Modulation of Mitochondrial Metabolism by Celastrol Enhances Apoptosis in FLT3-positive Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) remains a high-risk hematologic malignancy with substantial treatment-related toxicity, mortality, and poor prognosis. The success of chemotherapy is often hampered by drug resistance and relapse, affecting treatment response and long-term survival of patients. To find alternative drugs and targeted immunotherapies for AML remains an unmet need. Celastrol is a natural compound isolated from the medicinal plant Tripterygium wilfordii Hook F. It is a pentacyclic triterpenoid that has shown great potential for inducing apoptosis, yet the mechanism in FLT3-ITD AML is poorly understood. In the present study, we investigate the apoptotic mechanisms of celastrol using metabolomics and RNA sequencing analysis in FLT3-ITD AML cells and a xenograft mouse model. Initially, molecular modeling and molecular dynamics (MD) simulations indicate that celastrol binds to FLT3 in a stable and reproducible manner, comparable to established FLT3 inhibitors such as gilteritinib. Celastrol exhibits a persistent interaction network, characterized by stable Hydrogen Bonding and consistent contact patterns, which supports an efficient and mechanistically relevant binding mode to the FLT3 complex. In vitro, celastrol treatment in MV4-11 cells induced dose-dependent cytotoxic effects with an IC50 of 0.45 micro M. Exposure of MV4-11 cells to celastrol induces apoptotic cell death, as evidenced by Annexin V-positive staining and dual staining analysis with acridine orange and ethidium bromide, along with an increase in the formation of reactive oxygen species (ROS). Untargeted metabolomics analysis showed a significant reduction in glutathione pathway metabolites, including glutathione and L-glutamic acid, after celastrol treatment in MV4-11 cells. Furthermore, transcriptomics analysis revealed significant downregulation of mitochondrial respiration complex I-IV specific gene signatures upon celastrol treatment. The Seahorse assay confirmed that celastrol treatment significantly suppressed mitochondrial respiration by reducing the maximal oxygen consumption rate (OCR) and ATP production, as an indicator of OXPHOS, compared with giltertinib. Moreover, celastrol suppresses leukemic tumor growth in immunodeficient NSG mice engrafted with MV4-11-Luc cells, exhibiting in vivo anti-leukemic activity. Together, we demonstrated the anti-leukemic activity of celastrol through alterations in glutathione homeostasis and mitochondrial OXPHOS-mediated apoptotic cell death, providing a novel underlying mechanism that could be used to treat FLT3-ITD AML.