Genome-Wide Histone Acetylation Profiling in Chronic Lymphocytic Leukemia Reveals a Distinctive Signature in Stereotyped Subset #8

In CLL, subsets of patients carrying stereotyped B cell receptors (BcR) share similar biological and clinical features independently of IGHV gene somatic hypermutation status. Although the chromatin landscape of CLL as a whole has been recently characterized, it remains largely unexplored in stereotyped cases. Here, we analyzed the active chromatin regulatory landscape of 3 major CLL stereotyped subsets associated with clinical aggressiveness.

We performed chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac histone mark in sorted CLL cells from 19 cases, including clinically aggressive subsets #1 (clan I genes/IGKV(D)1-39, IG-unmutated CLL (U-CLL)(n=3)], #2 [IGHV3-21/IGLV3-21, IG-mutated CLL (M-CLL)(n=3)] and #8 [IGHV4-39/IGKV1(D)-39, U-CLL(n=3)] which we compared to non-stereotyped CLL cases [5 M-CLL|5 U-CLL]. In addition, a series of 15 normal B cell samples from different stages of B-cell differentiation were analyzed [naive B cells from peripheral blood (n=3), tonsillar naive B cells (n=3), germinal centre (GC) B cells (n=3), memory B cells (n=3), tonsillar plasma cells (n=3)].

Initial unsupervised principal component analysis (PCA) disclosed a distinct chromatin acetylation pattern in CLL, regardless of stereotypy status, versus normal B cells. CLL as a whole was found to be closer to naive and memory B cells rather than GC B cells and plasma cells. Detailed analysis of individual principal components (PC) revealed that PC4, which accounts for 5% of the total variability, segregated subset #8 cases and GC B cells from other CLLs and normal B cell subpopulations. Although PC4 accounts for only a small part of the total variability (5%), this suggests that subset #8 cases may share some chromatin features with proliferating GC B cells, in line with the fact that subset #8 BcR are IgG-switched.

We also investigated whether stereotyped CLLs have different chromatin acetylation features compared to non-stereotyped CLLs matched by IGHV somatic hypermutation status and identified 878 Differential Regions (DR) in subset #8 vs. U-CLL, 84 DR in subset #1 vs. U-CLL and 66 DR in #2 compared vs. M-CLL.

As subset #8 cases seemed to have the most distinct profile, we further characterized the detected regions. The 435 and 443 regions gaining and losing activation, respectively, mostly targeted promoters (29.5%) and regulatory elements located in introns (31%) and distal intergenic regions (21.8%). Hierarchical clustering based on the 878 DRs enabled the clear discrimination of subset #8 cases from U-CLL and normal B cells; however, it is worth noting that for several of these 878 DRs the acetylation patterns were shared between subset #8 and normal B cell subpopulations rather than subset #8 and U-CLL.

Of note, 11/435 regions gaining activity on subset #8 were found within the gene encoding for the EBF1 transcription factor (TF); additional regions were associated with genes significant to CLL pathogenesis, e.g. TCF4 and E2F1. Moreover, 3 DRs losing activity in subset #8 were located within the CTLA4 gene and 2 DRs within the IL21R gene, which we have recently reported as hypermethylated and not expressed in subset #8.

Next, we performed TF binding site analysis by MEME/AME suit, separately for regions gaining or losing activity, and identified significant enrichment (adj-p<0.001) on TFs such as AP-1, FOX, GATA, IRF. The regions losing activity in subset #8 showed a higher number of enriched TFs versus those gaining activity (165 vs 93 TFs), particularly displaying enrichment for many HOX family members . However, a cluster of TFs with enrichment on TF binding site analysis, such as FOXO1, FOXP1, MEF2D, PRDM1, RUNX1, RXRA, STAT6, were also located within the 878 DRs discriminating subset #8 from either U-CLL or normal B cell subpopulations.

Taken together, subset #8 cases have a distinct chromatin acetylation signature which includes both loss and gain of active elements, shared features with proliferating GC B cells, and specific changes in chromatin activity of several genes and TFs relevant to B cell/CLL biology. These findings further underscore the concept that BcR stereotypy defines subsets of patients with consistent biological profile, while they may also be relevant to the particular clinical behavior of subset #8, known to be associated with the highest risk of Richter's transformation amongst all CLL.