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.

Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.

Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage–associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.

Pharmacological modulators of AMP-dependent kinase (AMPK) have been suggested in treatment of cancer. The biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) have been reported to inhibit proliferation of solid tumors and hematological malignancies, but their role in differentiation is less explored. Our previous study demonstrated that AICAR alone induced AMPK-independent expression of differentiation markers in monocytic U937 leukemia cells, and no such effects were observed in response to metformin. The aim of this study was to determine the mechanism of AICAR-mediated effects and to test for the possible role of autophagy in differentiation of leukemia cells. The results showed that AICAR-mediated effects on the expression of differentiation markers were not mimicked by A769662, a more specific direct AMPK activator. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II, and these effects were not observed in response to metformin. Western blot and immunofluorescence analyses of U937 cells treated with bafilomycin A1 or transfected with mRFP-GFP-LC3 proved that the increase in the expression of LC3B-II was due to an increase in autophagy flux, and not to a decrease in lysosomal degradation. 3-Methyladenine inhibited the expression of differentiation markers in response to all inducers, but had stimulatory effects on autophagy flux at dose that effectively inhibited the production of phosphatidylinositol 3-phosphate. The small inhibitory RNA-mediated down-modulation of Beclin 1 and hVPS34 had no effects on AICAR and ATRA-mediated increase in the expression of differentiation markers. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that the effects of AICAR and ATRA on the expression of differentiation markers do not depend on the normal levels of key proteins of the classical or canonical autophagy pathway.