Gambogic acid inhibits tumor growth and induces HMGB1-mediated pyroptosis in AML models in vitro and in vivo.

[BACKGROUND] Acute myeloid leukemia (AML) is a highly aggressive hematologic malignancy with poor prognosis, high relapse rates, and frequent drug resistance, underscoring the urgent need for novel therapeutic strategies. Gambogic acid (GA) has demonstrated broad antitumor potential, though its mechanisms in AML remain unclear.

[PURPOSE] This study aimed to identify the direct molecular target of GA in AML and elucidate the mechanism by which it exerts its anti-leukemic effects, with a focus on HMGB1-mediated pathways.

[METHODS] We evaluated the anti-leukemic activity of GA both in vitro (using HL-60 and THP-1 cell lines) and in vivo (using xenograft mouse models). Activity-based protein profiling (ABPP) was employed to identify direct protein targets of GA. Binding interactions were validated through cellular thermal shift assay (CETSA), bio-layer interferometry (BLI), and immunofluorescence. Transcriptomic and proteomic analyses were conducted to explore downstream mechanisms. Functional assays including cell cycle, apoptosis, comet, TUNEL, and ROS detection were performed. HMGB1 knockdown was used to confirm its role in GA-induced pyroptosis.

[RESULTS] GA significantly inhibited tumor growth and prolonged survival in AML-bearing mice without obvious toxicity. ABPP identified HMGB1 as a direct target of GA, which was confirmed by CETSA and BLI. Multi-omics analysis revealed that GA treatment led to downregulation of pathways involved in cell cycle, DNA replication, and repair. GA induced DNA damage, ROS accumulation, and GSDMD-mediated pyroptosis via the AIM2 inflammasome pathway. GA did not aggravate HMGB1 knockdown induced cell cycle arrest, DNA damage, and pyroptosis.

[CONCLUSION] GA exerts its anti-AML effects by directly binding to HMGB1, disrupting DNA repair, and activating AIM2-mediated pyroptosis. These findings highlight HMGB1 as a promising therapeutic target and support the further development of GA-based treatments for AML.