Normoxic HIFα-mediated metabolic rewiring in cancer as a novel therapeutic target of tumor heterogeneity

Hypoxia-inducible factors (HIF1α, HIF2α) influence radiotherapy responses in non-small cell lung cancer (NSCLC) and glioblastoma (GBM), tumors characterized by oxygen and HIF expression heterogeneity. As the function of HIFs in normoxic metabolic function remained unexplored, we investigated how loss of HIF1α or HIF2α affects metabolism, redox homeostasis, and radiotherapy sensitivity in normoxia, aiming to identify opportunities for combined metabolic inhibition. NSCLC HIF1α or HIF2α knockout (KO) and HIFα wildtype (WT) models were analyzed using 13C-glucose mass spectrometry tracing before and after radiotherapy treatment. Metabolic phenotypes were validated using serine/glycine (ser/gly) synthesis enzyme expression by immunoblot and quantitative PCR, redox by ROS flow cytometry analysis, and DNA methylation by 5mC dot-blot assessment. Pharmacological inhibition of ser/gly metabolism was performed in both NSCLC and GBM models using the repurposed serine-glycine conversion inhibitor sertraline using incucyte confluency monitoring. Both HIF1α and HIF2α KO cells displayed reduced glycolysis and compensatory ser/gly pathway hyperactivation. HIF1α KO cells channeled ser/gly into nucleotide (particularly TTP) synthesis and glutathione (GSH)-mediated antioxidant defense, conferring radiotherapy resistance. In contrast, HIF2α KO cells preferentially used serine for α-ketoglutarate (α-KG) production, the enhanced NADH/methionine-dependent redox system and the methionine cycle to support enhanced DNA methylation. Subsequently, following irradiation, only the radiation resistant HIF1α KO cells further enhanced ser/gly metabolism, increasing AMP/ATP and GSH/GSSG (oxidized GSH) ratios, whereas HIF2α KO cells failed to adapt and accumulated oxidative stress. HIF1α KO cells were more sensitive to pharmacological inhibition of ser/gly metabolism by sertraline, particularly in combination with irradiation, which abrogated their radioresistant phenotype in both NSCLC and GBM models. HIF1α-deficient cells rely on ser/gly synthesis for nucleotide production and antioxidant defense, promoting radiotherapy resistance while creating vulnerability to sertraline plus irradiation. HIF2α-deficient cells favor α-KG production and methionine-driven alternative redox and methylation pathways. Targeting ser/gly synthesis may overcome HIF-gradient–dependent radiotherapy resistance.