795 or siRNA knockdown of TBK1 or IRF3 completely suppressed erlotinib-induced upregulation of IFN in EGFR mutant cell lines (Fig. 6A–B, Extended Data Fig. 9A–L) but not in EGFRwt cells (Fig. 6C–D, Extended Data Fig. 9M–S). Thus, EGFR inhibition-induced IFN upregulation requires TBK1/IRF3 in EGFR mutant but not in EGFRwt NSCLC lines. Previous studies have reported that EGFR inhibition leads to a rapid activation of NF-κB in both EGFR mutant and EGFRwt NSCLC^24,25. We found that a pharmacological inhibitor of NF-κB (BMS-345541) or a dominant negative IkBα mutant blocks EGFR inhibition-induced upregulation of IFN in EGFRwt expressing NSCLC cell lines (Fig. 6E–F, Extended Data Fig. 9T–EE) but not in EGFR mutant lines (Extended Data Fig. 9FF–OO). Next, we found that Etanercept, a specific TNF blocker, or siRNA knockdown of TNFR1 failed to inhibit erlotinib induced upregulation of type I IFNs in EGFR mutant lines, while it efficiently blocked TNF-induced activation of NF-κB (Extended Data Fig. 10A–BB).

A. HCC827 cells were treated with 1 µM BX795 and 0.1 µM erlotinib for 24h followed by qPCR for IFNB1 mRNA. B. HCC827 cells were transfected with siRNA for TBK1 or IRF3 for 48h. Then cells were exposed to 0.1 µM erlotinib for 24h followed by qPCR for IFNB1 mRNA. Western blot showing silencing of TBK1 and IRF3. **C-D**. Similar experiments were performed on A549 cells with erlotinib (1 µM). E. A549 cells were concurrently treated with 1 µM erlotinib and 0.1 µM BMS-345541 for 24h followed by qPCR for IFNB1 mRNA. F. A549 cells were infected with IκBα-DN/GFP adenoviruses for 24h followed by exposure to erlotinib (1 µM) for 48h and qPCR for IFNB1 mRNA. Western blot demonstrating expressing of mutant IκBα. **G-I.** RNA and protein samples from HCC827 cell line (parent) and its derived secondary erlotinib-resistant lines (ER3, ER4A, ER4B, ER5) were collected and analyzed by qPCR for IFNA1 and IFNB1 mRNA shown as three independently conducted experiments, or by Western blot shown as one representative of three experiments independently repeated with similar results. **J-S.** HCC827 derived ER3, ER4A, ER4B, and ER5 were concurrently treated with 10 µg/mL anifrolumab, or pre-transfected with IFNAR1 siRNA for 48 hours, then treated with erlotinib for 72 hours followed by AlamarBlue assay. IFNAR1 siRNA was confirmed by Western blot. **T-U.** H1975 cells were treated with 0.1µM Afatinib for indicated time points, IFNA1 and IFNB1 mRNA levels were detected by qPCR. **V-X.** H1975 cells were co-treated with 10 µg/mL anifrolumab, or pre-transfected with IFNAR1 siRNA for 48h and then treated with 0.1µM Afatinib for 72 hours followed by AlamarBlue assay. IFNAR1 siRNA was confirmed by Western blot. For experiments (A-H,J-M, O-R, and T-W), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are cropped and representative of three independent experiments with similar results. Uncropped Western blots are shown in Source_Data_Fig.6. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.6.

To investigate whether TNF-NF-κB signaling plays a role in the pro-survival effects of Type I IFNs in the context of EGFR inhibition, we examined whether etanercept is able to inhibit the pro-survival effect of exogenous Type I IFNs (shown in Fig. 1N–Q). Addition of etanercept or TNFR1 siRNA fails to block the pro-survival effects of IFNs in the presence of EGFR inhibition (Extended Data Fig. 10CC–II). Similarly, chemical inhibition of NF-κB using BMS-34551, or biological inhibition of NF-κB using a dominant negative IKBα mutant fail to abrogate the protective effect of type I IFNs in the context of EGFR inhibition (Extended Data Fig. 10JJ–PP).

The role of IFN signaling in secondary resistance to EGFR inhibition

To examine the role of Type I IFN signaling in NSCLCs in secondary resistance to EGFR inhibition, we examined NSCLC EGFR mutant cell lines rendered experimentally resistant to EGFR TKIs by two independent groups^50,51. Four independent clones were analyzed. We found that Type I IFN levels are high in all clones (Fig. 6G–H). The T790M mutation or Met amplification were not detected in these lines^50,51. Also, we found that TBK1/IRF3 is activated in these cell lines (Fig. 6I). Importantly, it is possible to restore sensitivity to erlotinib in these cell lines if IFN is inhibited using anifrolumab or TNFR1 siRNA (Fig. 6J–S). We also examined H1975 cells that have dual L858R/T790M mutations and found that type IFNs can be induced by afatinib (Fig. 6T–U). Furthermore, these cells are rendered sensitive to afatinib if IFN signaling is blocked (Fig. 6V–X). These data suggest that the TBK1-IRF3-IFN pathway activation may be an independent mechanism of secondary resistance to EGFR inhibition in NSCLC.

Type I interferons regulate sensitivity to EGFR inhibition in animal models

Next, we examined whether a combined inhibition of Type I IFNs and EGFR would influence sensitivity to erlotinib in mouse xenograft models. The Type I interferon receptor IFNAR is composed of two subunits IFNAR1 and IFNAR2. HCC827 cells with stable silencing of IFNAR1 or control shRNA were generated and tested in cell viability assays followed by injection into the flanks of athymic mice to form subcutaneous tumors (Fig. 7A). Once tumors became visible, treatment was started with control vehicle or low dose erlotinib. While low dose erlotinib failed to inhibit the growth of control tumors, there was a significant suppression of tumor growth in the IFNAR1 silenced group (Fig. 7A). Next, A549 cells with stable silencing of IFNA1R or control shRNA were generated and tested by cell viability assays followed by injection into the flanks of athymic mice to form subcutaneous tumors (Fig. 7B). While erlotinib failed to inhibit the growth of control tumors, there was a significant suppression of tumor growth in the IFNAR silenced group (Fig. 7B). Next we examined whether a combination of EGFR plus IFN inhibition acts synergistically controlling tumor growth in an EGFR mutant NSCLC PDX model (L858R, HCC4190). We used anifrolumab, a monoclonal antibody directed against the IFNAR, in this experiment. While erlotinib alone produced a minor suppression of tumor growth that was not statistically significant, a combination of erlotinib plus anifrolumab results in a significant suppression of tumor growth (Fig. 7C). We also found the combination of erotinib plus anifrolumab to be highly effective in inhibiting the growth of an EGFRwt/KRas mutant PDX model HCC4087 (Fig. 7D).

