ARID1A deficiency reverses the response to anti-PD(L)1 therapy in EGFR-mutant lung adenocarcinoma by enhancing autophagy-inhibited type I interferon production

Abstract

Introduction: EGFR mutations in non-small cell lung cancer (NSCLC) are associated with a poor response to immune checkpoint inhibitors (ICIs), and only 20% of NSCLC patients harboring EGFR mutations benefit from immunotherapy. Novel biomarkers or therapeutics are needed to predict NSCLC prognosis and enhance the efficacy of ICIs in NSCLC patients harboring EGFR mutations, especially lung adenocarcinoma (LUAD) patients, who account for approximately 40-50% of all NSCLC cases.

Methods: An ARID1A-knockdown (ARID1A-KD) EGFR-mutant LUAD cell line was constructed using lentivirus. RNA-seq and mass spectrometry were performed. Western blotting and IHC were used for protein expression evaluation. Effects of 3-MA and rapamycin on cells were explored. Immunofluorescence assays were used for immune cell infiltration examination.

Results: ARID1A expression was negatively associated with immune cell infiltration and immune scores for ICIs in LUAD with EGFR mutations. In vitro experiments suggested that ARID1A-KD activates the EGFR/PI3K/Akt/mTOR pathway and inhibits autophagy, which attenuates the inhibition of Rig-I-like receptor pathway activity and type I interferon production in EGFR-mutant LUAD cells. In addition, 3-MA upregulated production of type I interferon in EGFR-mutant LUAD cells, with an similar effect to ARID1A-KD. On the other hand, rapamycin attenuated the enhanced production of type I interferon in ARID1A-KD EGFR-mutant LUAD cells. ARID1A function appears to influence the tumor immune microenvironment and response to ICIs.

Conclusion: ARID1A deficiency reverses response to ICIs in EGFR-mutant LUAD by enhancing autophagy-inhibited type I interferon production. Video Abstract.

Keywords: ARID1A; Autophagy; EGFR-mutant lung adenocarcinoma; Immunotherapy; Type I interferon.

Fig. 1

ARID1A deficiency changes the immune phenotype and enhances immune cell infiltration in lung adenocarcinoma patients with or without EGFR mutations. A ARID1A mutations were associated with longer overall survival in pancancer patients who received immunotherapy. B ARID1A mutations were associated with higher TMB values in pancancer patients. C ARID1A mutations were associated with higher TMB values in lung adenocarcinoma patients; D1, D2 Low ARID1A expression was associated with higher ESTIMATE scores, immune scores and IPS scores in lung adenocarcinoma patients. E Low ARID1A expression was associated with higher ESTIMATE scores, immune scores and IPS scores (anti-PD1/PD-L1) in lung adenocarcinoma patients harboring EGFR mutation. F Enrichment analysis for lung adenocarcinoma patients with low ARID1A expression. G Lung adenocarcinoma patients with low ARID1A expression had a higher infiltrating immune cell abundance

Fig. 2

Exploration of biological processes and pathways related to low ARID1A expression in EGFR-mutant lung adenocarcinoma cell lines using a multiomics method. A Evaluation of ARID1A protein expression after construction of the ARID1A knockdown cell line. B PCA based on RNA samples for RNA-seq sequencing. C Volcano plot shows differentially expressed genes revealed by RNA-seq sequencing. D Heatmap for differentially expressed genes revealed by RNA-seq sequencing; E1, E2 Enrichment analysis based on upregulated genes in ARID1A knockdown cells revealed by RNA-seq sequencing. F Enrichment analysis based on differentially expressed proteins revealed by mass spectrometry for upregulated phosphorylated proteins in ARID1A-knockdown cells

Fig. 3

ARID1A-KD activates the EGFR/PI3K/Akt/mTOR signaling pathway and inhibits autophagy in EGFR-mutant lung adenocarcinoma cells. A–C Relative expression of phosphorylated proteins enriched in autophagy, the PI3K/Akt pathway and the mTOR pathway revealed by mass spectrometry. D Relative expression of autophagy-related biomarkers revealed by mass spectrometry. E Expression evaluation of targeted proteins revealed by western blotting

Fig. 4

ARID1A knockdown enhances production of type I interferon via the Rig-I-like receptor pathway in EGFR-mutant lung adenocarcinoma cells. A GSEA revealed activation of the Rig-I-like receptor pathway; RNA-seq demonstrated upregulation of IRF7. B Relative expression of phosphorylated proteins enriched in the MAPK pathway revealed by mass spectrometry. C Relative expression of phosphorylated NF-κB pathway biomarkers revealed by mass spectrometry. D RNA-seq sequencing and mass spectrometry revealed upregulation of interferon-induced proteins. E Expression evaluation of targeted proteins revealed by western blotting. F GSEA revealed the enhancement of the response to interferon γ in ARID1A knockdown lung adenocarcinoma cells

Fig. 5

Inhibition of autophagy in EGFR-mutant lung adenocarcinoma cells reverses production of type I interferon, with a similar effect as ARID1A knockdown. A Expression evaluation of targeted proteins revealed by western blotting (3-MA: 2 mM; rapamycin: 100 µM). B Schematic diagram elucidates the mechanism of ARID1A knockdown in enhancing production of type I interferon in EGFR-mutant lung adenocarcinoma cells

Fig. 6

EGFR-mutant lung adenocarcinoma patients with low ARID1A expression have enhanced immune cell filtration and a favorable response to anti-PD(L)1 therapy. A Representative images for IHC examination of ARID1A protein. B Survival analysis of progression-free survival of EGFR-mutant lung adenocarcinoma patients who received immune checkpoint inhibitors divided by ARID1A expression. C Representative images for the immunofluorescence assay to evaluate immune cell infiltration in EGFR-mutant lung adenocarcinoma patients. D Low ARID1A expression was associated with higher arbitrary units of CD3 and CD8, as revealed by the immunofluorescence assay. E ARID1A correlates negatively with immune cell infiltration in EGFR-mutant lung adenocarcinoma patients