Homoharringtonine and Gilteritinib Synergistically Induce Apoptosis and Suppress Viability in FLT3-ITD-Positive AML...

Background: The FLT3-ITD mutation is associated with a poor prognosis in acute myeloid leukemia (AML), particularly in relapsed or refractory (R/R) cases. Although Gilteritinib has been approved for the treatment of R/R AML with FLT3-ITD mutation, the emergence of resistance in clinical settings remains a major challenge. Homoharringtonine (HHT), a plant-derived alkaloid with antitumor properties, has also been used in AML treatment. However, the combination effects of HHT and gilteritinib have not been investigated. Methods: The cell viability and apoptosis of MV4-11 and MOLM-13 cells in the treatment of HHT, gilteritinib and the combination were assessed by CCK-8 assay and flow cytometry, respectively. Combination index (CI) values were calculated using CompuSyn 1.0. Western blotting was used to investigate the molecule mechanisms of HHT and gilteritinib mediated anti-leukemia effects in time- and dose-dependent experiments. To investigate the role of p53 status in drug responses, MV4-11-p53R248W and MV4-11-p53WT subclones were isolated and MV4-11-p53knockout cells was established through CRISPR/Cas9 system. The cell viability and apoptosis of MV4-11 cells with various p53 status were compared. Moreover, RNA-seq analysis was performed in MV4-11 cells treated with or without HHT. RT-qPCR and Western blotting were conducted to verify the mechanism underlying HHT-induced p53 upregulation. Results: HHT and gilteritinib exerted a significant synergistic effect on cell viability and apoptosis in MV4-11 and MOLM-13 cells, which was markedly diminished in the cells with the p53-R248W muta-tion or without p53. Mechanistically, HHT and gilteritinib both suppressed FLT3 signaling. Interestingly, HHT mediated the upregulation of p53 through HSPA8 downregulation, while gilteritinib downregulated the p53 level. The combination enhanced the p53 expression. Conclusions: Our findings elucidate the mechanism underlying this synergistic interaction and underscore the potential of p53 status as a predictive biomarker for identifying patients most likely to benefit from HHT and gilteritinib combination therapy.