A. HCC827 cells were stably infected with lentivirus control shRNA (shCtrl) or shRNA for IFNAR1 lentivirus and silencing was confirmed by Western blot. Silenced clones were studied in AlamarBlue cell survival assays following erlotinib exposure for 72h. Cells with stable silencing of IFNAR1 (clone #3) or control shRNA were subcutaneously injected into 8 nude mice per group. The rate of tumor formation was 5–8 per group as shown in the Source_Data_Fig.7 (n=5–8). Erlotinib was administered orally at 6.25 mg/kg/day. Tumor sizes were monitored as described in the Methods section. Representative tumor images are shown. B. A similar experiment was performed with A549 xenografts (shIFNAR1 clone #2). Eight nude mice were injected per group and the rate of tumor formation was 5–8 as shown in the Source_Data_Fig.7 (n=5–8). Erlotinib was used at 100 mg/kg/d. C. HCC4190 EGFR mutant PDX was subcutaneously implanted on NOD-SCID mice. Eight nude mice were implanted per group and the rate of tumor formation was 7–8 as shown in the Source_Data_Fig.7 (n=7–8). Mice were orally treated with 6.25 mg/kg/day erlotinib and/or i.p. injected with 2 mg/kg/day anifrolumab, a monoclonal IFNAR1 antibody. D. A similar PDX experiment was performed with HCC4087, which harbor mutant KRAS and wild-type EGFR. Eight nude mice were injected per group and all 8 mice formed tumors (n=8). Erlotinib was used at a dose of 100 mg/kg/d. E. KRAS LSL-G12D transgenic mice were generated as in the Methods section, and randomly divided into 4 groups (n=3–4 as the number of dots), receiving vehicle, oral erlotinib of 100 mg/kg/day, i.p. injection of mouse anti-mouse IFNAR1 antibody at 3 mg/kg/day, and combination administration of erlotinib plus IFNAR1 antibody for 28 continuous days. Bi-weekly MRI scanning was used to monitor tumor growth. Tumor sizes were calculated by ImageJ. Representative MRI images are shown, n=3–4 as indicated by the number of dots (mice). The tumors grow as diffuse lung opacities and “H” refers to heart. Data (A-E, *in vivo*) refers to mean ± S.E.M. of tumor sizes (n as above), *: p<0.05, **:p<0.01, ***:p<0.001, by two-way ANOVA adjusted by Bonferroni’s. For *in vitro* experiments (A-B), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are cropped and representative of three independent repeated experiments with similar results. Uncropped are in Source_Data_Fig.7. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.7.

Because the type I interferons play a key role in innate immunity, we also used a well-established immunocompetent transgenic mouse model of KRas mutant lung cancer that is driven by Adeno-CMV-Cre-mediated induction of KRas G12D expression⁵². These tumors express EGFRwt and previous studies have shown that ErbB receptors play an important role in driving the growth of KRas mutant tumors^53,54. Since anifrolumab is specifically directed against the human IFNAR, we used a Type I IFN neutralizing antibody directed against the mouse IFNAR. Once tumors were detected by magnetic resonance imaging (MRI) following adenovirus administration, treatment was started with control vehicle, erlotinib, IFN antibody, or erlotinib plus IFN antibody followed by periodic MRI imaging. While there is robust tumor growth in control, erlotinib alone, and IFN antibody treatment groups, a combination of erlotinib plus IFN antibody was highly effective in suppressing growth of these tumors resulting in significantly diminished tumor growth compared to other groups (Fig. 7E). Thus, a combination of EGFR+ IFN inhibition is an effective treatment strategy in this immunocompetent model.

The IFN adaptive response to EGFR inhibition in NSCLC

Next, we investigated whether EGFR inhibition induces a Type I interferon upregulation in tumor tissue derived from patients. We examined 13 TKI naive and 9 TKI treated NSCLC patients and found that erlotinib treated patients had higher Type I IFN levels compared to TKI naïve patients (Fig. 8A–B). Our experimental data indicate that a high level of Type I IFN protects cancer cells from the effects of EGFR TKIs. Thus, we examined tissue from a cohort of EGFR mutant NSCLC patients treated at UT Southwestern with EGFR TKIs. We measured Type I IFN levels in paraffin-embedded pre-treatment tumor tissue by qPCR and examined the effect of IFN levels on prognosis. Consistent with our experimental data, we found that increased levels of IFNA1 mRNA or IFNB1 mRNA confer resistance to EGFR TKIs and a worse prognosis in these patients (Fig. 8C–D). Importantly, an analysis of TCGA data also reveals a correlation between high Type I IFN DNA copy number and worse prognosis in EGFR mutant NSCLC (Fig. 8E). Furthermore, expression of IFN target genes may also correlate negatively with prognosis in EGFR mutant tumors (Fig. 8F–J). These data support our model that Type I IFN upregulation plays an important role in resistance to EGFR inhibition in NSCLC (Fig. 8K).

**A-B.** IFNA1 and IFNB1 mRNA was detected by qPCR from 23 NSCLC patients’ FFPE tissues (13 untreated and 10 TKI-treated), collected from UT Southwestern Medical Center and The Jackson Laboratory. Data represents median ± IQR, **: p<0.01, ***: p<0.001, by Kolmogorov-Smirnov test (KS test). **C-D.** We examined IFNA1 and IFNB1 mRNA from 30 advanced (stages IIIB & IV) NSCLC patients at UT Southwestern with either one of two classical TKI-sensitive mutations, L858R or exon 19 deletion, but without T790M mutation. We examined IFNA1 and IFNB1 mRNA by qPCR from these 30 patients’ FFPE tumor obtained before TKI-treatment. They were divided into high-50% and low-50% (n=15) by relative values and we examined the effect of IFN level on overall survival. E. 42 EGFR activating mutant NSCLC patients from TCGA-LUAD database were divided into high-50% and low-50% groups by IFNB1 DNA copy numbers and the effect on survival was examined. **F-J.** 41 EGFR activating mutant NSCLC patients (one patient has DNAseq but lacks RNAseq data) from TCGA-LUAD database were divided into high-50% and low-50% groups by mRNA levels of these erlotinib-induced Type I IFN target genes and the effect on survival was examined. Kaplan-Meier Overall-Survival curves (C-E) were drawn and compared by Log-rank test and Gehan’s test. K. A schematic representation of the EGFR inhibition induced Type I interferon driven adaptive response in NSCLC. Numerical source data for the experiments in this figure can be found in Source_Data_Fig.8.

