A novel therapeutic strategy for leukopenia: Miltefosine activates the Ras/MEK/ERK pathway to promote neutrophil differentiation.

Leukopenia, marked by diminished white blood cell (WBC) counts, presents significant challenges in the management of hematological malignancies and immunocompromised patients. This study evaluated the therapeutic potential of miltefosine (MFS), a phospholipid analogue, for treating leukopenia. In vitro studies using HL60 and NB4 cells revealed that MFS effectively promoted neutrophil differentiation and function, evidenced by the upregulation of surface markers CD11b, CD11c, CD14, and CD15, as well as enhanced bactericidal activity assessed through the NBT reduction assay. In a murine model of irradiation-induced leukopenia, MFS significantly restored both WBC and neutrophil counts, promoted neutrophil production, improved bone marrow (BM) cell proliferation, and mitigated radiation-induced apoptosis of BM cells while promoting the recovery of hematopoietic stem cells (HSCs). Transcriptomic analyses indicated that MFS mediated the regulation of key pathways, particularly the MAPK signaling pathway, which is vital for myeloid differentiation. A comprehensive integration of network pharmacology and RNA sequencing data identified common targets linking MFS to leukopenia, underscoring the critical role of the Ras/MEK/ERK signaling cascade in mediating MFS's effects. Furthermore, molecular docking and Western blot analyses confirmed MFS's interaction with and activation of Ras/MEK/ERK pathway, essential for facilitating neutrophil differentiation. Notably, pharmacological inhibition of ERK significantly diminished MFS-induced neutrophil differentiation. Collectively, these findings elucidate the molecular mechanisms underlying MFS-mediated therapy for leukopenia and provide a solid foundation for future clinical investigations.