Hedgehog Signaling Regulates Hypoxia-Associated Metabolic Adaptation in Myeloid Leukemia In Vitro Cell Models

Hedgehog (Hh) signaling regulates cell survival and microenvironmental responses in various cancers, but its role in metabolic adaptation to the hypoxic bone marrow microenvironment in myeloid malignancies remains insufficiently defined. K562, KU812, and U937 cells were cultured under normoxic or hypoxic conditions and treated with the Smoothened (Smo) antagonist Cyclopamine or the agonist SAG (Smoothened Agonist). Cell proliferation, apoptosis- and autophagy-related markers, glycolysis-associated proteins, and metabolic parameters were assessed by viability assays, Western blotting, immunofluorescence, and biochemical assays. Selected findings were further evaluated in CRISPR/Cas9-mediated Smo knockout cells. Smo inhibition reduced cell proliferation, increased PARP cleavage, and decreased BNIP3 expression under both oxygen conditions. It also downregulated glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase (LDH), monocarboxylate transporter 1 (MCT1), carbonic anhydrases IX and XII (CAIX and CAXII), and was associated with reduced glucose consumption, lactate production, and ATP levels. These changes were associated with modulation of the AMPK–mTOR axis and with reduced phosphorylation of mTOR downstream effectors. These findings support a role for Hh signaling in hypoxia-driven metabolic adaptation in myeloid malignancy cell models and suggest a functional link between Smo activity, glycolytic remodeling, and AMPK-mTOR-related signaling.