Project Abstract: More than 25 years ago we discovered the phosphoinositide 3-kinases or PI3K. To date more than 30 PI3K inhibitors have entered clinical trials and an inhibitor (idelalisib) that targets PI3K? was recently approved for treating B cell lymphomas. Our research and research from other labs over the past 25 years has revealed that the PI3K pathway evolved to control cell growth, primarily through regulation of cellular metabolism. The focus of this application is to understand the biochemical mechanisms by which phosphoinositide kinases control cellular metabolism. We expect to uncover new targets for pharmaceutical intervention in cancers, new biomarkers for predicting patients who are likely to respond to pathway inhibitors, and new insight into mechanisms of resistance to pathway inhibitors. The proposed research falls into three categories: 1) Evaluate the mechanism by which PI3K controls glucose metabolism and nucleotide synthesis and develop biomarkers to identify patients who are likely to respond to PI3K inhibitors and predict drug combinations that are likely to be more effective than single agents. We have recently found that the major effect of activating PI3K on glucose metabolism is activation of Rac1 and consequent activation of aldolase A due to release of aldolase A from the actin cytoskeleton. Importantly we find that aldolase A activation is required for deoxy-nucleotide triphosphate synthesis at rates needed for S phase progression in tumors with p53 and BRCA1 or PTEN mutations, explaining why these tumors can be dramatically shrunk by PI3K plus PARP inhibitors but not by AKT plus PARP inhibitors. 2) Evaluation of phosphatidylinositol-5- phosphate 4-kinases (PIP4K2A and PIP4K2B) as therapeutic targets in cancers lacking p53 function and determination of the biochemical mechanism by which these kinases become essential for tumor growth when p53 is defective. PIP4K2A and B generate PI-4,5-P2 from the low abundant and poorly characterized lipid PI-5-P. Recently we made the surprising observation that PIP4K2B?/? TP53-/- mice die as early embryos. Importantly, PIP4K2A-/-, PIP4K2B+/-, TP53-/- mice are viable and rarely develop cancers, suggesting that PIP4K2A/B inhibitors might be effective for treating cancers with genetic aberrations in p53. Our studies show that knocking down PIP4K2A and B causes metabolic stress in p53 mutant cancer cells. We propose to determine the mechanism by which loss of PIP4K2A and B only causes metabolic stress in the context of loss of p53. 3) Identification and characterization of PIP4K2A and B inhibitors and evaluation of inhibitors in pre-clinical models in order to provide pre-clinical proof of concept studies that will allow these inhibitors to progress into human cancer trials. We have identified inhibitors of PIP4K2A and B and shown that they mimic the effects of knockout or knockdown of these enzymes in regard to affecting growth of p53 mutant cell lines. We will evaluate whether these inhibitors are on target and determine whether they have an efficacy/toxicity ratio in vivo that would make them useful for treating cancers with p53 mutations.