Discussion

In this study we report that EGFR inhibition reprograms cellular signaling and results in a remarkable cooptation of antiviral signaling pathways in NSCLC. This antiviral response mediates resistance to EGFR inhibition in both EGFR wild type and EGFR mutant NSCLC. NSCLC cells respond to EGFR inhibition with a rapid increase in Type I interferon levels and the IFN upregulation was detected in all NSCLC cell lines examined, in animal tumor tissue, and in archival tissue from patients. In EGFRwt expressing NSCLCs, the increase in IFNs is sufficient to protect cells from loss of EGFR signaling. In NSCLCs with EGFR-activating mutations the IFN driven adaptive response is only partially protective and observed after treatment with low concentrations of EGFR inhibitors. This is also true for other adaptive bypass signaling mechanisms such as STAT3, or TNF-NF-κB that are triggered by EGFR inhibition in EGFR mutant NSCLC ^23–25 and do not inhibit the initial clinical response in patients but may play a role in the development of secondary resistance. Importantly, exogenous IFNα or IFNβ via activation of STAT1 protects NSCLC cells with mutant EGFR activating from cell death resulting from EGFR inhibition, further supporting an important role for Type I interferons in mediating resistance to EGFR inhibition in NSCLC.

The adaptive response to inhibition of RTK pathways is broad and leads to substantial reprogramming of signaling pathways that attempt to restore homeostasis ^22,55–60. However, targeted inhibition of one or a small number of pathways may cripple the adaptive response and overcome therapeutic resistance in such cancers. Here we show that a combined inhibition of EGFR and type I IFN signaling is highly effective in suppressing the growth of NSCLC tumors in multiple animal models.

Although most patients with EGFR activating mutations initially respond to EGFR TKIs, they inevitably develop resistance, implying the persistence of subsets of cancer cells. Primary or intrinsic resistance to inhibition of EGFRwt could occur because an adaptive response prevents cell death in response to EGFR inhibition. Currently the EGFRwt does not appear to be a useful target for treatment, because EGFR inhibition is ineffective in EGFRwt expressing NSCLC. However EGFRwt is widely expressed and recent studies suggest that targeting the EGFR signaling network inhibition may also hold promise in EGFRwt/KRas NSCLC ^25,53,54. We propose that the primary resistance to EGFR inhibition in EGFRwt NSCLC does not necessarily indicate that EGFR signaling is irrelevant to the malignant phenotype. Rather, EGFR inhibition may not work in because an adaptive survival mechanism triggered by EGFR inhibition negates its effect. A combined inhibition of EGFR+adaptive response either unmasks a requirement for EGFR signaling for survival and/or sets up synthetic lethal conditions.

EGFR inhibition results in an increase in Type I IFN levels via distinct mechanisms depending on whether EGFR is mutant or wild type (Extended Data Fig. 9a). In EGFR mutant tumors, a RIGI-TRIM32-TBK1-IRF3 axis mediates induction of IFNs and resistance to EGFR inhibition. We show that TRIM32, an E3-ubiquitin ligase, associates with TBK1 upon EGFR inhibition leading to K-63 linked ubiquitination of TBK1. TRIM32 is required for EGFR inhibition induced TBK1 and IRF3 phosphorylation and resistance to EGFR inhibition. In contrast, inhibition of EGFRwt tumors, upregulates Type I interferons via an NF-κB dependent pathway.

In a previous study we demonstrated that inhibition of the EGFR in lung cancer cells resulted in a rapid increase in TNF secretion via an effect on TNF mRNA stability mediated by miR-21and leading to a NF-κB driven survival pathway that protected cells from a loss of EGFR signaling²⁵. Here, we show that EGFR inhibition results in a distinct adaptive mechanism that activates an anti-viral signaling pathway mediated by upregulation of Type I IFNs. A combined inhibition of EGFR and type I interferons using the clinically available antibody anifrolumab enhances the effectiveness of EGFR inhibition in EGFR mutant cells and is able to overcome the primary resistance of EGFRwt NSCLC including the subset with KRas mutant. Intriguingly, we also found that EGFR inhibition may lead to an upregulation of PD-L1 via an interferon dependent pathway, providing a possible explanation for the failure of immunotherapy in EGFR mutant NSCLC, and the possibility that anifrolumab may render such tumors responsive to immunotherapy.

Our data indicate that Type I IFN signaling is a major targetable mechanism of resistance to EGFR TKI inhibition in EGFR mutant and EGFRwt NSCLC. The chromosomal locus for the Type I interferon genes is 9p23.1, one of the most common sites for homozygous deletions of tumor suppressive genes. Homozygous deletion of Type I IFN genes has been reported in multiple tumor types and in about 10% of NSCLC, and correlates with a worse prognosis¹⁰. However, our data indicate that in the context of EGFR inhibition, type I interferons mediate therapeutic resistance and confer a worse prognosis. We examined the combined effect of Type I IFN and EGFR inhibition is synergistic in highly resistant EGFRwt/KRas mutant models and in EGFR mutant models when a low concentration of erlotinib is used. These findings provide a therapeutic opportunity. Targeting a biologically significant upregulation of Type I interferons upon EGFR inhibition could greatly expand the reach and impact of EGFR targeted treatment in NSCLC. Thus, inhibiting the EGFR with a combination of TKI plus an IFN inhibitor such as the FDA approved anifrolumab may be effective in the treatment of NSCLCs that express EGFRwt. In tumors with EGFR activating mutations, a combined treatment with EGFR and IFN inhibition may result in a more effective elimination of tumor cells during the initial treatment and perhaps eliminate or delay secondary resistance to TKI treatment, and may also be useful in treating secondary resistance.

Methods

Cell lines:

A549 and U87MG cells were purchased from American Type Culture Collection (ATCC). HCC827/ER3, HCC827/ER4(A), and HCC827/ER5⁵¹ were obtained from Dr. Trever Bivona, University of California (San Francisco, CA). HCC827/ER4(B)⁵⁰ and were obtained from Dr. Eric Haura, Moffitt Cancer Center (Tampa, FL). All other NSCLC cell lines were from the Hamon Center for Therapeutic Oncology Research at the University of Texas Southwestern Medical Center. NSCLC cells were cultured in RPMI-1640 containing 5% FBS, and U87MG in DMEM medium with 10% FBS. Cell lines were authenticated by DNA fingerprints for cell-line individualization using Promega StemElite ID system, a short tandem repeat (STR)–based assay, at UT Southwestern genomics core. Cells were tested for mycoplasma contamination using an e-Myco kit (Boca Scientific).

