PROJECT SUMMARY PIP5K1A is amplified or mutated in approximately 5% of cancers, yet its potential as a drug target has not been realized to date. The enzyme is known to synthesize the key regulatory lipid PI(4,5)P2. Amplification of PIP5K1A therefore disrupts PI(4,5)P2 homeostasis, but the diaspora of plasma membrane function downstream of PI(4,5)P2 has made it difficult to identify the related function(s) disrupted in malignant disease. Our objective in this R03 is to determine the extent to which disrupted PI(4,5)P2 homeostasis and PIP5K1A amplification changes PI3K signaling, since PI3K is known to be a central pathway in tumorigenesis and PI(4,5)P2 is its substrate. Our central hypothesis is that PI(4,5)P2 homeostasis is a key determinant of PI3K signal strength in health and disease. Recent evidence indicates that PIP5K enzymes are negatively regulated by binding to with the related PIP4K enzymes, independent of PIP4K catalytic activity. Our own unpublished work shows that PIP4K proteins are also low-affinity PI(4,5)P2 binding proteins in cells, suggesting a tripartite PI(4,5)P2 homeostat; consequently, PIP5K1A over-expression is expected to upregulate PI(4,5)P2 levels but crucially, to do so in a catalytically independent manner, since the over-expressed PIP5K sequesters the available negative regulator, PIP4K. Elevated PI(4,5)P2 levels then drive enhanced PI3K signaling. The rationale for this work is that by identifying the mechanism by which PIP5K1A amplification disrupts PI(4,5)P2 homeostasis and enhances PI3K signal strength, we will illuminate the path to developing small molecule inhibitors of PIP5K1A. To this end, we will address the following specific aims: (1) Determine the extent to which PI3K signaling can be modulated by experimental manipulation of PI(4,5)P2 homeostasis. We hypothesize that elevated PI(4,5)P2 homeostasis via modulation of PIP5K-PIP4K-PI(4,5)P2 interactions will produce corresponding changes in PI3K signaling output. We will change the expression level and localization of endogenous PIP4K and PIP5K in 293A cells, and determine PI3K signaling using direct measures (PIP3 production and Akt phosphorylation). (2) Identify how changes in PIP5K1A expression in cancer cell lines activates PI3K signaling and drive proliferation. We will test the hypothesis that PIP5K1A overexpression in cancer cells upregulates PI3K signaling and proliferation. We will over-express PIP5K1A in non-transformed and diploid cells, as well as reducing expression in cell lines with PIP5K1A amplification. PI3K signaling will be determined as in aim 1, along with proliferation. Upon the completion of this project, we expect to find that elevated PI(4,5)P2 homeostasis enhances PI3K activity; moreover, we will show that this elevated homeostasis manifests in cancer cells with PIP5K1A amplification, causing increased oncogenic PI3K signaling and proliferation. This finding will be significant, because it will identify PIP5K1A as both a novel mar...