EACR26-1631

Nucleotide Imbalance and Ribonucleotide Reductase Isoforms Regulate Replication Stress–Induced Differentiation in Acute Myeloid Leukemia

A. Brcic¹, H. Lalic^1,2, T. Smoljo^1,3, K. Bardac¹, V. Dembitz¹, R. Penker⁴, G. Rodriguez Blanco⁴, D. Visnjic¹

¹Croatian Institute for Brain Research, University of Zagreb School of Medicine, Laboratory for Cell Biology, Department of Physiology, Zagreb, Croatia
²University Hospital Center Zagreb, Department of Laboratory Immunology, Clinical Department of Laboratory Diagnostics, Zagreb, Croatia
³University Hospital Center Split, Department of Oncology, Split, Croatia
⁴Medical University of Graz, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Graz, Austria

Introduction:

Replication stress can promote differentiation in acute myeloid leukemia (AML), yet the metabolic determinants of this response remain incompletely defined. We investigated whether disruption of nucleotide metabolism represents a central driver of replication stress–induced differentiation and examined the role of ribonucleotide reductase (RNR) subunits in modulating this process.

Material and method:

AML cell lines (U937, MOLM-13, THP-1) were treated with AICAr, the DHODH inhibitor brequinar, or low-dose cytarabine. Intracellular metabolites were quantified by targeted liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). Differentiation and cell cycle distribution were assessed by flow cytometry. RNR subunit expression was analyzed by immunoblotting and siRNA-mediated knockdown. Public proteomic datasets from AML cell lines were interrogated to evaluate RRM2/RRM2B abundance patterns.

Result and discussion:

All differentiation-inducing agents altered nucleotide homeostasis through distinct mechanisms but converged on S-phase arrest. Supplementation with high concentrations of ribo- and deoxyribonucleosides abrogated replication stress and differentiation indicating that a global nucleotide imbalance is required for this phenotype. Replication stress was accompanied by differential regulation of RNR subunits. In p53-mutant U937 cells, RRM2 was strongly induced, and its knockdown led to a dNTP imbalance and enhanced differentiation without reducing viability. In contrast, p53-wild-type MOLM-13 cells predominantly upregulated the p53-inducible isoform RRM2B, and RRM2 depletion impaired both survival and differentiation. Proteomic analysis showed a higher RRM2/RRM2B ratio in p53-mutant than in p53–wild-type AML cell lines.

Conclusion:

These findings identify nucleotide imbalance as a key metabolic regulator of AML differentiation and suggest that RNR isoform composition influences cellular responses to replication stress.

Acknowledgement:

We thank Ms. Marijana Andrijasevic for valuable technical assistance. This work has been funded by Croatian Science Foundation under the projects HRZZ IP-2022-10-9146, DOK-2020-01-2873, DOK-2025-02-6776 and DOK-NPOO-2023-10-9321 by the European Union through the ESF Operational Programme Efficient Human Resources (to DV).