Western blot, antibodies, plasmids and reagents:

Western blot and immunoprecipitation were performed according to standard protocols⁶¹. Western blot results are representative of at least 3 independent experiments. EGFR (06-847) antibody was from EMD Millipore (Billerica, MA); p-EGFR (Tyr1068) (2236), p-TBK1 (Ser172) (5483), TBK1 (3504), IKKε (2905), IRF3 (11904), K63-Ub (12930), RIG-I (3743), STAT1 (9172), p-STAT1 (Tyr701) (9167), AhR (83200), LAMIN A/C (4777), STING (3337), p-STING (Ser366) (19781), PD1 (86163), PD-L1 (13684), PD-L2 (82723) and IκBα (4814) antibodies were from Cell Signaling Technology (Danvers, MA); IFNAR1 (sc-7391), IFNGR1 (sc-12755), TNFR1 (sc-8346), and β-Actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX); TRIM32 (Mab6515) antibody was from R&D (Minneapolis, MN); p-IRF3 (Ser386) (ab76493) antibody was from Abcam (Cambridge, MA).

Recombinant human IFNα1 (z02866) was purchased from Genscript (Piscataway, NJ); IFNβ1 (300-02BC) and TNFα (300-01A) was obtained from PeproTech (Rocky Hill, NJ). Mouse anti-mouse IFNAR1 antibody (BE0241) was purchased from Bioxcell (West Lebanon, NH). Anifrolumab, an anti-IFNAR1 antibody was obtained from Creative-Biolabs (Shirley, NY) (TAB-722). Entanercept (Enbrel), a fusion protein of TNF receptor and IgG, was purchased from Mckesson Medical Supply (San Francisco CA). NF-κB inhibitor BMS-345541 was obtained from MilliporeSigma (Burlington, MA). LPS (19661), TBK1 inhibitor BX795, and EGFR inhibitor erlotinib and afatinib for in vitro studies were obtained from Cayman Chemical (Ann Arbor, MI). Erlotinib for animal treatment was purchased from LC Laboratories (Woburn, MA). pCMV2-IRF3 plasmid was a kind gift from Dr. John Hiscott (McGill University, Montreal, Canada). NFκB luciferase reporter plasmid was provided by Dr. Ezra Burstein (UT Southwestern).

Cell viability assay:

Cell viability assays were conducted with AlamarBlue Cell Viability Reagent from ThermoFisher (Waltham, MA), following the manufacturer’s protocol. Cells were cultured in Corning (Corning, NY) 96-well black plates with clear bottom and detected by the POLARstar Omega Microplate Reader (BMG LABTECH) (excitation at 544 nm and emission at 590 nm). At least 3 independent experiments were done. In oncogene addicted EGFR mutant cells, a lower dose of erlotinib 0.01 µM was used in cell viability assays to detect synergistic effects of combination therapy while a higher dose of 0.1 µM was used to detect the protection from erlotinib-induced cell death when IFNα or IFNβ were used.

Luciferase assays:

Cells were plated in 48 well dishes followed by plasmid transfection with two reporters detecting activation of IRF3, ISRE-luciferase reporter or IFI27-ISRE luciferase reporter¹⁸, using lipofectamine 2000. A dual-luciferase reporter assay system was used according to the instructions of the manufacturer (Promega, Madison WI). Firefly luciferase activity was measured in the POLARstar Omega Microplate Reader (BMG LABTECH) and normalized based on Renilla luciferase activity. Three independent experiments were done in triplicate.

Real-time PCR:

Total RNA was isolated by TRIzol Reagent (Fisher Scientific). cDNA Reverse Transcription was performed by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR primers were synthesized by IDT (Coralville, IA). Each PCR reaction was carried out in triplicate in a 20µl volume using SYBR Green Master Mix (Applied Biosystems) for 15 minutes at 95°C for initial denaturing, followed by 40 cycles of 95°C for 15 s and 60°C for 60s in ViiA 7 Real-Time PCR System (Applied Biosystems). At least three independent experiments were done. Values for each gene were normalized to expression levels of ACTB (β-Actin) mRNA. Primer sequences were as follows. IFNA1: 5’-GTGAGGAAATACTTCCAAAGAATCAC-3’ (forward), 5’-TCTCATGATTTCTGCTCTGACAA-3’ (reverse); IFNB1: 5’-AGCTGAAGCAGTTCCAGAAG-3’ (forward), 5’-AGTCTCATTCCAGCCAGTGC-3’ (reverse); IFNG: 5’-GGGTAACTGACTTGAATGTCC-3’ (forward), 5’-TTTTCGCTTCCCTGTTTTAG-3’ (reverse); ACTB: 5’-CATGTACGTTGCTATCCAGGC-3’ (forward), 5’-CTCCTTAATGTCACGCACGAT-3’ (reverse).

RNAseq:

RNA sequencing was performed at UT Southwestern Genomics and Microarray Core Facility. Total RNA was isolated by TRIzol Reagent (Fisher Scientific). RNA quality was determined by Agilent 2100 Bioanalyzer (RIN > 8), and quantity was measured by Qubit fluorometer. 1 µg RNA was then prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. Poly-A RNA (mRNAseq) is purified and fragmented before strand specific cDNA synthesis. cDNA are then a-tailed and indexed adapters are ligated. After adapter ligation, samples are PCR amplified and purified with Ampure XP beads, then run on the Illumina NextSeq 500/550 system (Kits V2.5) with 75 bp single end reads to product about 25 Million reads per sample.

Sequencing data were further processed at UT Southwestern Bioinformatics Core Facility. Differential expression was analyzed by DESeq2. Pathway analysis was performed based on Gene Set Enrichment Analysis (GSEA, http://software.broadinstitute.org/gsea/index.jsp).

ELISA (Enzyme-linked immunosorbent assay):

To detect IFNα and IFNβ levels in medium, cells were cultured in serum free medium and treated with indicated drugs for 48 hours. Supernatant was then collected and concentrated using a Pierce protein concentrator (Thermo-Fisher). To test IFNα and IFNβ in lysates, cell and tumor lysates were extracted using RIPA buffer. Total protein concentrations were determined by Pierce BCA Protein Assay Kit (Fisher Scientific). Then, the levels of IFNα and IFNβ protein were measured by ELISA using human IFNα ELISA kit (41100) and human IFNβ ELISA kit (41440), from PBL Assay Science (Piscataway, NJ) according to the manufacturer’s protocol.

