PROJECT SUMMARY The Cancer Genome Atlas (TCGA) studies have identified p53 as the most frequently mutated gene in breast cancer. In particular, 80% of triple-negative breast cancers (TNBCs) harbor p53 mutations. However, there is no specific therapy available for treating p53-mutant TNBC. Cancer cells have distinct metabolic needs compared to normal cells, and this observation has spurred the development of small molecule inhibitors to target metabolic enzymes that are specifically needed by cancer cells for survival. Notably, although there is evidence to support a role for metabolic pathway deregulation in breast cancer growth, the metabolic dependencies of p53-mutant TNBC growth and metastasis have not been comprehensively identified. Therefore, to specifically identify the metabolic dependencies of p53-mutant TNBCs, we performed an innovative and unbiased large-scale in vivo short-hairpin RNA (shRNA) screen by targeting 2000 genes with known metabolic functions. With this screen, we identified N-acylsphingosine amidohydrolase 1 (ASAH1) as an enzyme that is necessary for tumor-forming ability and metastatic activity of p53-mutant TNBCs. Additionally, we identified two small molecule inhibitors of ASAH1 with potent anti-p53-mutant TNBC activity, thereby indicating that ASAH1 is a potential drug target for the treatment of p53-mutant TNBCs. The central hypothesis of this proposal is that p53-mutant TNBC cells depend upon ASAH1 for their survival, and thus, ASAH1 inhibition selectively eradicates p53-mutant TNBCs. Our overall objectives are to determine the role of ASAH1 in driving p53-mutant TNBC tumor growth and metastasis, understand the mechanism underlying the dependency of p53-mutant TNBCs on ASAH1, and evaluate the translational potential of small molecule ASAH1 inhibitors for treating p53-mutant TNBC. In Aim 1, we will establish the role of ASAH1 in p53-mutant TNBC tumor growth and metastasis using a series of complementary, state-of-the-art mouse models that recapitulate characteristic features of TNBC growth and metastasis. These include a highly innovative humanized mouse model with a human immune system. In Aim 2, we will test our hypotheses that p53 represses ASAH1 activity by sequestering this protein in the nucleus. Additionally, based on our new preliminary results, we will confirm whether loss of ASAH1 activates the glucose starvation response, leading to AMP kinase pathway activation via a ceramide-mediated reduction in expression of the glucose transporter GLUT1 on the cell membrane. In Aim 3, we will evaluate the translational potential of ASAH1 inhibitors in immunocompromised and immunocompetent humanized mouse models of p53-mutant TNBC, either alone, or based on our preliminary findings, in combination with BET domain inhibitors. Collectively, we predict that the results of the experiments proposed in this application will establish ASAH1 as an important vulnerability inherent to p53-mutant TNBC cells, elucidate the mechanis...