Project Summary Diffuse large B-cell lymphoma (DLBCL) accounts for up to 40% of non-Hodgkins lymphoma (NHL) and there are few therapeutic options for patients who don’t respond to the standard of care. The transcription factor FOXO1 is recurrently mutated (mFOXO1) across DLBCL subtypes and enriched in relapsed/refractory patients. Unlike other tumor types, DLBCL FOXO1 mutations appear to be activating. While deletion of Foxo1 in mice demonstrated that it is required for proper B-cell development, the consequences of putative activating mutations have not been investigated. Importantly, genetic deletion and RNAi studies in cell lines cannot distinguish between direct and indirect transcriptional effects due to the lack of temporal analysis (analyzed days or weeks later). Furthermore, prior studies determining the mechanisms by which FOXO1 controls gene expression are fragmented, limited in scope, and the results are contradictory and cell type specific. To study FOXO1 in the context of DLBCL, we have developed in vitro and in vivo models using CRISPR/Cas9 that will answer a number of long-standing questions about FOXO1 biology. We engineered the endogenous loci for WT and mFOXO1 such that FOXO1 is fused to FKBP12F36V and a 2XHA epitope tag. These cell lines will allow us to rapidly inactivate the endogenous FOXO1 (FKBP12F36V) and use the analysis of nascent transcription over a 2hr time course after drug addition to identify the direct transcriptional targets of mFOXO1. The incorporation of an epitope tag into the endogenous FOXO1 will also allow us to generate robust Cut&Run libraries to unambiguously define the bound genomic loci. We will use xenografts of these cells to determine if mutant FOXO1 is a therapeutic target in vivo. We have also created a germline mouse model with a DLBCL-associated mutation in FOXO1 and crossed these mice with Pten-conditional deletion mice. These mice will allow us to directly test the hypothesis that mFOXO1 escapes activated PI3K/AKT signaling to maintain proliferation in the germinal center. In Aim 1, we will use techniques and informatic platforms already employed in the Hiebert lab to identify the direct transcriptional targets of FOXO1, determine the mechanism through which FOXO1 regulates transcription, and assess whether FOXO1 is a therapeutic target. We have already established mouse models containing mFOXO1 and determined that it caused a perturbation in B-cell development. Aim 2 will further interrogate the effects of mFOXO1 on the germinal center reaction and determine if mFoxo1 enhances lymphomagenesis. This proposal will test the hypothesis that FOXO1 mutations make a constitutively active form of FOXO1, which continuously activates target genes in the germinal center light zone, even in the face of high PI3K signaling, to trigger lymphomagenesis. Additionally, this project has the potential to identify novel therapeutic targets in mFOXO1 DLBCL.