Immunofluorescent staining:

Cells were cultured on coverslips in plates, after treatment cells were fixed with 4% Paraformaldehyde (PFA) followed by cell membrane permeabilization in 0.5 % Triton X-100/PBS and blocked in 1% BSA/PBS. The primary anti-AhR antibody (Santa Cruz Biotechnology, sc-101104, 1:200) was incubated at 4°C overnight, followed with Alexa555-conjugated secondary antibodies (Cell Signaling, 4413, 1:500) at room temperature for 2 hours. Cell nuclei were counterstained with DAPI (Invitrogen) at 0.1 μg/ml. The cells were examined using fluorescence microscope.

Mass Spectrometry:

HCC827 cells were treated with 0.1 µM erlotinib for 0, 2, 6, and 24 hours. Cell lysates were immunoprecipitated with TBK1 antibody. Antibody enriched protein samples were run on SDS-PAGE gels and submitted to UT Southwestern Proteomics Core Facility for Mass Spectrometry. Protein gel pieces were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma–Aldrich. The samples then underwent solid-phase extraction cleanup with Oasis HLB microelution plates (Waters) and the resulting samples were analyzed by LC/MS/MS, using an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex). Samples were injected onto a 75 μm i.d., 50-cm long EasySpray column (Thermo), and eluted with a gradient from 1–28% buffer B over 60 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.55 kV and an ion transfer tube temperature of 275 °C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2–7. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation.

Raw MS data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3. Peptide identification was performed using the X!Tandem and open MS search algorithm (OMSSA) search engines against the human protein database from Uniprot, with common contaminants and reversed decoy sequences appended. Fragment and precursor tolerances of 20 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification and oxidation of Met was set as a variable modification. Label-free quantitation of proteins across samples was performed using SINQ normalized spectral index Software⁶².

RNAi:

siRNA knockdown was conducted with siRIG-I(sc-61480), siTRIM32(sc-61714), siTBK1(sc-39058), siIRF3(sc-35710), siIFNAR1(sc-35637), siSTAT1(sc-44123), siIFNGR1(sc-29357), siTNFR1(sc-29507), siSTING(sc-92042), and Control siRNA (sc-37007), purchased from Santa Cruz Biotechnology (Dallas, TX).

Lentiviruses for establishing stable cell lines used for xenograft experiments were obtained from Santa Cruz Biotechnology (Dallas, TX), including shTBK1(sc-39058-V), shIRF3(sc-35710-V), shIFNAR1(sc-35637-V) Human Lentiviral Particles, and Control shRNA Lentiviral Particles-A(sc-108080). GFP adenovirus (1060) and IkBα (S32A/S36A)-DN (Dominant-negative) adenovirus (1028) were obtained from Vector Biolabs (Malvern, PA). A Multiplicity of infection (MOI) of 10 was used in the experiments. Cells were infected with shRNA lentiviral particles following the manufacturer’s protocol and 0.6 µg/mL puromycin was added for selecting stable clones.

Animal Experiments:

Cell lines: Female nude mice (088) at four- to six-week-old were purchased from Charles River Laboratories (Wilmington, MA). One million A549 cells (1×10⁶), or two million (2×10⁶) HCC827 cells (including stable cell lines derived) were injected subcutaneously (s.c.) into the flanks of nude mice. About two weeks later, mice would develop subcutaneous tumors. The mice were randomly divided into indicated groups. Mice were treated with drugs using the doses described in the figure legends. For combination treatment, both drugs were given concurrently for indicated periods. Tumor dimensions were measured every 4 days and tumor volumes calculated by the formula: volume = 0.5 × length × width × width. Mice were sacrificed when tumors reached over 20 millimeter (mm) of length or after the indicated number of days. Eight mice per group were used at injection, the rate of tumor formation was 5–8 per group as shown in Legends and Source_Data_Fig.3, Source_Data_Fig.7, and Source_Data_Extended_Data_Fig.6.

Patient-derived xenograft (PDX): The NSCLC specimens (P0) for HCC4087 and HCC4190 PDXs were surgically resected from a patient diagnosed with NSCLC at UT Southwestern, after obtaining Institutional Review Board approval and informed consent. HCC4087 has KRAS G13C mutation but no EGFR activating mutations, HCC4190 harbors EGFR L858R mutation identified by Exome sequencing. 4 to 6 weeks old female NOD SCID mice (394) were purchased from Charles River Laboratories. The PDX tumor tissues were cut into small pieces (~20 mm3) and subcutaneously implanted in NOD SCID mice of serial generations (P1, P2, etc.). P4 tumor bearing SCID mice were used in this study. Eight mice per group were used at implantation, the rate of tumor formation was 7–8 per group as shown in Legends and Source_Data_Fig.7.

LSL-Kras G12D mice (008179): were purchased from Jackson laboratories and the colony was expanded by breeding heterozygous LSL-Kras G12D mice with wildtype mice. Genotyping was performed per the protocol on Jackson website. Lung tumors were induced in mice carrying the LSL-Kras G12D allele with intranasal administration of 2.5 × 10⁸ PFU Adeno-CMV-Cre (University of Iowa). Treatments were initiated once the tumors were confirmed by with Magnetic resonance imaging (MRI) at about 10–12 weeks after tumor induction. Four mice per group were used at infection, the rate of tumor formation was 3–4 per group as shown in figure dots and Source_Data_Fig.7.

MRI imaging:

MRI Imaging was conducted at UT Southwestern Mouse MRI Core, Advanced Imaging Research Center, using a 7T small animal MRI scanner (Bruker, Rheinstetten, Germany) equipped with a 40 mm quadrature Radiofrequency (RF) coil (ExtendMR LLC, Milpitas, CA). Under anesthesia by inhalation of 1.5 – 3% isoflurane mixed in with medical-grade oxygen via nose-cone, the animals were placed supine on a mouse holder, with a pneumatic respiratory sensor and electrocardiography (ECG) electrodes for cardiac sensing, head first with the lung centered with respect to the center of the RF coil. The mice’s chests were shaved and conducting hydrogels were applied to optimize ECG contact between electrodes and mouse. All MRI acquisitions were gated using both cardiac and respiratory triggering. The bore temperature was kept at 23 ± 2 °C to assure adequate and constant heart rate. Two-dimensional (2D) scout images on three orthogonal planes (transverse, coronal and sagittal) were acquired to determine the positioning. Then, lower resolution gradient echo (T₁_FLASH) images were acquired on transverse plane to fine-adjust the slice position. Finally, higher resolution gradient echo images were recorded on the transverse plane, with the major parameters as follows: The repetition time (TR) = 200 ms (Note: the actual TR is changing according to ECG R-R interval, in range of 200 ms to 240 ms), the echo time (TE) = 1.966 ms, the flip angle (FA) = 45°, the number of average = 12, the field of view (FOV) = 32 × 32 mm², the matrix size = 256 × 256, the slice number = 17–21 (changed upon the mouse lung size), and the slice thickness = 1 mm without any gap. The image analyses were performed using ImageJ.

