PROJECT SUMMARY: 30 lines. The canonical phosphoinositide (PI) 3-kinase (PI3K)/Akt signaling pathway uses free membrane PI lipid to regulate cell growth and is frequently hyperactivated in cancer. Akt is also activated in the nucleus by poorly understood mechanisms. We discovered a nuclear PI3K/Akt pathway composed of PI kinases/phosphatases that modify phosphatidylinositol phosphates (PIPns) linked to p53 (p53-PIPn signalosome). PIPKI synthesizes p53-PIP2 that stabilizes it and regulates MDM2. The PI3K inositol polyphosphate multikinase (IPMK) converts p53-PIP2 to p53-PIP3, which is reversed by PTEN. p53-PIP3 recruits the full Akt pathway leading to nuclear Akt activation that regulates FOXOs, MDM2, genomic stress and cell survival. Hypothesis/Objective: Nuclear Akt is dose activated by p53-PIP3. The p53-PIPn complex is stabilized by MDM2 and MDM2 is also PIPn-linked. IPMK and PTEN control the generation of p53-PIP3 that activates nuclear Akt. Mutant p53-PIP3-Akt signaling, unlike p53wt, is constitutively activated and further stimulated by genotoxic stress, resulting in enhanced cell survival and promoting tumor initiation and promotion. We will test this in 4 aims. Aim 1. Determine the mechanisms of nuclear Akt activation by the p53-PIPn signalosome. Define the roles and mechanisms of IPMK kinase and PTEN activity toward the p53-PIPn complexes by elucidating the structural interaction of IPMK and PTEN with p53. Using this structural information, we will determine how genotoxic stress- induced PTMs on p53, IPMK and PTEN regulate the p53-PIPn signalosome and nuclear Akt activation. Aim 2. The residues that link PIPn to p53 and MDM2 will be defined, and mutants made that block PIPn linkage and nuclear Akt activation. We will determine if there are different PIP linkages for p53wt and p53mt. Aim 3. Define mechanisms for p53 stabilization by MDM2, sHSPs and PIPns. The stability of p53 is key for p53-PIP3 activation of Akt and is regulated by MDM2. MDM2 is also PIPn-linked and the mechanisms by which PIPns and sHSPs regulate the p53-MDM2 pathway will be studied. MDM2 PIPn linkage and binding mutants will be used to investigate the role of PIPns in regulating MDM2 ligase activity, interactions with p53 and p53 stability. The PIP kinases and PTEN that generate the MDM2-PIPn complexes will be defined and studied. Aim 4. Define roles of the p53-PIPn signalosome in regulating tumor growth. Using cellular and murine models of breast cancer, we will determine the functional role of the p53-PIPn signalosome in tumor suppression by p53wt and transformation/tumor progression by p53mt. We will also determine whether the expression of p53- PIPn and p53-PIP3-Akt complexes in clinical breast cancer specimens correlates with clinical outcomes. Significance: We have discovered a novel nuclear PI3K/Akt pathway scaffolded on p53 that is independent of the canonical membrane-localized pathway and insensitive to PI3K inhibitors in the clinic, underscoring its therapeu...