Acute myeloid leukemia (AML) is a hematological malignancy characterized by the proliferation of immature blasts arrested at specific differentiation stages, making differentiation-based therapies a promising alternative to traditional cytotoxic treatments. Our previous study demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induces AML differentiation through pyrimidine synthesis inhibition, similar to the DHODH inhibitor brequinar. We also found that cytarabine (AraC), a standard chemotherapy agent, promotes differentiation via replication stress and Chk1 activation, a mechanism shared with pyrimidine synthesis inhibitors. However, the precise link between DNA damage, replication stress, and differentiation remains incompletely understood.

Human AML cell lines U937, THP-1, and MOLM-13 were cultured in RPMI-1640 or αMEM medium supplemented with ribonucleosides (rNs) and deoxyribonucleosides (dNs). Metabolomic profiling was performed using liquid chromatography-mass spectrometry (LC-MS). Cell viability, cell cycle distribution, and differentiation were assessed using multiparametric flow cytometry, while Western blotting was used to evaluate ribonucleotide reductase subunit M2 (RRM2) and Wee1 signaling. Inhibition studies were conducted using COH29 and hydroxyurea (HU) as inhibitors of ribonucleotide reductase (RNR), MK1775 as a Wee1 inhibitor, and siRNA targeting Wee1.

Metabolomic analysis revealed that pyrimidine and purine nucleotides were among the most differentially regulated metabolites, decreasing in brequinar- and AICAr-treated samples while increasing in AraC-treated samples. When cells were cultured in αMEM supplemented with 10 mg/ml of both rNs and dNs, the effects of all tested agents on proliferation, differentiation, and cell cycle arrest were completely abolished. All agents increased RRM2 expression, suggesting a role for RNR in differentiation. Inhibition of Wee1 kinase with MK1775 blocked RRM2 upregulation and AML differentiation without preventing S-phase arrest. Similar effects on differentiation and the cell cycle were observed in cells treated with COH29, a recently described RNR inhibitor. However, HU, a well-known RNR inhibitor, failed to prevent differentiation. Experiments are underway to further investigate the role of RNR in cells with downregulated expression.

Our findings suggest that ribonucleotide metabolism regulates AML differentiation, with Wee1 and RNR activation occurring downstream of replication stress.