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

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 interferon (IFN)-I upregulation via a RIG-I–TANK-binding kinase 1 (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 IFNs via a NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-tyrosine kinase inhibitor (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 patients with NSCLC.

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Fig. 1: EGFR inhibition upregulates IFN, which promotes resistance to EGFR inhibition in NSCLC.
Fig. 2: STAT1 activation is involved in prosurvival effect of type I IFNs in the context of EGFR inhibition.
Fig. 3: EGFR inhibition triggers a biologically notable TBK1–IRF3 pathway in EGFR-mutant NSCLC.
Fig. 4: TRIM32 is required for EGFR inhibition-induced activation of TBK1 and IRF3.
Fig. 5: RIG-I is upregulated when EGFR is inhibited in EGFR-mutant NSCLC lines.
Fig. 6: EGFRwt and EGFR-mutant NSCLC upregulate type I IFNs via distinct pathways: the role of IFN signaling in secondary resistance to EGFR inhibition.
Fig. 7: A synergistic effect of EGFR plus type I IFN inhibition in mouse models of NSCLC.
Fig. 8: The type I IFN level inversely correlates with response to TKI treatment in NSCLC.

Data availability

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

MS data have been deposited in ProteomeXchange with primary accession code PXD016558.

Human LUAD data were derived from the TCGA Research Network: http://cancergenome.nih.gov/. Unprocessed western blot images for Figs. 17 and Extended Data Figs. 110, have been provided as source data files, Source Data Figs. 17 and Source Data Extended Data Figs. 110. Raw digital source data for Figs. 13, 58 and Extended Data Figs. 17, 910 have been provided as source data files, Source Data Figs. 13, 58 and Source Data Extended Data Figs. 17, 910. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

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

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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 A.A.H. This work was also supported by National Cancer Institute (NCI) Lung Cancer SPORE (P50CA70907), U01CA176284 and Cancer Prevention and Research Institute of Texas (CPRIT) (RP110708 and RP160652) to J.D.M. D.E.G. was supported by a NCI Midcareer Investigator Award in Patient-Oriented Research, K24CA201543-01. S.B. was supported by grants from the National Institutes of Health (NIH) (R01CA197796) and the National Aeronautics and Space Administration (NNX16AD78G). C.-M.C.’s research was supported by NIH (CA103867), CPRIT (RP180349 and RP190077) and the Welch Foundation (I-1805). D.Z. was supported by NIH grant R01CA194578. E.A.A. was supported by CPRIT Scholar Award RR160080 and a Career Enhancement Award through NIH 5P50CA070907. Research reported in this publication was supported in part by the Harold C. Simmons Comprehensive Cancer Center’s Biomarker Research Core, which is supported by NCI Cancer Center Support grant 1P30 CA142543–03. We acknowledge NIH shared instrumentation grant 1S10OD023552-01 that funded MRI equipment. We thank Dr J. Hiscott for providing IRF3 plasmid and J. Saltarski (UT Southwestern Medical Center) for assistance in obtaining FFPE tissues.

Author information

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Authors

Contributions

K.G., G.G. and A.A.H. designed experiments. K.G., G.G., N.P., M.E.B., S.Z. and E.A.A. performed or assisted with experiments. D.E.G., J.D.M., F.F., B.G., M.P., K.K. and K.H.D. provided cell lines, PDX or human tissue specimens. K.G., G.G., D.E.G., B.M., S.B., C.-M.C., A.A.S., C.X., K.H.D., D.Z. and A.A.H. analyzed data. K.G. and A.A.H. wrote the manuscript with contributions from G.G., J.D.M. and C.-M.C. Study conception and supervision was conducted by A.A.H.

Corresponding author

Correspondence to Amyn A. Habib.

