Lung metastasis is the leading cause of death in patients with osteosarcoma (OS), and new drugs are urgently needed. Epigenetic reprogramming is a recently proposed hallmark of malignancy; therefore, targeting epigenetic enzymes might provide a novel therapeutic strategy for OS lung metastasis. We recently reported that ailanthone (AIL), a natural product isolated from the Chinese medicinal plant Ailanthus altissima, inhibits OS cell growth and induces substantial metabolic changes; however, its direct targets remain unclear.To identify the direct targets of AIL in OS and to explore the effects of AIL on OS lung metastasis in vivo.Direct target proteins of AIL and downstream signaling pathways were identified in Saos-2 and U-2OS OS cells. The in vivo effects of AIL on OS lung metastasis were investigated using a mouse model.A novel surface plasmon resonance-high-performance liquid chromatography-mass spectrometry (SPR-HPLC-MS) assay was used to determine direct targets of AIL in OS. A cellular thermal shift assay, molecular docking analysis, enzyme activity assay, qRT-PCR, western blotting, chromatin immunoprecipitation assay, and reverse tests were performed to confirm the target and downstream pathway of AIL. A tumor xenograft model was used to verify the efficacy and mechanisms in vivo.Histone-lysine N-methyltransferase 2A (KMT2A) together with its scaffold protein menin (MEN1) were identified as direct target proteins of AIL in OS. AIL induced the autophagic degradation of the KMT2A-MEN1 complex. Moreover, AIL inhibited intracellular H3K4 methyltransferase activity and epigenetically inhibited the transcription of genes in the serine biosynthetic pathway (SSP). Furthermore, AIL suppressed OS lung metastasis and downregulated KMT2A, MEN1, and SSP in mouse models.This work showed that AIL targets the KMT2A-MEN1 complex and inhibits SSP to suppress OS lung metastasis. Notably, AIL exhibits new mechanisms of action, distinct from those of existing anti-OS drugs. On the basis of these findings, we proposed a novel strategy to treat OS by targeting epigenetic enzymes and cancer metabolism.Copyright © 2024 Elsevier GmbH. All rights reserved.