Activation of a pyruvate-acetaldehyde-acetate metabolic shunt supports redox homeostasis in osimertinib-resistant EGFR-mutant non-small cell lung cancer

Resistance to epidermal growth factor receptor (EGFR)-targeted therapies remains a major obstacle in the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). Here, we report a metabolic adaptation in osimertinib-resistant (OsiR) cells characterized by elevated acetate levels and activation of an unconventional pyruvate-acetaldehyde-acetate (PAA) shunt. While our previous work explored the PAA pathway from a functional perspective, including mitochondrial remodeling and in vivo analyses in transgenic models, the present study focuses on detailed biochemical and metabolite-level adaptations occurring in osimertinib-resistant NSCLC cells. Integrated transcriptomic, exometabolomic, and targeted protein analyses were performed in parental (Par) and OsiR H1975 NSCLC cells. Extracellular metabolites were quantified using high-resolution chromatographic platforms including LC-HRMS, HPLC-DAD, and GC-MS, and pathway analysis was performed using MetaboAnalyst. Resistant cells displayed suppression of canonical NADPH-producing pathways, particularly the oxidative branch of the pentose phosphate pathway (PPP), concomitant with activation of an alternative metabolic route marked by elevated extracellular pyruvate, acetaldehyde, and acetate. Transcriptomic and protein analyses revealed upregulation of the aldehyde dehydrogenases ALDH2 and ALDH7A1, enzymes capable of catalyzing NADP⁺-dependent oxidation of acetaldehyde to acetate. These findings support engagement of a PAA metabolic shunt that contributes to adaptive NADPH turnover and supports redox balance despite severe NADPH depletion when canonical pathways are compromised. Resistant cells also exhibited enhanced glycolytic flux, increased acetate production, and elevated expression of enzymes involved in acetate utilization and acetyl-CoA metabolism. Together, these findings identify a resistance-associated metabolic adaptation involving activation of an unconventional PAA pathway that may serve as a de novo alternative source of reducing power. By focusing on specific metabolic intermediates and enzyme-level changes, this work provides mechanistic insights complementary to our previous functional study. The PAA pathway may represent a metabolic vulnerability in osimertinib-resistant EGFR-mutant NSCLC.