2644 - AARS1-Mediated Lactylation of XRCC5 Regulates the Stability of FOXO1 to Control Radioresistance In Nasopharyngeal Carcinoma

Purpose/Objective(s): The hypothesis is that AARS1-mediated lactylation of XRCC5 stabilizes FOXO1, pre-programming nasopharyngeal carcinoma (NPC) cells for radioresistance. NPC relies heavily on radiotherapy, yet radioresistance drives recurrences. EBV-associated NPC exhibits enhanced glycolysis and lactate accumulation, promoting progression via protein lactylation. This study examines AARS1 in radioresistant NPC cells to define its role in metabolic reprogramming and lactylation-mediated radioresistance, focusing on the novel AARS1/XRCC5/FOXO1 signaling axis.

Materials/Methods: AARS1 expression was examined using public NPC datasets and tumor biopsies. Survival outcomes were analyzed via Kaplan-Meier and Cox regression models. Stable AARS1 knockdown and overexpression models were generated in CNE-2 and 5-8F cells. Radioresistance (0-8 Gy) was evaluated by CCK-8 and colony formation assays. DNA damage response was monitored via ?-H2AX foci. Protein interactions and stability under basal conditions were evaluated via Mass Spectrometry (MS), Co-Immunoprecipitation, and Cycloheximide/MG132 assays. Phenotypic rescue experiments were performed by either pretreating AARS1-knockdown cells with exogenous lactic acid or genetically overexpressing FOXO1.

Results: AARS1 was significantly upregulated in NPC tissues, and strikingly enriched in radioresistant clinical specimens compared to radiosensitive cohorts. Furthermore, high AARS1 expression independently predicted poorer progression-free (P<0.01) and overall survival (P<0.05). AARS1 knockdown sensitized cells to irradiation and delayed ?-H2AX foci clearance. Mechanistically, MS and Co-IP revealed a constitutive basal interaction between AARS1 and the DNA repair scaffold XRCC5 (Ku80). AARS1-mediated lactylation of XRCC5 altered its conformation, enhancing its physical binding to the transcription factor FOXO1. This interaction robustly shielded FOXO1 from proteasomal degradation, leading to its stabilization and subsequent transcriptional activation of downstream survival networks upon radiation. Crucially, supplementation with exogenous lactate driven residual enzyme kinetics to restore XRCC5 lactylation, and both lactate pretreatment and FOXO1 overexpression fully rescued the radiosensitizing effects induced by AARS1 knockdown.

Conclusion: AARS1 serves as a robust prognostic marker for NPC radiotherapy failure. It confers radioresistance by basally lactylating XRCC5, creating a protective scaffold that stabilizes FOXO1 to orchestrate a rapid response to radiation-induced DNA damage. Both metabolic restoration via exogenous lactate and genetic restoration of FOXO1 effectively reverse AARS1 depletion-induced radiosensitivity. These findings prove that AARS1 drives radioresistance strictly through this epigenetic-metabolic axis, highlighting the AARS1/XRCC5/FOXO1 pathway as a promising therapeutic target for NPC.