Chromosomal Instability Is Associated with cGAS-STING Activation in EGFR-TKI Refractory Non-Small-Cell Lung Cancer

Abstract

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are standard therapies for EGFR-mutated non-small-cell lung cancer (NSCLC); however, their efficacy is inconsistent. Secondary mutations in the EGFR or other genes that lead to resistance have been identified, but resistance mechanisms have not been fully identified. Chromosomal instability (CIN) is a hallmark of cancer and results in genetic diversity. In this study, we demonstrated by transcriptomic analysis that CIN activates the cGAS-STING signaling pathway, which leads to EGFR-TKI refractoriness in a subset of EGFR-mutated NSCLC patients. Furthermore, EGFR-mutated H1975dnMCAK cells, which frequently underwent chromosomal mis-segregation, demonstrated refractoriness to the EGFR-TKI osimertinib compared to control cells. Second, H1975dnMCAK cells exhibited activation of cGAS-STING signaling and its downstream signaling, including tumor-promoting cytokine IL-6. Finally, chromosomally unstable EGFR-mutated NSCLC exhibited enhanced epithelial-mesenchymal transition (EMT). Blockade of cGAS-STING-TBK1 signaling reversed EMT, resulting in restored susceptibility to EGFR-TKIs in vitro and in vivo. These results suggest that CIN may lead to the activation of cGAS-STING signaling in some EGFR-mutated NSCLC, resulting in EMT-associated EGFR-TKI resistance.

Keywords: EGFR-mutated non-small-cell lung cancer; chromosomal instability; epidermal growth factor receptor tyrosine kinase inhibitors.

Figure 1

CIN in EGFR-mutated non-small-cell lung cancer is associated with the progression-free survival of EGFR-TKIs. (A) Schema of sampling at pretreatment (n = 32) as the “Pretreatment sample” and after the acquisition of EGFR-TKI resistance (n = 35) as the “Posttreatment sample”. (B) Scattered plot of CIN z-scores of individuals pre- and posttreatment samples. Clinical characteristics are analyzed as indicated. Data represent the mean ± SEM. (C) Progression-free survival curves for patients treated with EGFR-TKIs, separated into CIN-high and CIN-low groups. (D) Cox proportional hazards model for progression-free survival for EGFR-TKI adjusted by factors comprising CIN, type of EGFR mutation, performance status, EGFR-TKI generation, sex, age, smoking, and TP53 mutation. (E) Gene set enrichment analysis (GSEA) plots showing the gene enrichment pattern of CIN between pretreatment and posttreatment samples. Normalized enrichment score (NES) and p value are shown on the plot. (F) Scattered plot of CIN z-scores of individual samples is shown and compared between pre- and posttreatment. Data represent the mean ± SEM. * p < 0.05; CIN, chromosomal instability; EGFR-TKI, epidermal growth factor receptor tyrosine kinase inhibitor; SEM, standard error of the mean.

Figure 2

CIN correlated with cGAS–STING and its downstream signaling in EGFR-mutated non-small-cell lung cancer with acquired resistance to EGFR-TKIs. (A) A heatmap sorted by CIN z-score of hallmark-based gene signatures in the EGFR-mutated tumors with acquired resistance to EGFR-TKIs. (B) A heatmap sorted by CIN z-score of gene expression or gene signatures for cGAS–STING and its downstream signaling in the EGFR-mutated NSCLC tumors that have acquired resistance to EGFR-TKIs. (C) A schematic diagram showing a signaling pathway thought to be caused by CIN in EGFR-mutated NSCLC tumors with acquired resistance to EGFR-TKIs. CIN, chromosomal instability; EGFR, epidermal growth factor receptor; r, Pearson correlation coefficient; IL-6, interleukin 6; STING, stimulator of interferon response cGAMP interactor 1; TBK1, TANK-binding kinase 1; IFN, interferon; cGAS, cyclic GMP–AMP synthase; NF-κB, nuclear factor-kappa B.

