PROJECT SUMMARY The class I phosphoinositide-3 kinase α (PI3Kα), which produces the signaling lipid phosphatidylinositol 3,4,5- trisphosphate (PIP3), is overactive in over 40% of breast cancer cases. Results from recent clinical trials have shown that targeted inhibition of PI3Kα is effective in prolonging survival in these patients. PI3Ks produce PIP3 by phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2), which is synthesized by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5K1A has been implicated as a driver in breast cancer and is found amplified in over 18% of tumors, though often with inactivating mutations. This amplification is found independent of PI3Kα mutations in half of these cases. However, it is not yet understood whether amplification of PIP5K1A and elevated PIP2 levels will drive tumor growth by increasing PIP3 production. The objective in this particular application is to determine the mechanism by which increased PIP5K1A expression disrupts PIP2 homeostasis, and how subsequently elevated PIP2 levels drive PI3K signaling. The central hypothesis is that amplification of PIP5K1A saturates a negative regulator, leading to elevated PIP2 and increased PI3K signaling, regardless of PIP5K catalytic activity. The rationale for the proposed research is that this will reveal how amplified PIP5K1A drives PI3K signaling in breast cancer. Guided by strong preliminary data, this hypothesis will be tested using two specific aims: 1) Identify the mechanism of PIP2 homeostasis at the plasma membrane; and 2) Define the role of altered PIP2 homeostasis in driving PI3K signaling, and its effects breast cancer cell proliferation. Under the first aim, the interaction between PIP5K and a negative regulator will be determined and the effects on catalytic activity will be identified. Under the second aim, the effect of PIP5K1A over-expression and elevated PIP2 levels on PI3K signaling and breast cancer cell proliferation will be determined. The proposed research is significant because it will identify PIP5K1A as a novel biomarker of breast cancer sensitivity to PI3Kα inhibitors. This will be a critical first step in establishing treatment options for ~10% of breast cancer cases without activation of PI3Kα.