Mapping of WT FOXO1 and M1L FOXO1 binding sites in SUDHL4 cells

The transcription factor FOXO1 directs germinal center (GC) polarity and supports affinity maturation by silencing immune activation programs in GC B cells. Recurrent somatic mutations in GC-derived B cell non-Hodgkin lymphomas (B-NHL) often target and reportedly activate FOXO1 by preventing its negative regulation by PI3K. Herein, using ad hoc mouse models, engineered cell lines and primary tumor specimens, we show instead that FOXO1 mutant proteins display altered transcriptional activities suggestive of partial loss-of-function. These defects drive simultaneous hyperactivation of signaling pathways (Stress Activated Protein Kinase -SAPK/JNK, Phosphoinositide 3-kinase -PI3K/AKT) and gene expression programs typically activated in GC B cells upon positive selection. Such changes confer mutant B cells with competitive advantages in response to key immune signals, leading to abnormal amplification of GC responses. Evidence of these FOXO1 'mutant' gene programs can be found in a fraction of human B-NHL and predict clinical outcome, thus implying the frequent co-option of positive selection programs during GC-derived B-NHL pathogenesis. Overall design: SUDHL4 were edited using CRISPR-Cas9 to introduce biallic M1L mutations in the FOXO1 locus. Two independent single cell clones were expanded in culture and used to compare binding repertoires between WT and M1L FOXO1.