PROJECT SUMMARY/ABSTRACTFOXO transcription factors are regulators of cellular homeostasis that are activated in response to a variety ofenvironmental stresses. Upon activation FOXOs control the expression of genes in multiple and often conflictingcellular processes. For example FOXOs activate the expression of transcriptional programs involved in celldeath but also in cytoprotective processes like DNA repair and cell-cycle arrest. How FOXOs determine whichof these outcomes a cell will undergo is not known.Cells often utilize a single transcription factor to decide between multiple often opposing cellular outcomes asseen with FOXO transcription factors. Previous single cell studies in the p53 and NF-kB transcription factorsystems have revealed that the dynamics of these transcription factors or how their levels or location changeover time following a stimulus controls which outcome the cells enact. Like p53 and NF-kB FOXO has the samemultiple input and multiple output motif making it a good candidate for a dynamically regulated system.FOXO transcription factors are largely regulated by subcellular location. In unstressed conditions FOXOs arelocated in the cytoplasm rendering them inactive. Cell stresses like growth factor depletion or DNA damagelead to shuttling of FOXOs into the nucleus where they actively transcribe target genes. In this proposal wewill use live fluorescence microscopy of single cells to elucidate whether FOXO nuclear/cytoplasmic shuttlingdynamics can induce different cellular outcomes in cancer cells. In the first aim we will take advantage of themultiple cellular phenotypes (arrest and apoptosis) that arise after treating cancer cells with Epidermal GrowthFactor Receptor (EGFR) tyrosine kinase inhibitors (TKIs). EGFR TKIs are clinically approved drugs used totreat patients with advanced Non-Small Cell Lung Cancers with hyperactivating mutations in EGFR; Foxo3ahas been strongly implemented in both cancer cell death and arrest after treatment. We will stimulate botharrest and death outcomes in cancer cells using EGFR TKIs and determine whether each fate is correlatedwith a specific Foxo3a dynamic pattern. Our preliminary data has suggested that Foxo3a is pulsatile followingEGFR TKI treatment. We will focus on development of an experimentally validated mathematical model toelucidate the structure of the PI3K/Akt/Foxo3a signaling network that causes Foxo3a pulses. Overall thisresearch will further our understanding of how protein dynamics regulate cellular fate outcomes.