PLCG1 and RAC1 orchestrate adaptive cellular programs driving osimertinib-tolerant persister cell formation in malignant pleural effusion-associated non-small cell lung cancer.

Acquired resistance to epidermal growth factor receptor (EGFR)-targeted therapies remain a major challenge in non-small cell lung cancer (NSCLC), particularly in patients with malignant pleural effusion (MPE). The MPE microenvironment, characterized by acidic, cytokine- and metabolite-rich conditions, promotes the emergence of osimertinib-tolerant persister cells (OTPCs), contributing to disease relapse. In this study, we investigated the role of MPE in driving OTPC formation and identified key molecular regulators underlying this adaptive phenotype. MPE samples from advanced EGFR-mutant NSCLC were used to generate OTPCs through coculture with PC9 and H1975 cell lines. In contrast, non-malignant pleural effusions induced only limited tolerance. Transcriptomic profiling revealed extensive reprogramming in OTPCs, with PLCG1 and RAC1 among the most significantly upregulated genes, enriched in pathways related to glycolysis, hypoxia, and epithelial-mesenchymal transition. Functional analyses demonstrated that OTPCs exhibit enhanced macropinocytosis, metabolic flexibility, and invasive capacity. Mechanistically, PLCG1 and RAC1 formed a co-dependent signaling network, as supported by reciprocal knockdown and protein interaction studies. Inhibition of PLCG1 significantly impaired both mitochondrial respiration and glycolytic activity, reduced mesenchymal marker expression, and decreased OTPC viability by >60%, thereby restoring sensitivity to osimertinib. In vivo, combined inhibition of EGFR and PLCG1 resulted in sustained tumor suppression and improved survival without detectable toxicity. Collectively, these findings identify a co-dependent PLCG1-RAC1 signaling network that integrates metabolic adaptation and phenotypic plasticity to sustain drug tolerance in MPE-associated NSCLC. Targeting this pathway represents a promising strategy to overcome resistance to EGFR-directed therapies.