ABSTRACT
Background: De novo nucleotide synthesis is a dynamic process that can address the enormous demand for nucleotides and other macromolecules required in acute myeloid leukemia (AML) proliferation. Hence, we hypothesized that targeting de novo nucleotide synthesis would lead to the depletion of the nucleotide pool and pyrimidine starvation in leukemic cells compared to their non-malignant counterparts, impacting proliferative and differentiation pathways. Emvododstat (PTC299) is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Objectives: The goals of these studies were to demonstrate that emvododstat effects leukemia growth due to the inhibition of de novo pyrimidine nucleotide synthesis. Comprehensive analyses of mitochondrial function, metabolic signaling in PI3K/AKT pathways, apoptotic signatures, and DNA damage responses were evaluated. The clinical relevance of emvododstat efficacy was confirmed in an AML-PDX model. Results: Emvododstat treatment in cytarabine-resistant AML cells and primary AML blasts induced apoptosis, differentiation, and reduced proliferation, with corresponding increases in annexin V- and CD14-positive cells. Indeed, the inhibition of de novo nucleotide synthesis compromises the dynamic metabolic landscape and mitochondrial function, as indicated by decreases in the oxygen consumption rate (OCR) and mitochondrial ROS/membrane potential. These effects can be reversed by the addition of exogenous uridine and orotate. Further immunoblotting and mass cytometry (CyTOF) analyses demonstrated changes in apoptotic and cell signaling proteins (cleaved PARP, cleaved caspase-3) and DNA damage responses (TP53, γH2AX) and PI3/AKT pathway downregulation in response to emvododstat. Finally, in a PDX mouse model of human AML, emvododstat treatment improved survival compared to mice treated with vehicle (median survival 40 days vs 30 days, P=0.0002). This corresponded with a reduction in the bone marrow burden of leukemia and increased expression of differentiation markers in mice treated with PTC299. Conclusion: Inhibition of de novo pyrimidine synthesis triggers differentiation, apoptosis, and/or inhibition of proliferation in AML models. Emvododstat is a novel dihydroorotate dehydrogenase inhibitor being tested in a clinical trial for the treatment of myeloid malignancies and COVID-19.