Halofuginone exerts broad-spectrum cytotoxic effects by regulating p-eIF2α-S100A8/A9-calcium signaling, inhibiting global protein synthesis, and reversing the resistance of idarubicin in acute myeloid leukemia.

[BACKGROUND] Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with poor overall survival (OS). Resistance to chemotherapeutic drugs such as idarubicin (IDA) remains a major cause of treatment failure. This study investigated the anti-leukemic activity of halofuginone (HF) a synthetic derivative of the natural compound from hydrangea Dichroa febrifuge and its potential to overcome IDA resistance in AML cells.

[METHODS] Apoptosis, proliferation, cell cycle, and colony formation were assessed in AML cells treated with HF. RNA sequencing (RNA-seq) was performed to identify the potential molecular targets of HF. The anti-leukemic efficacy of HF was further assessed in NOD/SCID-IL2Rγ (NSG) mice xenografted with human relapsed/refractory (R/R) AML samples.

[RESULTS] HF treatment significantly inhibited cell proliferation, reduced colony formation, and induced apoptosis in AML cells. By RNA-seq analysis, S100A8 and S100A9 (S100A8/A9) were identified as potential targets of HF, and HF treatment markedly suppressed their expression. Overexpression of S100A8/A9 abrogated the anti-leukemic effects of HF, indicating that S100A8/A9 are critical mediators of HF activity. Mechanistically, HF activated the amino acid starvation response (AAR), leading to phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α), subsequent downregulation of S100A8/A9, and elevation of cytoplasmic Ca⁺ levels. Knockdown of eIF2α prevented HF-induced downregulation of S100A8/A9, confirming that HF regulates S100A8/A9 expression via the eIF2α pathway. Furthermore, HF treatment inhibited global protein synthesis, enhanced the cytotoxicity of chemotherapeutic drugs, and reversed IDA resistance by suppressing S100A8/A9 expression. Finally, HF inhibits leukemic infiltration and extended OS in MLL-AF9-transduced AML mice and enhanced IDA-induced anti-leukemic effects in R/R AML-xenografted NSG mice model.

[CONCLUSIONS] These findings reveal that HF exerts anti-leukemic effects by modulating the p-eIF2α-S100A8/A9-Ca⁺ signaling axis in AML cells. HF represents a promising therapeutic candidate for AML, particularly for patients with IDA-resistant disease.