5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most commonly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK-independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway.

This study is the first to demonstrate that human stromal cell lines and primary mesenchymal stromal cells from patients enhance the in vitro differentiation of acute myeloid leukemia (AML) cells induced by pyrimidine synthesis inhibitors, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), and brequinar. Furthermore, this is the first report to show that AICAr affects mouse bone marrow stromal cells by activating AMP-activated protein kinase (AMPK) and that human stromal cells are superior to mouse cells for testing the effects of drugs on AML differentiation.

Background and Objectives: Hereditary cancer predisposition is associated with pathogenic germline variants in numerous genes. Identification of inherited cancer susceptibility enables personalized screening, preventive interventions, and targeted therapies. It also facilitates cascade testing of relatives who may be at increased risk and benefit from tailored clinical management. We describe the first results of hereditary cancer patient identification, selection, and genetic testing at the University Hospital of Split. Materials and Methods: Next-generation sequencing and hereditary cancer gene panel analysis were performed in individuals who met the National Comprehensive Cancer Network clinical criteria for genetic testing at the University Hospital of Split between May 2025 and July 2026. A comprehensive cancer gene panel covering 101 genes was used. Results: A total of 297 individuals underwent genetic testing, including 268 (90.24%) females and 29 (9.76%) males. Of these, 138 (46.46%) were unaffected individuals who met testing criteria based on a family history of cancer. Among individuals with a personal history of malignancy, breast cancer was the most common diagnosis, occurring in 130 patients (43.77%). Pathogenic or likely pathogenic (P/LP) variants, as well as risk variants, were identified in 80 individuals (26.94%), including seven individuals carrying two P/LP variants. Variants were detected in 29 different genes, most frequently BRCA1 (n = 14), BRCA2 (n = 11), CHEK2 (n = 9) and PALB2 (n = 7). The detected variant types included frameshift (n = 30), missense (n = 22), nonsense (n = 21), splice-site variants (n = 4), start loss variants (n = 4), large deletions (n = 4), and in-frame deletions (n = 2). Conclusions: To our knowledge, this is the first study to report the results of panel germline testing for hereditary cancer syndromes in a Croatian population. These findings provide initial epidemiological data on the spectrum and distribution of P/LP variants. The relatively high detection rate may reflect the recent implementation of genetic testing and the careful selection of individuals with a strong clinical suspicion of hereditary cancer predisposition.

Differentiation therapy offers a promising approach in acute myeloid leukemia (AML) by overcoming the developmental block that maintains leukemic blasts. Increasing evidence indicates that DNA replication stress can promote differentiation rather than cytotoxicity; however, the metabolic mechanisms linking replication stress to differentiation remain poorly defined. Here, we investigated how perturbations in nucleotide metabolism regulate replication stress–driven differentiation. Using metabolomic and functional analyses in AML cell lines, we show that agents inducing differentiation through replication stress, including 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), dihydroorotate dehydrogenase (DHODH) inhibition, and low-dose cytarabine, converge on disruption of nucleotide pool balance. Low-dose AICAr induced a pyrimidine–purine imbalance, S phase arrest, and enhanced differentiation, whereas high-dose reduced these effects. Although brequinar and cytarabine altered nucleotide metabolism through distinct mechanisms, differentiation induced by all agents was abolished by supplementation with high levels of ribo- and deoxyribonucleosides, confirming that nucleotide imbalance is a central driver. We further identify ribonucleotide reductase (RNR) as a critical modulator of this process. Replication stress induced context-dependent regulation of RNR subunits, with RRM2 upregulated in p53-mutant U937 cells and the p53-responsive RRM2B isoform predominating in p53-wild-type MOLM-13 cells. Consistent with these differences, RRM2 depletion enhanced differentiation in U937 cells without affecting viability but impaired differentiation and survival in MOLM-13 cells. These findings position nucleotide metabolism as a key regulator of AML differentiation and suggest that combining RNR-targeted and checkpoint-modulating strategies could optimize therapeutic responses.