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Extended data

Extended Data Fig. 1 EGFR inhibition upregulates IFN mRNA levels in multiple NSCLC cell lines.

a–h. Four EGFR mutant cell lines, H3255, PC9, HCC2279, and H1650 were treated with 0.1 µM Erlotinib for the indicated time points. IFNα1 and IFNβ1 mRNA levels were detected by qPCR with β-Actin as the loading control. i–p. A similar experiment was conducted in four EGFR wt cell lines: H441, H2122, H1373, and H1573, exposed to 1 µM erlotinib. q. Two EGFR mutant cells (HCC827 and PC9), and two EGFRwt cell lines (A549 and H441) were treated with 0.1 µM or 1 µM erlotinib respectively for the indicated time points. Cell lysates were collected for detecting IFNAR1 expression by Western blot. r-x. Four NSCLC cell lines carrying the indicated drivers (EML4/ALK, ROS1, MET, BRAF) were treated with 1 µM Erlotinib for the indicated time points. IFNα1 and IFNβ1 mRNA levels were detected by qPCR. β-Actin was used as the loading control. For experiments (ap, rx), n = 3 technical replicates, representative of 3 independent repeats with similar results. H1666 is reported to harbor IFNA1 homo-deletion in COSMIC (Catalogue Of Somatic Mutations In Cancer)-v90 http://cancer.sanger.ac.uk/cosmic (Updated 5 September 2019), also in Data from a CPRIT (Cancer Prevention & Research Institute of Texas)-funded NGS (next generation sequencing) project by Dr. John Minna, UT Southwestern Medical Center, and Data from Dr. Adi Gazdar, UT Southwestern Medical Center. All other cell lines used in this research were searched on those databases above and confirmed to harbor neither IFNA1 nor IFNB1 homo-deletion. 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.1. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.1.

Source data

Extended Data Fig. 2 EGFR inhibition upregulates IFNs in multiple NSCLC cell lines and in vivo.

a, b. HCC827 and A549 were treated with 0.1 µM or 1 µM Erlotinib for 2 days. The protein concentration of IFNα1 and IFNβ1 in cell lysates were measured by ELISA. c–j. EGFR mutant cell lines were treated with 0.1 µM Erlotinib. 48 hours later cell lysates (cf) and supernatants (gi) were collected for IFN ELISA. k–r. A similar experiment was conducted in four EGFR wt cell lines. s–z. Nude mice were injected subcutaneously (s.c.) with HCC827 or A549 cells. NOD-SCID mice were s.c. implanted with HCC4190 (EGFR mutant) or HCC4087 (EGFR wt) NSCLC PDX. After tumor formation, erlotinib at 50 mg/kg for EGFR mutant or 100 mg/kg for EGFR wt was given to mice daily for indicated days. Tumors were removed and subjected to ELISA for IFNα1 and IFNβ1. aa–ff. EGFR mutant and EGFRwt NSCLC cell lines were treated with 0.1 or 1 µM Erlotinib for the indicated time points. IFNG mRNA levels were detected by qPCR. β-Actin was used as the loading control. gg–mm. NSCLC cell lines were transfected with IFNGR1 siRNA or control siRNA for 48 hours followed by exposure to erlotinib for 72 h, followed by AlamarBlue assay. siRNA knockdown of IFNGR1 was confirmed with Western blot. N = 3, data (a-z) refers to mean ± s.e.m. from three independent experiments. ELISA (az) was analyzed by two-sided t-test (ar) and one-way ANOVA with Dunnett’s test (sz) for animal tumors. #: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001 (az). For experiments (aall), 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.2. Numerical source data for the experiments in this figure can be found in Source_Data_Extended_Data_Fig.2.

Source data

Extended Data Fig. 3 Type I IFNs promote resistance to EGFR inhibition in multiple NSCLC cell lines.

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 72 h, 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 72 h, 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 72 h, 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 72 h, or concurrently treated with erlotinib together with 10 µg/mL Anifrolumab for 72 h, 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.

Source data

Extended Data Fig. 4 STAT1 activation is involved in pro-survival effect of Type I IFNs in the context of EGFR inhibition.

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 48 h, followed by 0.1 Erlotinib for 24 h. 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 48 h, followed by indicated doses of Erlotinib for 72 h, 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.

