Venetoclax (VEN) resistance remains a major obstacle to durable responses in acute myeloid leukemia (AML). Although the core circadian regulator brain and muscle ARNT-like protein 1 (BMAL1) has been implicated in AML progression and drug response, its role in acquired VEN resistance remains unclear. In this study, VEN-resistant R-THP1 and R-MOLM13 cells were established, and the role of BMAL1 was evaluated using public datasets, clinical samples, cell experiments, and in vivo xenograft models. BMAL1 was upregulated in relapsed/refractory AML samples and VEN-resistant cells, and high BMAL1 expression was associated with adverse clinical outcomes. BMAL1 knockdown increased VEN sensitivity, promoted apoptosis, and reduced proliferation and clonogenic capacity in resistant cells, whereas BMAL1 overexpression produced the opposite effects in parental cells. Consistently, BMAL1 depletion reduced leukemia burden and enhanced the response to VEN in vivo. Integrated transcriptomic and proteomic analyses identified tumor protein D52 (TPD52) as a downstream candidate associated with BMAL1-mediated VEN resistance. TPD52 was upregulated in resistant AML cells, and its genetic modulation recapitulated the effects of BMAL1 on VEN sensitivity and malignant phenotypes of AML. Co-immunoprecipitation supported an association between BMAL1 and TPD52, while cycloheximide chase assays showed that BMAL1 enhanced TPD52 protein stability. Modulation of BMAL1 or TPD52 also altered p-PI3K and p-AKT without markedly affecting on total PI3K or AKT expression. In conclusion, these findings indicate that BMAL1 contributes to acquired VEN resistance by interacting with and stabilizing TPD52, accompanied by PI3K/AKT activation. BMAL1-TPD52 axis maybe a novel therapeutic target to overcome VEN resistance in AML.