Patient Data:

Before vs After treatment: Formalin fixed paraffin-embedded (FFPE) tissues from 23 NSCLC patients were obtained from The Jackson Laboratory or UT Southwestern according to IRB-approved protocols. Thirteen specimens were obtained from UT Southwestern and ten from The Jackson Laboratory. Thirteen patients had no EGFR TKI treatment, and ten patients had undergone EGFR TKI treatment.

To assess the effects of Interferons on Overall Survival we reviewed the medical records of NSCLC patients treated at UT Southwestern. 30 advanced (stages IIIB & IV) NSCLC patients harbored classical TKI-sensitive mutations, L858R or exon 19 deletion, but no T790M mutation at initial diagnosis, and all of them had TKI treatment history. Their FFPE tissues from initial diagnosis were collected for this study.

TCGA:

Data were downloaded from https://portal.gdc.cancer.gov/. 42 TCGA-LUAD patients (any stages) with classical TKI-sensitive mutations, L858R or exon 19 deletion, but without T790M mutation, were achieved with their Copy Number Variation (CNV) and Survival data. 41/42 have RNAseq data.

Study approval:

All animal studies were done under IACUC-approved protocols at UT Southwestern and North Texas VA Medical Center (Dallas, Texas, USA). Patient tissues and medical records were obtained from UT Southwestern with IRB approval.

Statistics and Reproducibility:

Error bars represent the means ± S.E.M. of 3 independent experiments unless indicated otherwise. The combination effects in vivo were analyzed by two-way ANOVA with Bonferroni’s correction to adjust the significance level for multiple comparisons. ELISA experiments were analyzed by two-tailed one-sample Student’s t-test, two-tailed two-sample Student’s t-test, or One-way ANOVA with Dunnett’s test. The familywise error rate (FWER) set at 0.05. Kaplan-Meier survival curves were constructed and compared by log-rank test and Gehan’s test. The patient data comparison was shown as median ± IQR, analyzed by Kolmogorov-Smirnov test. RNAseq data were analyzed by DESeq2 and GSEA. All analyses above were performed using GraphPad Prism 8 software. A P value or an adjusted P value for multiple comparison less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #: p>0.05, not statistically significant). The data from cell survival assay, qPCR assay, and luciferase assay were presented as mean lines with three dots from three cell cultures within one experiment, representative of 3 independent repeats with similar results.

The Reproducibility information is provided in Reporting Summary, including sample size predetermination, randomization, blinding, and replication. There are no data exclusions.

Extended Data

Extended Data Fig. 3 — **A-D**. EGFR mutant NSCLC cell lines PC9, H3255 and HCC2279 were transfected with IFNAR1 siRNA or control siRNA for 48 hours followed by exposure to 0.01 µM erlotinib for 72h, followed by AlamarBlue assay. siRNA knockdown of IFNAR1 was confirmed with Western blot. **E-H**. EGFR wt NSCLC cell lines H441, H2122 and H1373 were transfected with IFNAR1 siRNA or control siRNA for 48 hours followed by exposure to 1 µM erlotinib for 72h, followed by AlamarBlue assay. siRNA knockdown of IFNAR1 was confirmed with Western blot. **I-N**. NSCLC cells were concurrently treated by Erlotinib at 0.01 µM (EGFR mutant), or 1 µM (EGFR wt), together with 10 µg/mL Anifrolumab for 72h, followed by AlamarBlue assay. **O-W**. NSCLC cell lines carrying the indicated drivers (EML4/ALK, ROS1, MET, BRAF) were transfected with IFNAR1 siRNA or control siRNA for 48 hours followed by exposure to erlotinib for 72h, or concurrently treated with erlotinib together with 10 µg/mL Anifrolumab for 72h, followed by AlamarBlue assay. siRNA knockdown of IFNAR1 was confirmed with Western blot. For experiments (A-C, E-G, I-V), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are representative of 3 independent experiments with similar results. Cropped images are shown. Uncropped Western blot images are shown in Source_Data_Extended_Data_Fig.3. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.3.

Extended Data Fig. 4 — **A-B.** Two EGFR mutant NSCLC cell lines H3255 and HCC2279 were concurrently treated by 0.1 µM Erlotinib with or without 10 µg/mL Anifrolumab. **C-D.** H3255 and HCC2279 were transfected with IFNAR1 or control siRNA for 48h, followed by 0.1 Erlotinib for 24h. Western blot was performed to detect total and phosphorylated STAT1. **E-H.** Similar experiments were performed on two EGFRwt NSCLC cell lines H441 and H1573, while Erlotinib was used at 1 µM Erlotinib for the indicated time points. **I-N.** EGFR mutant and EGFRwt cells were transfected with STAT1 or control siRNA for 48h, followed by indicated doses of Erlotinib for 72h, and then cell viabilities were measured by AlamarBlue assay. STAT1 siRNA was confirmed by Western blot. For experiments (I-J, L-M), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are representative of three independent experiments with similar results. Cropped images are shown. Uncropped Western blot images are shown in Source_Data_Extended_Data_Fig.4. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.4.