Source data

Extended Data Fig. 5 EGFR inhibition activate TBK1-IRF3 axis in EGFR mutant but not in EGFR wt NSCLC; Lack of IKKε expression in lung cancer cell lines.

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 24 h, 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 72 h, 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 48 h followed by 0.1 µM erlotinib for an additional 24 h, followed by Western blot. v–x. EGFR mutant cells were transfected with ISRE reporter for 48 h followed by treatment with erlotinib 0.1 µM and/or 1 µM BX795 for an additional 24 h 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 48 h followed by 0.1 µM Erlotinib and for an additional 24 h 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 (fn, vaa), 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.

Source data

Extended Data Fig. 6 Biological importance of EGFR induced TBK1/IRF3 activation in EGFR mutant NSCLC.

a–d. EGFR mutant cells were transfected with the TBK1 siRNA for 48 h 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 48 h 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 72 h. 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 (ac, ek, mq), 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.

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Extended Data Fig. 7 STING is not involved in response to EGFR inhibition.

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 48 h, and then exposed to 0.1 µM Erlotinib for 24 h, 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 48 h and then treated with the indicated doses of erlotinib for 72 h followed by AlamarBlue assay. STING siRNA were confirmed by Western blot. For experiments (gv, 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.

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Extended Data Fig. 8 AhR is not activated in response to EGFR inhibition; Regulation of PD-L1 by EGFR inhibition.

a–d. Cells were treated by Erlotinib, followed by Western blot for AhR. e–h. Cells were treated by Erlotinib for 24 h. AhR nuclear translocation was detected by Western blot. Lamin A/C was the loading control. LPS treatment at 10 μg/mL for 2 h was the positive control. i, j. PC9 and H2122 cells were treated with Erlotinib for 24 h. Cells were then fixed, stained with the AhR antibody (red) and counterstained with the DAPI (blue). LPS treatment at 10 μg/mL for 2 h 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 24 h, with or without 10 µg/mL Anifrolumab. bb, cc. HCC827 and H3255 cells were transfected with IFNAR1 or control siRNA for 48 h, and then treated with erlotinib for 24 h. 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 PD-1/PDL1/PDL2 band, +: intensity ratio between PD-1/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.

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Extended Data Fig. 9 Distinguished mechanisms of EGFR inhibition induced Type I IFN regulation.

a, b. HCC827 cells were concurrently treated by 1 µM BX795 and 0.1 µM erlotinib for 24 h, or pre-transfected with siRNA for TBK1 or IRF3 for 48 hours followed by 0.1 µM erlotinib for 24 h, followed by qPCR for IFNA1 mRNA. IFNB1 mRNA was shown in Fig. 6a. Silencing of TBK1 and IRF3 were confirmed in Fig. 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 Fig. 6c. siRNA knockdown was confirmed in Fig. 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 24 h or infected with IκBα-DN/GFP adenoviruses for 48 h followed by Erlotinib (1 µM) for 24 h, followed by qPCR for IFNA1 mRNA. IFNB1 mRNA was shown in Fig. 6e. Expression of mutant IκBα is shown in Fig. 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 (af, hk, mr, ty, aadd, ffii, kknn), 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.

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Extended Data Fig. 10 Mechanisms and biological effects of EGFR inhibition induced Type I IFN regulation.

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 48 h, followed by 10 µg/mL Etanercept for 1 h and then 10 ng/mL TNF for 24 h, 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 24 h 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 72 h. 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 72 h, 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 72 h followed by AlamarBlue assay. mm–pp. EGFR mutant cells were infected with or IκBα-DN or GFP adenoviruses for 48 h, then concurrently treated with 0.1 µM Erlotinib, 50 ng/mL IFNα1 or IFNβ1 for 72 h followed by AlamarBlue assay. IκBα-DN overexpression was confirmed by Western blot. For experiments (ag, in, paa, cchh, 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.

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Gong, K., Guo, G., Panchani, N. et al. EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer. Nat Cancer 1, 394–409 (2020). https://doi.org/10.1038/s43018-020-0048-0

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