Aberrant coexpression of FOXP1 and AID characterizes NF-κB-driven murine and human ABC-DLBCL. (A) Representative immunohistochemical staining of FOXP1 (nuclear) and AID (cytoplasmic) in normal splenic GCs and lymphomas. Scale bars, 200 μm or 10 μm at insets. (B) Expression analysis by qRT-PCR of FACS-sorted reporter-positive normal resting or GCBs, and lymphoma B cells (n5 animals). Relative values were normalized to resting or GCB expression levels for mFoxp1 or mAID, respectively. (C) Expression analysis by qRT-PCR of paired NBC or GCBs magnetically sorted from human tonsils or FACS sorted from normal murine spleens (n4). Relative values are normalized to endogenous levels h/mGAPDH. (D) Representative images of inverse correlation of FOXP1 and AID expression in reactive human tonsil or murine spleen examined by IHC, using combined labeling of anti-FOXP1 (brown) and anti-AID (red). Scale bars, 200 μm; magnification, ×20. (E) ChIP-seq data from OCI-Ly1 cells showing enrichment of FOXP1 on the hAID locus. Distribution of FOXP1 occupancy is overlapped with chromatin-state models predicted by ENCODE ChromHMM from GM12878 (human B lymphoblastoid) or CH12 (mouse B-cell lymphoma) cells, and with the predicted conserved mAID gene locus according to Genome Browser comparison of hg19 and mm9 sequence data. Black boxes on top indicate genomic regions (R1-4) associated with transcriptional regulation of AID expression. (F) Comparison of hFOXP1 ChIP-seq peaks at intronic region 2 of the hAID locus observed here in OCI-Ly1 cells or previously in other GCB-DLBCL or ABC-DLBCL cell lines (GSE69009). (G) Validation of FOXP1 occupancy at AID intronic peaks and measured by ChIP-qPCR in human OCI-Ly1 DLBCL cells, as well as in resting or activated murine cells (ie, CH12 lymphoma cells and primary magnetically sorted CD19⁺ splenic cells). CIT, combination of anti-CD40 plus IL-4 and TNF-β; LPS, lipopolysaccharides. (H) Scatter plot of gene expression array data from OCI-Ly1 cells showing average log₂ fold-changes (n = 3 replicates) in the expression of FOXP1-bound genes (according to ChIP-seq data from OCI-LY1) relative to scramble control after FOXP1 silencing with 2 different siRNAs. (I) Heat map of average fold-changes (n = 3 replicates) in the expression of FOXP1 and AID relative to scramble control after siRNA-mediated silencing of FOXP1 in DLBCL cell lines and in CIT-activated CH12 cells. (J) Forest graph plot of Pearson r coefficients measuring the correlation of hFOXP1 and hAID expression in previously published GSE series. (K) Scatter plot of FOXP1 and AID gene expression array data from R-CHOP-treated patients with DLBCL (n = 233, GSE10846). Median expression levels for FOXP1 (223287_s_at) and AID (219841_at) are indicated and were used as cutoff values for patient stratification. (L) Overall survival of R-CHOP-treated patients with DLBCL stratified by FOXP1/AID expression levels in panel K. (M) Distribution of COO-based subtypes in the FOXP1/AID HiHi and LoLo expression DLBCL subgroups stratified in panel K. COO subtypes were defined by expression signatures and were available in metadata from GSE10846. (N) Scatter plot of FOXP1 and AID protein expression data as measured by IHC scoring from CHOP-treated patients with DLBCL (n = 112). Median IHC scores are indicated and were used as cutoff values for patient stratification. (O) Overall survival of CHOP-treated patients with DLBCL stratified by FOXP1/AID IHC scores in panel N. (P) Distribution of COO-based subtypes in the FOXP1/AID HiHi and LoLo expression DLBCL subgroups stratified in panel N. COO subtypes were defined by the Hans IHC algorithm. HiHi, FOXP1^highAID^high; LoLo, FOXP1^lowAID^low; NBC, naive B cells; UNC, unclassified.