Metabolomics Identifies Pyrimidine Starvation as the Mechanism of 5-Aminoimidazole-4-Carboxamide-1-β-Riboside-Induced Apoptosis in Multiple Myeloma Cells

To investigate the mechanism by which AICAr induces apoptosis in multiple myeloma (MM) cells, we performed an unbiased metabolomics screen. AICAr had selective effects on nucleotide metabolism, resulting in an increase in purine metabolites and a decrease in pyrimidine metabolites. The most striking abnormality was a 26 x fold increase in orotate associated with a decrease in UMP levels, indicating an inhibition of UMP synthetase (UMPS), the last enzyme in the de novo pyrimidine biosynthetic pathway, which produces UMP from orotate and PRPP. As all pyrimidine nucleotides can be synthesized from UMP, this suggested the decrease in UMP would lead to pyrimidine starvation as a possible cause of AICAr-induced apoptosis. Exogenous pyrimidines uridine, cytidine and thymidine, but not purines adenosine or guanosine, rescued MM cells from AICAr-induced apoptosis, supporting this notion. In contrast, exogenous uridine had no protective effect on apoptosis resulting from bortezomib, melphalan or metformin. Rescue resulting from thymidine add-back indicated apoptosis was induced by limiting DNA synthesis rather than RNA synthesis. DNA replicative stress was identified by associated H2A.X phosphorylation in AICAr-treated cells, which was also prevented by uridine add-back. Although phosphorylation of AICAr by adenosine kinase was required to induce MM cell death, apoptosis was not associated with AMP-activated kinase activation or mTORC1 inhibition. A possible explanation for inhibition of UMP synthase activity by AICAr was a depression in cellular levels of PRPP, a substrate of UMP synthase. These data identify pyrimidine biosynthesis as a potential molecular target for future therapeutics in MM cells.