BACKGROUND: Drug resistance and relapse are still major challenges in acute myeloid leukemia (AML) because of the inability to effectively eradicate leukemia stem cells (LSCs). Senescence induction combined with immune killing may offer promising strategies for LSC eradication. However, whether and how drug-resistant LSCs retain stemness via senescence and immune regulation remains unknown. METHODS: The immunoproteasome subunit PSMB10 expression levels were analyzed by single-cell RNA-seq data, along with the bioinformatics analysis of publicly available AML datasets, and quantified using RT-qPCR and flow cytometry (FCM) analysis on clinical samples from AML patients. The cellular senescence was evaluated by the assays of cell proliferation, cell cycle, senescence-associated β-galactosidase activity, and senescence-associated secretory phenotype factors. In vitro T-cell killing assay was played to determine immune escape reprogramming of AML cells. FCM was conducted to estimate intracellular drug concentration and cellular apoptosis rates. Human AML xenografts and PSMB10 knockout syngeneic mouse bone marrow transplantation models were utilized to investigate the function of PSMB10. Various techniques were employed for mechanism studies, including Lentivirus transduction or siRNA transfection, western blotting, co-immunoprecipitation assays, luciferase reporter assays, polysome profiling assays, quantitative proteomics, etc. RESULTS: PSMB10 mRNA was significantly upregulated in the surviving nonsenescent LSCs, exhibiting a 13-fold increase compared to senescent LSCs following chemotherapy. The specific high expression of PSMB10 in post-chemotherapy nonsenescent LSCs predicts a poor AML prognosis. The genetic inactivation of PSMB10 resulted in increased senescence and cytotoxic T lymphocyte (CTL) killing, as well as increased intracellular drug concentrations and drug-induced cellular senescence in different types of human AML cells, which also impeded human and murine leukemia initiation and stemness maintenance in vivo with a 19-fold decrease in the frequency of human LSCs and a 7.6-fold decrease of drug-resistant mouse LSCs, while normal hematopoietic cells remained unaffected. Mechanistically, the downregulation of PSMB10 boosted SLC22A16-mediated drug endocytosis and further induced chemotherapy drug-mediated senescence through the RPL6/RPS6-MDM2-P21 pathway in AML cells. Additionally, downregulating PSMB10 also impeded MHC-I protein degradation-induced escape of CTL killing. CONCLUSIONS: PSMB10 is a key candidate molecular target for eradicating drug-resistant LSCs via senescence and immune regulation.