ANXA2 + Small Extracellular Vesicles Drive Chemoresistance in Anaplastic Thyroid Cancer by Promoting XRCC5 Lactylation and Enhancing Non‐Homologous End‐Joining Repair

ABSTRACT Anaplastic thyroid carcinoma (ATC) is an exceptionally aggressive malignancy with dismal survival, largely due to intrinsic cisplatin resistance. This study identifies a novel mechanism by which small extracellular vesicles (sEVs) promote chemoresistance by enhancing DNA repair via protein lactylation. ATC cells secrete sEVs enriched with Annexin A2 (ANXA2). Upon delivery to recipient ATC cells, ANXA2 stabilizes the interaction between SRC kinase and lactate dehydrogenase A (LDHA), leading to increased LDHA phosphorylation (Y10), enzyme activity, and lactate production. The resulting lactate surge serves as a substrate for lysine lactylation. Ku80 (XRCC5) is identified as a key lactylation target at K265, catalyzed by the acyltransferase KAT5. This lactylation modification strengthens the interaction between Ku80 and its partner Ku70 (XRCC6), stabilizing the initial DNA‐end binding complex in the non‐homologous end‐joining (NHEJ) repair pathway. Consequently, NHEJ efficiency is significantly enhanced, enabling ATC cells to rapidly repair cisplatin‐induced DNA double‐strand breaks and survive treatment. Genetic disruption of the XRCC5‐K265 lactylation site or pharmacological inhibition of LDHA sensitizes ATC xenograft tumors to cisplatin, while in vitro, inhibition of the SRC/LDHA axis produces a similar chemosensitizing effect. This work unveils the ANXA2 + sEV/SRC/LDHA/lactate/XRCC5‐lactylation axis as a critical driver of NHEJ‐mediated chemoresistance in ATC, offering new potential therapeutic targets.