Reprogramming of Osimertinib-Resistant EGFR-mutant NSCLC: The Pyruvate-Acetaldehyde-Acetate Pathway As a Key Driver of Resistance

Osimertinib (Osi) resistance remains a significant challenge in EGFR mutant non-small-cell lung cancer (NSCLC). This study investigates the metabolic reprogramming associated with Osi resistance, identifying key metabolic vulnerabilities that may be targeted for therapeutic intervention. Employing the EGFR-mutant H1975 parental (Par) cell line and its Osi-resistant (OsiR) counterpart, we integrated transcriptomics, metabolomics, nuclear and mitochondrial genomics, functional assays and bioanalytical techniques, as well as advanced 3D imaging to comprehensively define the resistant phenotype. We found that OsiR cells exhibit mitochondrial dysfunction, including impaired oxidative phosphorylation (OXPHOS), mitochondrial DNA mutations, and altered mitochondrial gene expression. To describe this systems-level characterization, we introduce the concept of mitochondromics, a comprehensive profiling of mitochondrial genomic, transcriptomic, structural, and functional changes contributing to therapeutic resistance. Metabolomic profiling revealed a significant accumulation of glycolytic intermediates (lactate, pyruvate, acetate, and acetaldehyde) in the extracellular medium, indicating a shift toward glycolysis and activation of alternative metabolic pathways, including the Warburg effect. Notably, we identified the pyruvate-acetaldehyde-acetate (PAA) pathway as a functionally repurposed metabolic route that facilitates NADPH production, which is critical for antioxidant defense and anabolic processes in OsiR cells. Additionally, although the pentose phosphate pathway (PPP) is not the primary source of NADPH in OsiR cells, it plays a supporting role in biosynthesis, contributing to the production of amino acids, nucleotides, and vitamins. Altered expression of enzymes involved in glycolysis, the TCA cycle, and both oxidative and non-oxidative arms of the PPP further supports an adaptive metabolic network promoting cell growth and resistance to Osi. This study reveals a complex metabolic reprogramming in Osi-resistant EGFR-mutant NSCLC, where a newly identified role for the PAA pathway, alongside integrated mitochondromic alterations emerges as key driver of resistance. These insights uncover potential metabolic vulnerabilities of Osi-resistant tumors and provide a foundation for developing therapeutic strategies to counteract resistance and improve osimertinib efficacy. Targeting these metabolic pathways may offer promising avenues for overcoming resistance in clinical settings.