Multi-Omics and Single-Cell Insights Reveal a Lysophosphatidic Acid (LPA)-Mediated Resistant Mechanism to Third Generation EGFR-TKI in Non-Small Cell Lung Cancer

Introduction: Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of EGFR-sensitive-mutant non-small cell lung cancer (NSCLC). However, acquired resistance remains a significant challenge. This study investigates the metabolic mechanisms driving third-generation EGFR-TKI resistance. Methods: We conducted plasma metabolomics analysis on 216 longitudinal samples from 186 NSCLC patients enrolled in the clinical trial of rezivertinib (NCT03386955). Additionally, multi-omics profiling of rezivertinib-resistant cell lines, functional in vitro experiments, and single-cell RNA sequencing (scRNA-seq) analyses of 215 NSCLC patients were integrated to reveal underlying mechanisms. Results: Non-responder patients exhibited elevated glycerophospholipids and dysregulated lysophospholipid (LPL) metabolism. Unsupervised clustering identified two patient subgroups, with Cluster 1 (characterized by high LPL levels) associated with poorer survival (P = 0.022). Metabolite-based predictive model achieved robust performance (AUC: 0.7762 [training], 0.7485 [test]). Longitudinal analyses demonstrated LPLs and lysophosphatidic acid (LPA) accumulation during the resistance process. Integrated multi-omics analyses highlighted epithelial-mesenchymal transition (EMT) and glycerophospholipid reprogramming in rezivertinib-resistant cells. Functional assays confirmed that LPA promoted cell migration and invasion, and attenuated the efficacy of third-generation EGFR-TKI, while disruption of the LPA-LPAR signaling axis reversed LPA-mediated resistance. scRNA-seq identified an LPA-secreting malignant subset (cluster c4), characterized by enhanced EMT activation and extensive microenvironmental crosstalk through Wnt, TGF-β, and ECM signals. Conclusions: Our study highlights the pivotal role of LPA-mediated signaling and metabolic reprogramming in third-generation EGFR-TKI resistance. Targeting LPA production or its downstream pathways may offer novel therapeutic strategies to overcome resistance. This study provides critical metabolic insights for managing EGFR-sensitive-mutant NSCLC.