The discovery of crotonoside as a nucleoside analogue demonstrates its potential to inhibit acute myeloid leukemia  activity through multiple pathways

Abstract Acute myeloid leukemia (AML) is a type of malignant neoplasm that affects hematopoietic stem and progenitor cells, characterized by the clonal proliferation of immature blast cells exceeding 10% in the bone marrow and peripheral blood. Despite considerable advancements in AML therapeutics in recent years, as evidenced by the U.S. Food and Drug Administration's approval of ten novel therapies since 2017, relapsed or refractory AML remains a particularly challenging subset, necessitating the development of improved treatment strategies. Traditional Chinese Medicine (TCM) and natural products have garnered increasing attention as adjuncts to conventional chemotherapy for leukemia. Crotonoside, a guanosine analog originally isolated from Croton tiglium L., is a key component in various traditional treatments for conditions such as constipation, headache, abdominal and stomach pain, inflammation, and rheumatism. Preliminary reports have indicated that crotonoside exhibits activity against the activation of FLT3 and reduces the expression of HDAC3/6 in AML. In our research, we identified crotonoside as an anti-tumor compound through its regulation of two distinct pathways. On one hand, our study revealed that the expression of APOBEC3G is significantly upregulated in acute myeloid leukemia (AML), and elevated levels of APOBEC3G are strongly correlated with reduced overall survival (OS) and adverse cytogenetic profiles. This suggests that APOBEC3G represents a critical risk factor for OS in AML patients. Additionally, we discovered that crotonoside specifically reduces APOBEC3G expression and disrupts DNA synthesis by interfering with DNA bases. Consequently, crotonoside inhibits the viability of various AML cell lines in vitro, induces cell cycle arrest in the S phase for KG-1 and MV-4-11 cells, alters the expression of cell cycle-related proteins, and promotes apoptosis. On the other hand, our observations indicate that crotonoside causes mitochondrial damage, leading to mitochondrial dysfunction and the accumulation of reactive oxygen species (ROS), which subsequently initiate ferroptosis in AML cells. Molecular docking simulations, based on the structural properties of crotonoside, suggest that this mitochondrial damage is mediated through the inhibition of mitochondrial DNA polymerase γ. Furthermore, the induction of ferroptosis is closely associated with crotonoside's modulation of autophagy in AML cells via the p62/KEAP1 signaling pathway. Our subsequent research will focus on elucidating the direct inhibitory effects of crotonoside on mitochondrial DNA polymerase γ and exploring the detailed mechanisms related to autophagy.