Imatinib Resistance in Chronic Myeloid Leukemia Is Associated with Distinct Metabolic, Oxidative, and Autophagic Adaptations in K562-Derived Cell Sublines

Background/Objectives: Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy initiated by the BCR::ABL1 oncoprotein. Although tyrosine kinase inhibitors (TKIs) have revolutionized CML therapy, resistance remains a major clinical challenge. Emerging evidence suggests that metabolic reprogramming contributes to TKI resistance, yet its relationship with oxidative stress and autophagy remains poorly characterized. Methods: Metabolic profiles, redox-related parameters, and autophagy markers were assessed in parental K562, imatinib-sensitive K562-S, and imatinib-resistant K562-R cells. Metabolic activity was first assessed via the MTT assay to determine the half-maximal inhibitory concentration (IC50) for each line, which was subsequently used as the treatment dose. Glycolytic and mitochondrial functions were profiled via Seahorse extracellular flux analysis under basal conditions. Oxidative stress was assessed via reactive oxygen species (ROS) and nitric oxide (NO) levels, and the levels of autophagy markers (p62 and LC3-I/II) were measured via Western blotting. Results: Imatinib reduced metabolic activity in K562 and K562-S cells (IC50 = 0.445 and 0.137 µM, respectively), whereas K562-R cells retained high metabolic activity across the tested range (0.1–1 µM), which was consistent with a resistant phenotype. Compared with parental K562 cells, both K562-S and K562-R cells presented reduced glycolytic activity. K562-S cells displayed greater basal respiration than K562-R cells, which presented reduced basal respiration and proton leakage but preserved spare respiratory capacity, indicating a distinct mitochondrial respiratory phenotype. Imatinib increased ROS in K562 and K562-S cells but not in K562-R cells, whereas NO increased in all three lines. Autophagy-related alterations occurred in K562 and K562-S cells, while K562-R cells presented minimal changes. Conclusions: Imatinib resistance involves distinct metabolic and autophagic adaptations across K562-derived cell lines. These pathways may represent potential therapeutic targets warranting further investigation to overcome TKI resistance in CML.