S2637
Radiobiology - Tumour radiobiology
ESTRO 2026
These tumors actually show oxygen heterogeneity, highlighting the need to explore their roles in normoxic conditions as well. We investigated how loss of HIF1 α or HIF2 α in a normoxic state can affect metabolism, redox homeostasis, and sensitivity to radiotherapy combined with metabolic inhibition. Material/Methods: We used NSCLC and GBM cell models with HIF1 α or HIF2 α knockout (KO) and HIF α wildtype (WT) controls. Metabolomics using 13C6-glucose mass spectrometry tracing was performed to assess glycolytic flux, ser/gly synthesis, nucleotide, TCA cycle metabolite production, and redox balance. Cancer models were exposed to 6 Gy irradiation to assess radiation- induced metabolic changes. Additional pharmacological inhibition of serine/glycine (ser/gly) synthesis was tested using the repurposed SHMT — serine to glycine catalyzing enzyme — inhibitor sertraline (Figure 1A) using clonogenic assays and incucyte growth curves. Results: Both HIF1 α and HIF2 α KO cells displayed reduced glycolysis and compensatory ser/gly pathway hyperactivation. HIF1 α KO cells channeled ser/gly into nucleotide (particularly TTP) synthesis and glutathione (GSH)-mediated antioxidant defense, conferring radiotherapy resistance. In contrast, HIF2 α KO cells preferentially used serine for α -ketoglutarate ( α -KG) production, fueling TCA cycle intermediates, glutamate, and proline synthesis, and relied on NADH/methionine-dependent redox systems. Following irradiation, only the radiation resistant HIF1 α KO cells further enhanced ser/gly metabolism, increasing AMP/ATP and GSH/GSSG ratios, whereas HIF2 α KO cells failed to adapt and accumulated oxidative stress. Functionally, HIF1 α KO cancer cells were more sensitive to pharmacological inhibition of ser/gly metabolism by sertraline, particularly in combination with irradiation, which abrogated their radioresistant phenotype (Figure 1B/C). Conclusion: Loss of HIF1 α or HIF2 α drives distinct metabolic dependencies in cancer cells under normoxic conditions. HIF1 α KO cells rely on ser/gly synthesis for nucleotide and antioxidant defense, promoting radiotherapy resistance but rendering them vulnerable to sertraline plus radiotherapy. HIF2 α KO cells favor α -KG metabolism and alternative redox systems. Targeting ser/gly synthesis may overcome HIF-gradient dependent radiotherapy resistance and improve radiotherapy efficacy in tumors that are characterized by low HIF expression.
Clonogenic survival was determined using colony formation assays (CFAs): Survival was assessed following the treatment with different concentrations of the NQO1 inhibitor dicoumarol (10-40 µM), Keap1 inhibitor ML334 (1-10 µM) and the NQO1 prodrugs β - lapachone (0.25-1 µM) and streptonigrin (20-40 nM) and irradiated after an incubation period of one hour (0; 2; 4; 8 Gy). To further investigate the role of NQO1, four murine PDAC cell lines were transfected with a non-viral ORF clone to increase NQO1 expression. Results: Proteomic analysis revealed significantly increased NQO1 expression in radioresistant PDAC cell lines. These results were validated by Western blotting, showing increased NQO1 and the highest Keap1 expression in radioresistant murine and human PDAC cell lines. Combination treatments of radiation with NQO1 inhibition, Keap1 inhibition or prodrug activation were established but did not result in significant radiosensitizing effects in CFAs. Stable transfection of NQO1-overexpressing murine PDAC cell lines was generated for further functional analyses. Conclusion: NQO1 and Keap1 are upregulated in radioresistant PDAC cell lines, indicating their central role in radiation resistance in PDAC. The established NQO1- overexpressing models will be used to investigate redox regulation, reactive oxygen species (ROS) generation using flow cytometry and clonogenic survival following irradiation to further elucidate the role of NQO1 in PDAC radioresistance. This may uncover novel therapeutic opportunities to sensitize PDAC to radiotherapy and improve treatment outcomes. Keywords: pancreatic cancer, NQO1, ROS Proffered Paper 4140 Distinct serine/glycine pathway usage in HIF1α and HIF2α deficient cancer models determines radiotherapy resistance and therapeutic opportunities Anais Sanchez-Castillo 1,2 , Jairo Lommen 1 , Denise Consten 1 , Kim Savelkouls 1 , Kasper Rouschop 1 , Marc Vooijs 1 , Kim Rosalie Kampen 1,2 1 Radiation Oncology, University Maastricht, Maastricht, Netherlands. 2 Oncology, KU Leuven, Leuven, Belgium Purpose/Objective: Hypoxia-inducible factors (HIF1 α , HIF2 α ) are both implicated to affect radiotherapy responses in non- small cell lung cancer (NSCLC) and glioblastoma (GBM). While the role of HIFs is well studied in relation to tumor hypoxia, little is known about their function in normoxic conditions related to metabolic rewiring.
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