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

ARID1A deficiency changes the immune phenotype and enhances immune cell infiltration in LUAD patients with or without EGFR mutations

Pan-cancer analysis (N = 1904) revealed that patients harboring ARID1A mutation (N = 291) had a longer OS than those with a wild-type gene after ICI administration (28.0 months versus 18.0 months, P = 0.0052), as displayed in Fig. 1A. ARID1A mutation was found to be related to the genomic instability of malignancy represented by a higher TMB both in pancancer patients (18.14/Mb versus 5.27/Mb, P < 0.0001, Fig. 1B) and LUAD patients (13.03/Mb versus 7.14/Mb, P = 0.0071, Fig. 1C). We then explored the role of ARID1A expression, which may be downregulated by ARID1A mutations, in cancer immunity and immunotherapy. Low ARID1A expression was related to the immunotherapy-sensitive phenotype in LUAD patients (N = 461), as characterized by higher ESTIMATE scores, immune scores (Fig. 1D1), and IPS scores for single agents of PD-1/PDL1 or CTLA-4 inhibitors or combined therapy (Fig. 1D2). Specifically, for LUAD patients harboring EGFR mutation (N = 64), low ARID1A expression still predicted a favorable response to immunotherapy, as represented by a higher ESTIMATE score (P = 0.0372), immune score (P = 0.0072) and IPS score for PD-1/PD-L1 inhibitors (P = 0.0406), as shown in Fig. 1E.

Fig. 1figure 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

Mechanistically, enrichment analysis demonstrated that low ARID1A expression activates a variety of biological processes related to immunity, including antigen processing, immune cell chemotaxis and T-cell-mediated cytotoxicity upregulation (Fig. 1F). ssGSEA was performed to compare different statuses of immune cell infiltration between different groups of LUAD patients divided by ARID1A expression, as shown in Fig. 1G. The abundance of the majority of infiltrating immune cells was higher in the ARID1A low expression group, revealing a phenotype of abundant immune cell infiltration in these LUAD patients.

Exploration of biological processes and pathways related to low ARID1A expression in LUAD cell lines using a multiomics method

Bioinformatic analysis revealed the potential role of low ARID1A expression in LUAD immunotherapy. We thus further explored differences and underlying mechanisms induced by ARID1A expression changes using RNA-seq and MS for total proteins and phosphorylated proteins. The EGFR-mutant human LUAD cell line HCC4006 was implanted with sh-RNA for ARID1A and the NC vector after lentivirus infection. The expression level of ARID1A protein was significantly downregulated in ARID1A-KD compared with NC (P = 0.0013), as displayed in Fig. 2A. Principal component analysis (PCA) of RNA samples indicated that they can be separated well by PC1 and PC2 (Fig. 2B). The volcano plot in Fig. 2C demonstrates the DEGs between ARID1A-KD and NC cells based on RNA-seq. According to the DEGs revealed by RNA-seq (Fig. 2D), enrichment analysis was performed, as displayed in Fig. 2E1, E2, based on the GO database and REACTOM database, respectively. The DEGs were mainly enriched in interferon-related pathways, especially the type I IFN pathway, response to type I IFN, and activation of the interferon α/β signaling pathway. Other immune-related pathways, including the IFN γ signaling pathway and enhancement of antigen processing and immune response, were all significantly enriched in ARID1A-KD cells. To further clarify the specific signaling pathway altered in EGFR-mutant LUAD cells harboring ARID1A-KD, MS assays were performed to quantify phosphorylated proteins, and enrichment analysis was also performed based on DEPs revealed by MS, as shown in Fig. 2F. Interestingly, a significant change in phosphorylation level was detected in the PI3K/Akt/mTOR signaling pathway and the activity change of autophagy, which was revealed through enrichment analysis based on the KEGG database and REACTOM database. The results of RNA-seq based on RNA samples from the A549 cell line verified the findings above, as displayed in Additional file 2: Figure S1. The DEGs induced by ARID1A-KD in the A549 cell line are shown in Additional file 2: Figure S1A in a volcano plot format. ARID1A-KD significantly downregulated autophagy-related genes and upregulated IFN-related proteins (Additional file 2: Figure S1B). The enrichment analysis (Additional file 2: Figure S1C-S1D) based on DEGs revealed by RNA-seq in the A549 cell line also indicated that ARID1A-KD played an important role in influencing autophagy activity, anticancer immunity, type I IFN production and related pathways, according to the GO database and REACTOM database.

