EGFR and IRE1α pathways are associated with distinct immunomodulatory gene expression profiles in NSCLC cells with acquired resistance to EGFR TKIs.

EGFR-TKI-resistant cell lines were established by long-term exposure to gefitinib, afatinib, and osimertinib via the PC9 model. This model helps study EGFR-TKI resistance mechanisms in non-small cell lung cancer (NSCLC) and may offer insights to improve treatment outcomes, particularly for patients unresponsive to anti-PD-1/PD-L1 therapies. We investigated molecular alterations in these resistant cell lines using multi-omics techniques, including genome sequencing, proteomics, and transcriptomics. Differential dependencies on EGFR downstream pathways were observed among resistant cell lines. Immune evasion mechanisms were analyzed to identify alterations in EGFR downstream pathways and unfolded protein response (UPR) elements contributing to immune-related transcriptional patterns. Afatinib-resistant cells exhibited the most pronounced immunosuppressive-like transcriptional features compared to gefitinib- and osimertinib-resistant cells. This was characterized by elevated PD-L1 expression, minimal changes in MHC class 1 levels, down-regulation of shared immune-modulated gene sets, and a cytokine profile favoring immunosuppression (e.g., lower IL-6, CXCL10, IFN-γ, CCL22; higher IL-8). These findings suggest a poor prognosis and limited efficacy of anti-PD-1/PD-L1 immunotherapy in NSCLC patients with similar resistance profiles. Notably, inhibition of IRE1α signaling partially reversed this immunosuppressive-like transcriptional signature, indicating a potential strategy to restore immune responsiveness in afatinib-resistant NSCLC. Our study underscores the distinct remodeling effects of different EGFR-TKIs on tumor-cell-intrinsic immunoregulatory pathways. Targeting endoplasmic reticulum (ER) stress pathways alongside immune checkpoint inhibitors may be crucial for overcoming resistance mechanisms identified here. These insights provide a rationale for personalized treatment strategies tailored to the immune-related gene-expression profiles observed in EGFR-TKI-resistant NSCLC models, aiming to enhance therapeutic responses and improve clinical outcomes.