TLR9 agonists as a potential therapeutic option for B-ALL patients with low P53 expression.

6526 Background: B-cell acute lymphoblastic leukemia (B-ALL) is a highly malignant hematologic cancer with poor prognosis, especially in relapsed or refractory cases. Abnormal P53 expression is more prevalent in relapsed/refractory B-ALL and is associated with increased drug resistance. B-ALL cells in the bone marrow (BM) promote the survival of malignant cells by suppressing P53 accumulation. Therefore, identifying drugs that can reactivate P53 signaling may provide novel therapeutic strategies for patients with low P53 expression, particularly by targeting BM-resident B-ALL cells. Methods: Building on our previous findings that TLR9 agonists can activate the P53 signaling pathway and preferentially eliminate BM-resident B-ALL cells over peripheral B-ALL cells, we investigated the mechanisms by which TLR9 agonists regulate glucose metabolism through P53 to induce B-ALL apoptosis. We constructed P53 knockdown cell lines, conducted seahorse assays to analyze glucose metabolism, and performed RNA sequencing to identify key molecules. Mechanistic studies focused on the regulation of glucose metabolism and its P53-dependent pathways. Furthermore, we elucidated the role of glucose metabolism in TLR9 agonist-induced reactive oxygen species (ROS) production and mitochondrial apoptosis. Results: TLR9 agonists preferentially eliminate BM-resident B-ALL cells via P53-dependent pathways. Specifically, TLR9 agonists suppress glycolysis while enhancing oxidative phosphorylation and ROS generation through the P38-P53-TIGAR signaling axis. Elevated ROS levels further facilitate the formation of the BAX/BAK/TOM20 complex, promote TOM20 oxidation and accumulation, and activate BAX. The P53-TIGAR-ROS-MOMP axis was identified as the critical mechanism underlying TLR9 agonist-induced apoptosis in B-ALL. Additionally, patient-derived xenograft models confirmed that ROS generation is key to the efficient clearance of BM-resident B-ALL cells by TLR9 agonists. Conclusions: As P53-reactivating agents, TLR9 agonists selectively eliminate B-ALL cells while preserving immune cell anti-tumor function. This study highlights the role of glucose metabolism in the TLR9 agonist-mediated clearance of BM-residual B-ALL cells, providing a potential maintenance or combination therapy strategy for relapsed/refractory B-ALL patients, particularly those with low P53 expression.