Figure 3

CIN-induced EGFR-TKI resistance in EGFR-mutated NSCLC may be associated with EMT. (A) A representative figure shows normal and abnormal chromosomes, including lagging chromosome, anaphase bridge, and isolation, in H1975dnMCAK cells. (B) A ratio of mitotic abnormalities in H1975Cont and H1975dnMCAK cells is shown. (C) In vitro growth inhibition assay showing CIN-induced EGFR-TKI resistance. H1975Cont or H1975dnMCAK cells were treated with the indicated concentrations of EGFR-TKI osimertinib for 3 d. Cell viability is shown relative to untreated control cells (mean ± SD of 6 replicate and 2 independent experiments). (D) In vivo study showing CIN-induced EGFR-TKI resistance. In a mouse xenograft model using H1975Cont or H1975dnMCAK cells, tumors were treated with osimertinib 5 mg/kg or vehicle 5 times per week. Tumor growth curves are shown. Each group consisted of eight mice. Data are the mean ± SEM. (E) Cellular signaling analysis for CIN-induced, EGFR-mutated NSCLC under EGFR-TKI exposure. H1975Cont or H1975dnMCAK cells were treated with 100 nM osimertinib for 3 h and then probed for the indicated proteins. (F) A heatmap of an epithelial marker, EMT-TFs, and mesenchymal markers in three clones of H1975Cont and H1975dnMCAK cells. (G) Immune blotting in H1975Cont or H1975dnMCAK cells of the indicated EMT-related proteins. EGFR, epidermal growth factor receptor; EMT-TFs, epithelial–mesenchymal transition transcription factors.

Figure 4

cGAS–STING–TBK1 signaling is associated with EMT in chromosomally unstable EGFR-activating NSCLC that is resistant to EGFR-TKIs. (A) A heatmap sorted by CIN z-score of Reactome pathway analysis, including STING, type I IFN, and IL-6 signaling. (B) CIN-dependent activation of TBK1 in EGFR-mutated NSCLC. Immune blotting of the indicated proteins in H1975Cont or H1975dnMCAK cells. (C) The cGAS inhibitor G150 reversed EMT associated with CIN. H1975Cont or H1975dnMCAK cells were treated with 100 nM osimertinib, 10 μM G150, or combination of these agents for 48 h and then probed for the indicated proteins. (D,E) The cGAS inhibitor G150 decreased EGFR-TKI resistance associated with CIN. Clonogenic assay for H1975Cont (D) and H1975dnMCAK (E) cells treated with/without 100 nM osimertinib, 10 μM G150, or a combination of both drugs for 10 d. The colony-formation area was measured and quantified using Image J. (F) The TBK1 inhibitor MRT67307 reversed EMT associated with CIN. H1975dnMCAK cells were treated with 100 nM osimertinib, 1 μM MRT67307, or a combination of these agents for 48 h and then probed for the indicated proteins. (G) The TBK1 inhibitor MRT67307 decreased EGFR-TKI resistance associated with CIN. Clonogenic assay for H1975dnMCAK cells treated with/without 100 nM osimertinib, 1 μM MRT67307, or a combination of both drugs for 10 d. (H) cGAS repression using siRNA reversed EMT associated with CIN. H1975dnMCAK cells were transiently transfected with siRNA targeting cGAS or a mock siRNA (si-Mock) and probed for the indicated proteins. (I) cGAS repression using siRNA decreased EGFR-TKI resistance associated with CIN. H1975dnMCAK cells were transiently transfected with siRNA targeting cGAS or mock siRNA (si-Mock) and treated with 100 nM osimertinib for 72 h. Cell viability is shown relative to untreated control cells (mean ± SD of five replicates). (J) The cGAS inhibitor G150 increase the susceptibility to EGFR-TKI osimertinib in chromosomally unstable EGFR-mutated NSCLC in vivo. In a mouse H1975dnMCAK xenograft model, tumors were treated with osimertinib 5 mg/kg or vehicle 5 times per week with/without G150 (5 mg/kg every 2 d). Tumor growth curves are shown. Each group consisted of seven mice. Data are the mean ± SEM. * p < 0.05; STING, stimulator of interferon response cGAMP interactor; cGAS, cyclic GMP–AMP synthase; TBK1, TANK-binding kinase 1.

Figure 5

EMT is correlated with CIN in EGFR-mutated NSCLC tumors with acquired EGFR-TKI resistance. (A) A heatmap sorted by CIN z-score of Hallmark gene signature for epithelial–mesenchymal transition in EGFR-mutated NSCLC tumors with acquired EGFR-TKI resistance. (B) A heatmap sorted by CIN z-score of epithelial markers, EMT TFs, and mesenchymal markers as indicated. (C) Representative images of hematoxylin–eosin staining and vimentin immunohistochemistry for four EGFR-mutated NSCLC tumors with acquired resistance to EGFR-TKIs. (D) CIN is correlated with the ratio of vimentin-positive cells in EGFR-mutated NSCLC tumors. Correlation of CIN z-score and vimentin positive ratio in nine EGFR-mutated NSCLC tumors with acquired resistance to EGFR-TKIs. (E) Schematic figure of the relationship between CIN and cGAS–STING activation, inflammation, EMT, and sensitivity to EGFR-TKIs. CIN, chromosomal instability; EGFR-TKI, epidermal growth factor receptor tyrosine kinase inhibitor; NSCLC, non-small-cell lung cancer; EMT-TFs, epithelial–mesenchymal transition transcription factors.