PROJECT SUMMARY Triple negative breast cancer (TNBC) accounts for 15–20% of all breast cancers. Women with TNBC are three times more likely to experience death compared to other subtypes. The poor prognosis of TNBC can be attributed to the lack of effective targeted therapy. Although TP53 mutations are frequently found in TNBC, it is difficult to target p53-deficiency with drugs. We have identified a family of non-canonical phosphatidylinositol kinases, the phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks) and their crucial role in the growth of cancers with TP53 aberrations. Furthermore, we discovered a role for the PI5P4Ks in autophagy, and for the first time, showed that these enzymes localize to lysosomes, suggesting that the PI5P4Ks may be important for lysosome function, as well as shedding light on the anti-cancer mechanism of PI5P4K inhibition in p53 mutant cancers. Using preclinical studies in novel genetically engineered mouse breast tumor models, human breast cancer cell lines, and patient breast tumor samples we propose to investigate the biochemical basis for the function of these ‘druggable’ enzymes. We will also validate them as targets for pharmaceutical intervention in human cancers, especially for TNBC where targeted therapies have not been successful. Our overarching hypothesis is that the PI5P4Ks act as sensors of metabolic stress that allow cells to modulate lysosomal events, including autophagy, in order to maintain cellular homeostasis. Further, we posit that the metabolic rewiring associated with p53 mutations, create a novel dependency on the PI5P4Ks for TNBC cell growth and survival. In Aim 1 we will determine the mechanism of synthetic vulnerability by which the PI5P4Ks become essential for tumor cell growth when p53 is defective. To this end we will characterize how mutant p53, both loss of function and gain of function, contributes to the synthetic lethality as well as dissect the metabolic consequences of PI5P4K loss in p53 mutant TNBC cells. In Aim 2 we will test the therapeutic effectiveness of targeting the PI5P4Ks in TNBC using our genetically engineered mouse breast tumor models and patient breast tumor samples. The ultimate goal and the overall impact of this project are to characterize PI5P4Ks as a novel liability for tumors with TP53 mutations in order to define opportunities for therapeutic intervention for TNBC patients with these mutations, which currently lack targeted therapy.