Fig. 2figure 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

ARID1A-KD activates the EGFR/PI3K/Akt/mTOR signaling pathway and inhibits autophagy in EGFR-mutant LUAD cells

All of the phosphorylated proteins displayed in the heatmap in Fig. 3A–C were significantly upregulated in ARID1A-KD cells. The results demonstrated that phosphorylated RAPTOR in mTOR complex 1 (mTORC1) and RICTOR in mTOR complex 2 (mTORC2) were upregulated in ARID1A-KD cells. In addition, LAMTOR1 (an activator of mTOR) and DEPTOR (an inhibitor of mTOR) were activated in ARID1A-KD cells, creating confusion regarding the actual effects on mTOR signaling and autophagy. Therefore, subsequent MS for total proteins (Fig. 3D) and WB analysis for target proteins (Fig. 3E) were performed. ARID1A-KD significantly increased mTOR (P = 0.0011) and phosphorylated mTOR (p-mTOR, P = 0.0184) levels in HCC4006 cells (Fig. 3E). In ARID1A-KD cells, Beclin-1 (P = 0.0184) and autophagy-related 5 (Atg5, P = 0.0069) were downregulated (Fig. 3D), as was expression of LC3-II (P = 0.0080, Fig. 3E), but P62 (also called Sequestosome 1 [SQSTM1], P = 0.0406) was upregulated (Fig. 3E). All expression-level changes of the listed proteins suggest activation of the mTOR signaling pathway but inhibition of autophagy. MS indicated activation of the PI3K/Akt signaling pathway, which is an upstream pathway of mTOR, in ARID1A-KD cells (Fig. 3B). The members of the ErbB family, including EGFR, HER2 and ERBB3, were also activated in ARID1A-KD cells (Fig. 3B). WB analysis also confirmed increases in phosphorylated EGFR (p-EGFR, P = 0.0149), phosphorylated PI3K (p-PI3K, P = 0.0136) and phosphorylated Akt (p-Akt, P = 0.0091) proteins (Fig. 3E). Hence, ARID1A-KD activates the EGFR/PI3K/Akt/mTOR signaling pathway, which inhibits autophagy in EGFR-mutant LUAD cells.

Fig. 3figure 3

ARID1A-KD activates the EGFR/PI3K/Akt/mTOR signaling pathway and inhibits autophagy in EGFR-mutant lung adenocarcinoma cells. AC 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

ARID1A-KD enhances production of type I interferon via the Rig-I-like receptor pathway in EGFR-mutant LUAD cells

GSEA based on the KEGG database suggested activation of the Rig-I-like receptor pathway (P < 0.0001). The three main downstream signaling pathways of the Rig-I-like receptor pathway, including the IRF7 pathway (Fig. 4A), MAPK pathway (Fig. 4B) and NF-κB pathway (Fig. 4C), were all stimulated in ARID1A-KD cells. The proteins listed in the heatmap in Fig. 4B, D were also significantly upregulated in ARID1A-KD cells. WB results for relative proteins are displayed in Fig. 4E. MS for total proteins showed significant upregulation of IRF7 (P < 0.0001, Fig. 4A). MS for phosphorylated proteins and WB suggested activation of the MAPK pathway and especially upregulation of phosphorylated MAP2K4 (Fig. 4B) as well as P38 MAPK (P = 0.0360, Fig. 4E). Phosphorylated IKBKG (P = 0.0024, Fig. 4C) and NF-κB2 (P = 0.0148, Fig. 4C) were upregulated in ARID1A-KD cells based on MS, which indicated attenuation of NF-κB inhibition and activation of the pathway, respectively. Given this evidence, the Rig-I-like receptor was activated, and one of the effects of this pathway is production of type I IFN. Through WB, expression of IFN-α (P = 0.0016) and IFN-β (P = 0.0105) was significantly upregulated in ARID1A-KD cells (Fig. 4E). RNA-seq and MS for phosphorylated proteins also revealed that IFN-induced proteins, such as IFI16, IFI27, IFI35, and IFI6, as well as phosphorylated IFIH1 (P = 0.0360) and IFIT1 (P = 0.0070), were obviously upregulated in ARID1A-KD cells, as displayed in Fig. 4D. Interestingly, we found that IFNGR2, which associates with IFNGR1 to form a receptor for the cytokine IFN-γ, was upregulated in ARID1A-KD cells (P = 0.0011, Fig. 4E). GSEA for DEGs based on the GO database suggested significantly improved response to IFN-γ in ARID1A-KD cells with EGFR mutation (P < 0.0001, Fig. 4F).

