Background: NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I/II clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. We have recently shown in cell lines a potential therapeutic role for NUC-7738 in patients with acute myeloid leukemia (AML), whereby NUC-7738 induced myeloid cell differentiation and mitochondrial-mediated apoptosis. AML leukemia stem cells (LSCs) are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs and therefore, targeting β-catenin is a potential therapeutic strategy. Aims: The aim of this study was to determine whether NUC-7738 regulates β-catenin and the expression of its target genes in AML cells Methods: AML cell lines KG1a, OCI-AML3, HL-60 and U937 were treated with NUC-7738 for 48 or 72 hours. LSCs were determined by the expression of surface markers CD34, CD38 and CD123 via flow cytometry. Self-renewal was assessed by performing the colony forming assay using MethoCult Enriched media. Cell proliferation was determined by the Alamar Blue assay. Immunoblotting was performed for β-catenin, PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 Results: NUC-7738 reduced PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 resulting in reduced β-catenin expression. NUC-7738 decreased the percentage of CD34+ CD38- CD123+ (LSCs) from 15% to 7% and significantly reduced the total number and size of leukemic colonies. NUC-7738 inhibited cell proliferation in U937, OCI-AML3 and HL-60 cells, with IC50’s of 1.55 µM, 7 µM and 16 µM, respectively. Summary/Conclusion: Self-renewal pathways, including Wnt/β-catenin, are key for the initiation and long-term maintenance of LSCs. Through the ability to reduce PI3K-p110, phosphorylated AktSer473, and phosphorylated GSK3βSer9 and suppress β-catenin signalling, NUC-7738 exploits a key developmental process of AML, supressing the expansion and survival of LSCs. These results indicate that targeting LSCs with NUC-7738 may offer a successful therapeutic strategy for treating patients with leukemia.