Transcriptional profiling of SUDHL4 cells edited by CRISPR-cas9 to induce point mutations in the FOXO1 locus

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. SUDHL4 cells which are normally WT for FOXO1 were edited with CRISPR-Cas9 to intruduce M1L point mutations in homozygosity or S22P point mutations in heterozygosity. Singles clones were expanded in culture and profiled by RNA-seq