Extended Data Fig. 5 — **A-C.** Cells were treated with 0.1 (A-B) or 1 µM (C) Erlotinib. D. Nude mice bearing A549 xenografts and E. NOD-SCID mice with HCC4087 PDX were treated with erlotinib 100 mg/kg, followed by Western blot. **F-K.** Cells were transfected with ISRE or IFI27-ISRE reporter for 48 hours and treated with erlotinib for 24h, followed by a luciferase reporter assay. **L-O.** EGFR mutant NSCLC lines were transfected with TBK1 siRNA for 48 hours followed by 0.1 µM erlotinib for 72h, concurrently with exogenous IFNα1 or IFNβ1 at 50 ng/mL, followed by AlamarBlue assay. TBK1 siRNA was confirmed with Western blot. **P-R.** EGFR mutant cells were concurrently treated with 0.1 µM Erlotinib and/or 1 µM BX795 for 24 hours, followed by Western blot. **S-U**. EGFR mutant lines were transfected with TBK1 siRNA for 48h followed by 0.1 µM erlotinib for an additional 24h, followed by Western blot. **V-X.** EGFR mutant cells were transfected with ISRE reporter for 48h followed by treatment with erlotinib 0.1 µM and/or 1 µM BX795 for an additional 24h followed by a luciferase assay. **Y-BB**. EGFR mutant cell lines were transfected with siRNA for TBK1 or control siRNA and a luciferase reporter for ISRE for 48h followed by 0.1 µM Erlotinib and for an additional 24h followed by a luciferase assay. Silencing of TBK1 was confirmed by Western blot. **CC.** Western blotting for IKKε expression in NSCLC lines. U87MG cells were used as a positive control. For experiments (F-N, V-AA), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are representative of three independent experiments with similar results. Cropped images are shown. Uncropped images are shown in Source_Data_Extended_Data_Fig.5. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.5.

Extended Data Fig. 6 — **A-D**. EGFR mutant cells were transfected with the TBK1 siRNA for 48h followed by exposure to erlotinib 0.1 µM Erlotinib for 72 hours and AlamarBlue assay. siRNA knockdown of TBK1 was confirmed with Western blot. **E-H.** AlamarBlue assay was done on four EGFR mutant cells after co-treatment with 0.01 µM Erlotinib and/or 1 µM BX795 for 72 hours. **I-L.** EGFR mutant cells were transfected with IRF3 or control siRNA for 48h followed by treatment with 0.01 µM Erlotinib for 72 hours and AlamarBlue assay. Silencing of IRF3 was confirmed with Western blot. **M-O.** EGFR mutant cells were transfected with IRF3 expressing plasmid or empty vector for 48 hours, followed by incubation with 0.1 µM Erlotinib for 72 hours. Cell viability was detected by AlamarBlue assay. Overexpression of IRF3 was confirmed with Western blot. **P-Q.** PC9 cells were stably infected with lentivirus control shRNA (shCtrl) or shRNA for TBK1 or IRF3 lentivirus and Western blot was conducted to confirm silencing. Silenced clones were studied in AlamarBlue cell survival assays following erlotinib exposure for 72h. R. PC9 cells with stable silencing of TBK1 (clone #9) or IRF3 (clone #9) were injected subcutaneously into eight nude mice per group and the rate of tumor formation was 5–8 per group as shown in Source_Data_Extended_Data_Fig.6. Erlotinib was administered daily at 6.25 mg/kg by oral gavage. Tumor sizes were monitored as described in the Methods section. Representative tumor images are shown. For experiments (A-C, E-K, M-Q), n=3 technical replicates, representative of 3 independent repeats with similar results. Data (R) refers to mean tumor size ± S.E.M. (n=5–8 tumors per condition). *: p<0.05, **: p<0.01, ***: p<0.001, by two-way ANOVA adjusted by Bonferroni’s correction (R). Western blots are representative of three independent experiments with similar results. Cropped images are shown. Uncropped Western blot images are shown in Source_Data_Extended_Data_Fig.6. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.6.

Extended Data Fig. 7 — **A-F.** Three EGFR mutant and three EGFRwt NSCLC cell lines were treated by 0.1 or 1 µM Erlotinib for the indicated time points and cell lysates were analyzed by Western blot for detection of total and phosphorylated STING expression. STING was undetectable in PC9 and A549 cells. Phosphorylated STING can only be detected in HCC2279 cell line. **G-H.** HCC827 cells were transfected with STING or control siRNA for 48h, and then exposed to 0.1 µM Erlotinib for 24h, followed by qPCR for detection of IFNA1 and IFNB1 mRNA. **I-J.** As STING siRNA alone was found to be able to decrease the basal IFN mRNA levels by two-way ANOVA adjusted by Bonferroni’s correction, the baseline correction set on Erlotinib untreated groups was performed via GraphPad Prism 8. **K-V.** Similar experiments and corrections were performed on other three NSCLC cell lines, while Erlotinib was used at 0.1 or 1 µM for EGFR mutant and EGFRwt cells respectively. **W-BB.** Two EGFR mutant and two EGFR wt NSCLC cell lines which do have STING expression were transfected with STING or control siRNA for 48h and then treated with the indicated doses of erlotinib for 72h followed by AlamarBlue assay. STING siRNA were confirmed by Western blot. For experiments (G-V, X-Y, AA-BB), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are representative of three independent experiments with similar results. Cropped images are shown. Uncropped Western blot images are shown in Source_Data_Extended_Data_Fig.7. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.7.

Extended Data Fig. 8 — **A-D.** Cells were treated by Erlotinib, followed by Western blot for AhR. **E-H.** Cells were treated by Erlotinib for 24h. AhR nuclear translocation was detected by Western blot. Lamin A/C was the loading control. LPS treatment at 10 μg/mL for 2h was the positive control. **I-J.** PC9 and H2122 cells were treated with Erlotinib for 24h. Cells were then fixed, stained with the AhR antibody (red) and counterstained with the DAPI (blue). LPS treatment at 10 μg/mL for 2h was the positive control. Scale bar represents 25 µm. **K-P.** Three EGFR mutant NSCLC cell lines were treated as indicated, Q. Nude mice bearing HCC827 xenografts were treated with erlotinib 50 mg/kg, and **R-Y.** Four EGFRwt NSCLC cell lines were treated as indicated, PD-L1 expression was detected by Western blot. **Z-AA.** HCC827 and H3255 cells were concurrently treated witherlotinib for 24h, with or without 10 µg/mL Anifrolumab. **BB-CC.** HCC827 and H3255 cells were transfected with IFNAR1 or control siRNA for 48h, and then treated with erlotinib for 24h. PD-L1 expression in cell lysates above, and effects of IFNAR1 siRNA were detected by Western blot. **DD-EE.** Four EGFR mutant and four EGFRwt cells were treated by indicated doses of Erlotinib for 24 hours. PD-1, PD-L1, and PD-L2 expression was detected by Western blot. PD-L1 expression levels were partly shown above. Signal strength was quantified by ImageJ and represented by symbols. -: undetected PD1/PDL1/PDL2 band, +: intensity ratio between PD1/PDL1/PDL2 and β-Actin, set as 100%, ++: ratio>200%, +++: ratio>500%. If more than one + shown, the basal ratio was set as 100%, and the other ratios were between 50% and 200%. Two-fold change threshold determines significance. Western blot and Immunofluorescent staining images are representative of three independent experiments with similar results. Cropped images are shown. Uncropped Western blot images are shown in Source_Data_Extended_Data_Fig.8.

