Amino acid metabolism in tumor cells is very different from that in normal cells. The tumor-specific metabolic pathway “glutaminolysis” focuses on glutamine. Recently, another fundamental pathway was discovered for cancer cell proliferation: the “serine-glycine-methionine-one-carbon” pathway. Tumor cells upregulate specific amino acid transporters to satisfy their increased need for glutamine, glycine, serine, and methionine to fuel these pathways. Blocking the entry of these amino acids into tumor cells has potential for cancer therapy. Recently, we provided evidence for this approach by identifying SLC6A14, an amino acid transporter with broad specificity, that is induced in ER-positive (ER+) breast cancer (BC) and by showing that blockade of this transporter reduces ER+ BC. But, SLC6A14 is not expressed in triple-negative breast cancer (TNBC). TNBC must rely upon a different transporter as a “fix” to its “addiction” to glutamine/serine/glycine/methionine. We have now identified this transporter as SLC38A5, a Na+-coupled uptake system exclusively for these four amino acids. SLC38A5 is upregulated in TNBC by WNT/DVL1. Furthermore, SLC38A5 promotes macropinocytosis, a unique feature essential for tumor growth. Here we will show that SLC38A5 drives the glutamine- and one-carbon metabolic pathways in TNBC and evaluate in preclinical models the potential of this transporter as a logical drug target for TNBC. We have identified the FDA-approved drug niclosamide as a potent inhibitor of SLC38A5 function and expression. This drug can be used pharmacologically to interrogate the role of SLC38A5 in TNBC. We also have Slc38a5-null mice as a genetic tool for these studies. Hypothesis: SLC38A5 drives the glutamine-dependent and one-carbon metabolic pathways in TNBC; as such, pharmacologic inhibition or genetic deletion of SLC38A5 will block TNBC. We will test this hypothesis with two aims: (Aim 1) Demonstrate that SLC38A5 is obligatory for glutaminolysis and one-carbon metabolic pathway to promote cell proliferation and remodeling of the epigenetic landscape in TNBC cells and elucidate the molecular mechanisms by which WNT/DVL1 induces SLC38A5 in these cells. Here we will use classical as well as patient-derived xenograft TNBC cell lines. (Aim 2) Evaluate the impact of SLC38A5 loss on tumor growth in mouse models of TNBC using Slc38a5-/- mice and niclosamide as a proof-of-concept for future use of this transporter as a selective drug target for TNBC. Here we will use xenografts of human TNBC cell lines in nude mice to evaluate the anticancer efficacy of niclosamide, and also compare the development/growth of spontaneous mammary tumors in a TNBC mouse model [C3(1)-TAg mouse] with and without niclosamide treatment (pharmacologic) and on Slc38a5+/+ and Slc38a5-/- backgrounds (genetic). Impact: With no known targeted therapy, chemotherapy is the only option for TNBC. Establishment of SLC38A5 as a drug target would aid future use of pharmacologics (e.g., niclosa...