Disruption of NAD⁺ and gsh/GPX4 axis via NAMPT inhibition by elaiophylin sensitizes AML to ferroptosis

Abstract

Abstract Objective Acute myeloid leukemia (AML) is a highly heterogeneous and aggressive hematologic malignancy, with therapeutic outcomes frequently limited by relapse and drug resistance. Natural products have emerged as promising candidates for anti-AML drug development due to their structural diversity, multitarget capabilities, and relatively low toxicity. This study aimed to screen polyketide compounds derived from Streptomyces and investigate their therapeutic potential and underlying mechanisms in AML. Methods Three polyketide compounds—Azalomycin F3a, Azalomycin F4a, and Elaiophylin—were isolated from Streptomyces sp. 211726. Their cytotoxic effects were evaluated in AML cell lines (THP-1, MOLM-13, and MV4-11). The potential molecular target of Elaiophylin was identified using thermal proteome profiling (TPP) and further validated by cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and molecular docking (MD) analysis. The anti-leukemic efficacy of Elaiophylin was confirmed in vivo using both an MLL-AF9 transgenic mouse model and a patient-derived xenograft (PDX) model. Results Among the compounds tested, Elaiophylin demonstrated the most potent anti-AML activity, with IC₅₀ values ranging from 0.51 to 0.74 μM after 24 hours of treatment. Elaiophylin induced apoptosis and caused cell cycle arrest at the G0/G1 phase. Transmission electron microscopy revealed hallmark features of ferroptosis, including condensed mitochondrial membranes, reduced mitochondrial volume, outer membrane rupture, and cristae loss. Mitochondrial membrane potential assays (JC-1/TMRE) indicated marked mitochondrial depolarization. Elaiophylin also elevated intracellular reactive oxygen species (ROS) levels and enhanced lipid peroxidation, ultimately triggering ferroptosis. The ferroptosis inhibitor Ferrostatin-1 (Fer-1) partially rescued Elaiophylin-induced cell death, confirming ferroptosis involvement. Through TPP, CETSA, SPR, and molecular docking, nicotinamide phosphoribosyltransferase (NAMPT) was identified as a direct molecular target of Elaiophylin. Supplementation with nicotinamide mononucleotide (NMN), the downstream product of NAMPT, significantly attenuated the cytotoxic effects of Elaiophylin, underscoring the critical role of NAMPT inhibition in its mechanism of action. These results were further validated in a PDX mouse model. Additionally, Elaiophylin exhibited synergistic anti-leukemic effects in combination with Venetoclax both in vitro and in the MLL-AF9 mouse model, resulting in superior tumor suppression compared to monotherapy. Conclusion Elaiophylin induces ferroptosis in AML cells by promoting lipid peroxidation and mitochondrial dysfunction. Mechanistically, it directly binds to NAMPT, inhibits NAD⁺ biosynthesis, and suppresses the GSH/GPX4 antioxidant pathway, thereby triggering ferroptotic cell death. Moreover, its combination with Venetoclax represents a promising therapeutic strategy for AML. This study provides novel insights into the development of natural product-based therapies and identifies Elaiophylin as a potential anti-AML candidate.