Extended Data Fig. 9 — **A-B**. HCC827 cells were concurrently treated by 1 µM BX795 and 0.1 µM erlotinib for 24h, or pre-transfected with siRNA for TBK1 or IRF3 for 48 hours followed by 0.1 µM erlotinib for 24h, followed by qPCR for IFNA1 mRNA. IFNB1 mRNA was shown in Figure 6A. Silencing of TBK1 and IRF3 were confirmed in Figure 6B. **C-L.** Similar experiments were performed on PC9 and H3255 cells. Silencing of TBK1 and IRF3 was confirmed by Western blot. **M-N**. Similar experiments were performed on A549 cells with 1 µM erlotinib. IFNB1 mRNA was shown in Figure 6C. siRNA knockdown was confirmed in Figure 6D. **O-S.** Similar experiments with H441 cells including siRNA confirmed by Western blot. **T-U.** A549 cells were concurrently treated by 1 µM erlotinib and 0.1 µM BMS-345541 for 24h or infected with IκBα-DN/GFP adenoviruses for 48h followed by Erlotinib (1 µM) for 24h, followed by qPCR for IFNA1 mRNA. IFNB1 mRNA was shown in Figure 6E. Expression of mutant IκBα is shown in Figure 6F. **V-EE.** Similar experiments were performed on H441 and H2122 cells. IκBα-DN overexpression was detected by Western blot. **FF-OO.** Similar experiments were performed on HCC827 and PC9 cells, with 100 nM Erlotinib. For experiments (A-F, H-K, M-R, T-Y, AA-DD, FF-II, KK-NN), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are representative of three independent experiments with similar results and cropped. Uncropped images are shown in Source_Data_Extended_Data_Fig.9. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.9.

Extended Data Fig. 10 — **A-F.** EGFR mutant lines were concurrently treated with 0.1 µM Erlotinib and 10 µg/mL Etanercept (Enbrel) for 24 hours, followed by qPCR for detection of IFNA1 and IFNB1 mRNA. **G-H.** EGFR mutant lines were transfected with an NF-κB reporter for 48h, followed by 10 µg/mL Etanercept for 1h and then 10 ng/mL TNF for 24h, followed by a luciferase assay or Western blot. **I-O.** EGFR mutant cell lines were transfected with TNFR1 siRNA for 48 hours, then treated with 0.1 µM erlotinib for 24h followed by qPCR for IFNA1 and IFNB1 mRNA. Silencing of TNFR1 was confirmed by Western blot. **P-BB.** Similar experiments were performed on EGFRwt cell lines while 1 µM Erlotinib was used. **CC-EE.** Three EGFR mutant NSCLC cell lines were concurrently treated with 50 µg/mL Etanercept, 0.1 µM Erlotinib, 50 ng/mL IFNα1 or IFNβ1 for 72h. **FF-II.** EGFR mutant cells were transfected with TNFR1 for 48 hours, then treated with 0.1 µM erlotinib, 50 ng/mL IFNα1 or IFNβ1 for 72h, followed by AlamarBlue assay. Silencing of TNFR1 was confirmed by Western blot. **JJ-LL.** EGFR mutant cells were concurrently treated with 0.1 µM Erlotinib, 0.1 µM BMS-345541, 50 ng/mL IFNα1 or IFNβ1 as indicated for 72h followed by AlamarBlue assay. **MM-PP.** EGFR mutant cells were infected with or IκBα-DN or GFP adenoviruses for 48h, then concurrently treated with 0.1 µM Erlotinib, 50 ng/mL IFNα1 or IFNβ1 for 72h followed by AlamarBlue assay. IκBα-DN overexpression was confirmed by Western blot. For experiments (A-G, I-N, P-AA, CC-HH, JJ-OO), n=3 technical replicates, representative of 3 independent repeats with similar results. Western blots are representative of three independent experiments with similar results. Cropped images are shown. Uncropped Western blot images are shown in Source_Data_Extended_Data_Fig.10. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.10.

Supplementary Material

NR-Reporting-Summary

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SourceData_Fig1(RawData)

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Acknowledgements:

This work was supported in part by the Office of Medical Research, Departments of Veterans Affairs, a Lung Cancer SPORE Career Enhancement Program Award, and support from the Dallas VA Research Corporation to AAH. This work was also supported by NCI Lung Cancer SPORE (P50CA70907), U01CA176284, and CPRIT (RP110708 and RP160652) to JDM. DEG is supported by a National Cancer Institute (NCI) Midcareer Investigator Award in Patient-Oriented Research, K24CA201543-01. SB is supported by grants from the National Institutes of Health (RO1CA197796) and the National Aeronautics and Space Administration (NNX16AD78G). C-MC’s research was supported by NIH (CA103867), CPRIT (RP180349 and RP190077) and the Welch Foundation (I-1805). EAA was supported by Cancer Prevention and Research Institute of Texas (CPRIT) Scholar Award RR160080 and a Career Enhancement Award through National Institutes of Health 5P50CA070907. Research reported in this publication was supported in part by the Harold C. Simmons Comprehensive Cancer Center’s Biomarker Research Core, which are supported by NCI Cancer Center Support Grant 1P30 CA142543-03. We acknowledge NIH shared instrumentation grant 1S10OD023552-01 that funded the MRI equipment. We thank Dr. John Hiscott for providing the IRF3 plasmid and Jessica Saltarski (UT Southwestern) for assistance in obtaining FFPE tissues.

Footnotes

Competing Interests Statement The authors declare no competing interests.

Code Availability Statement

Information regarding codes used in this study have been proved in Reporting Summary. They are either commercially available or open-source.

Data Availability Statement

RNA-seq data that support the findings of this study have been deposited in the Sequence Read Archive (SRA) under accession code PRJNA593064.

Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD016558.

The human lung adenocarcinoma data were derived from the TCGA Research Network: http://cancergenome.nih.gov/. Unprocessed WB images for Figures 1–7 and Extended Data Figures 1–10, have been provided as Source Data file Source_Data_Fig.1–7 and Source_Data_Extended_Data_Fig.1–10. Raw digital source data for Figures 1–3, 5–8 and Extended Data Figure 1–7, 9–10 have been provided as Source Data file Source_Data_Fig.1–3, 5–8 and Source_Data_Extended_Data_Fig.1–7, 9–10. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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