Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles

Abstract

Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.

Figure 1. Unsupervised hierarchical clustering (A) and three-dimensional principal component analysis (PCA) of DNA methylation data (B) comparing samples belonging to subset #1, subset #2 and subset #4. The S refers to the subset number while the following number refers to the specific case as detailed in Table S1.

Figure 2. Supervised hierarchical clustering of differentially methylated genes (methylation index geometric mean difference > 0.35, p < 0.05). Comparisons were made between subsets #1 and #4 (A), subsets #1 and #2 (B) and subsets #2 and #4 (C). A gradient color scale represents the methylation index values; green as unmethylated and red as methylated.

Figure 3. Pyrosequencing (A) and RQ-PCR (B) data on CD80 and CD86 in subset #1 and #4 patient samples. Boxes indicate the interquartile range (25–75%) while the small inner square indicates the median value. The whiskers represent the minimum and maximum values, except for outliers (circles) and extremes (stars).