3458 - Glutamine Synthetase and Radiosensitivity in Non-Small Cell Lung Cancer via a STAT5-Regulated Metabolic-DNA Repair Axis

Purpose/Objective(s):

Enhancing radiosensitivity remains a critical challenge in the treatment of non-small cell lung cancer (NSCLC). Metabolic reprogramming, particularly dysregulated glutamine metabolism, is increasingly recognized as a key driver of tumor response to radiation. Glutamine synthetase (GS), a central enzyme in this pathway, represents a potential target for modulating radiosensitivity, yet its specific role in the DNA damage response (DDR) remains elusive. This study aims to evaluate the impact of GS knockdown on NSCLC radiosensitivity and to elucidate the underlying mechanism linking GS-mediated metabolism to DDR efficiency.

Materials/Methods:

Stable GS-knockdown models were established in A549 and H1299 NSCLC cell lines. Changes in radiosensitivity were quantified using clonogenic survival assays following exposure to graded radiation doses (0, 2, 4, 6, and 8 Gy). To dissect the molecular mechanism, immunofluorescence staining for ?-H2AX foci was performed at various time points post-irradiation to monitor the kinetics of DNA double-strand break repair. Additionally, bioinformatic promoter analysis (MatInspector, JASPAR) was conducted to identify potential binding sites for stress-responsive transcription factors, with a focus on the STAT family, that may regulate GS expression and consequently influence radiosensitivity.

Results:

GS knockdown significantly enhanced radiosensitivity in both A549 and H1299 cells, evidenced by a marked reduction in clonogenic survival compared to controls. Mechanistically, while initial DNA damage induction was comparable, GS-deficient cells exhibited a significant delay in ?-H2AX foci clearance, indicating a compromised capacity for DNA repair. Although direct metabolic profiling was not performed in this study, this repair defect is highly consistent with established literature suggesting that GS deficiency leads to depleted -ketoglutarate ( -KG) levels and elevated reactive oxygen species (ROS). These metabolic perturbations are known to impair the activity of -KG-dependent dioxygenases and hinder the recruitment of essential repair complexes, thereby sensitizing cells to radiation. Furthermore, bioinformatic analysis identified conserved STAT5 binding motifs in the GS promoter region, suggesting that targeting the STAT5-GS axis could be a viable strategy to modulate radiosensitivity.

Conclusion:

This study identifies GS as a critical regulator of radiosensitivity in NSCLC, functioning as a metabolic checkpoint for efficient DNA damage repair. Our findings suggest that GS likely sustains DDR fidelity by maintaining the metabolic milieu (e.g., -KG/ROS homeostasis) necessary for repair protein function, under the potential transcriptional control of STAT5. Consequently, targeting the STAT5-GS metabolic axis represents a promising therapeutic strategy to sensitize NSCLC tumors to radiotherapy by disrupting their metabolic support for DNA repair.