Fig. 4figure 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

Inhibition of autophagy in EGFR-mutant LUAD cells reverse type I IFN production, with an effect similar to ARID1A-KD

According to the KEGG database (https://www.genome.jp/pathway/map04622), the process of autophagy, especially the protein Atg5, inhibits signaling from Rig-I to downstream pathways and type I IFN production. Therefore, we propose that inhibition of the autophagy process reverses type I IFN production in EGFR-mutant LUAD cells, similar to the effect of ARID1A-KD. Then, 2 mM 3-methyladenine (3-MA, SELLECK, S2767) and 100 µM rapamycin (SELLECK, S1039) were administered to NC and ARID1A-KD cells for 24 h [30]; total protein was harvested, and WB experiments were performed (Fig. 5A). Expression levels of p-mTOR and P62 were used to verify the drug effect on cells. Rapamycin inhibited the mTOR pathway in ARID1A-KD cells, downregulated expression of p-mTOR, and promoted autophagy, as indicated by downregulation of P62 expression. 3-MA inhibited the autophagy process and upregulated expression of P62 in NC cells. After treatment with the corresponding drug, production of type I IFN changed significantly. As displayed in Fig. 5A, inhibition of autophagy significantly elevated expression of IFN-α and IFN-β in NC cells, approaching the expression level in ARID1-KD cells. In contrast, autophagy promotion in ARID1A-KD cells obviously downregulated expression of IFN-α and IFN-β. In addition, after reversion of autophagy activity, production of IFN-α and IFN-β in NC cells was significantly higher than that in ARID1A-KD cells. In summary, autophagy activity in EGFR-mutant LUAD cells determines production of type I IFN.

Fig. 5figure 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

To better elucidate the mechanism by which ARID1A-KD enhances production of type I IFN in EGFR-mutant LUAD cells, we plotted a schematic diagram, as depicted in Fig. 5B. Distinct activation of the EGFR/PI3K/Akt/mTOR pathway in ARID1A-KD LUAD cells harboring EGFR mutation inhibits autophagy activity and downregulates expression of autophagy-related proteins, such as Atg5. Moreover, downregulation of autophagy-related proteins attenuates inhibition of the Rig-I-like receptor pathway and downstream pathways, including the P38 MAPK, IRF7 and NF-κB pathways, and elevates type I IFN gene transcription, translation and secretion. Ultimately, type I IFN in the LUAD stroma increases immune cell infiltration, remodels the immune phenotype and reverses response to immunotherapy.

EGFR-mutant LUAD patients with low ARID1A expression have enhanced immune filtration and a favorable response to anti-PD(L)1 therapy

The evidence above indicates the mechanisms related to ARID1A-KD through which a better response to ICIs occurs, and further verification was performed using EGFR-mutant LUAD cohorts from our cancer center and TCGA. In total, 10 EGFR-mutant LUAD patients who received ICIs combined with chemotherapy and/or antiangiogenic therapy were enrolled for survival analysis based on ARID1A expression, and representative images of IHC for ARID1A are displayed in Fig. 6A. EGFR-mutant LUAD patients with low ARID1A expression had better progression-free survival (PFS) than those with high ARID1A expression (5.63 months versus 1.59 months, P = 0.0458), and the hazard ratio (HR) was 0.16 (95% confidential interval [CI]: 0.03–0.97) (Fig. 6B). The ARID1A low expression group had a higher disease control rate (DCR) than the high expression group (80% versus 25%) in the best response to ICI treatment. Furthermore, we estimated immune cell infiltration, mainly including IFA signals (AU) of CD3 and CD8, in the enrolled EGFR-mutant LUAD patients (n = 49). Low ARID1A expression was associated with more abundant AU of both CD3 and CD8 (Fig. 6C). Quantitatively, as shown in Fig. 6D, EGFR-mutant LUAD patients with low ARID1A expression had higher AU of CD3 (P < 0.0001) and CD8 (P = 0.0003) compared with the ARID1A high expression group. The linear correlation between ARID1A expression and immune cell infiltration was then detected in EGFR-mutant LUAD patients, as illustrated in Fig. 6E. ARID1A expression correlated negatively with CD3-positive T cells (P < 0.0010, r = − 0.542) and CD8-positive T cells (P < 0.0010, r = − 0.551) in our cohort of patients and with activated CD4-positive T cells (P = 0.0300, r = − 0.272) and effector memory CD4-positive T cells (P = 0.0100, r = − 0.318) in the cohort of patients from TCGA.

Fig. 6figure 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

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