Large-scale analysis of DNA methylation in chronic lymphocytic leukemia

Abstract

Aims: B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy that clinically ranges from indolent to rapidly progressive. CLL, like other cancers, can be affected by epigenetic alterations.

Materials & methods: A microarray discovery-based study was initiated to determine DNA methylation in CLL cases with a range of CD38 expression (1–92%).

Results: Many loci were either methylated or unmethylated across all CD38 levels, but differential methylation was also observed for some genes. Genomic sequencing of DLEU7 confirmed extensive cytosine methylation preferentially in patient samples with low CD38 expression, whereas NRP2, SFRP2 and ADAM12 were more commonly methylated in those with high CD38 expression.

Conclusion: This study demonstrates that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The CpG island methylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. It will now be useful to investigate the effectiveness of epigenetic therapeutic reversal of these alterations to develop effective treatments for the disease.

Keywords: CD38 expression; CLL; DNA methylation; epigenetics; leukemia.

Figure 1. Hierarchical clustering analysis of DNA methylation

This cluster is based on a 10% threshold where there are 12 samples with less than 10% of the cells expressing CD38 and 26 samples with more than 10% CD38 expression. This illustrates a measure of relatedness of DNA methylation across all loci for each sample. Each column represents a patient sample and each row represents a clone/locus on microarray chip. The florescence ratios of cy3/cy5 are measures of DNA methylation and are depicted as a color intensity (-0.5 to +0.5) in log base 2; yellow indicates loci that have a higher level of DNA methylation in chronic lymphocytic leukemia compared with normal controls, blue indicates a lower level of methylation and black indicates no change. Graded colors across the spectrum represent various levels of methylation. The dendrogram from the top of the cluster (rotated 90° and enlarged on the right) represents the CD38 expression level of each sample. Not every patient sample clustered with the expected group. The numbers shown in blue color are seven patients with CD38^high that are clustered in the group of 10% or less, and one sample with 6% CD38 clustered with the CD38^high group.

Figure 2. COBRA or MSP validation of DNA methylation

The DNA methylation status of primary chronic lymphocytic leukemia (CLL) tumor samples was validated using either combined bisulfite restriction analysis (COBRA) or methylation-specific PCR (MSP) from the samples above, as well as primary CLL samples that were divided to three groups (1–10%, 11–30% and 31–91%) based on CD38 positivity. Results are summarized as methylated (dark square) or unmethylated (open square). Each square represents an individual patient sample and each specific gene. The right-side panel represents the results of control samples, while the left-side panel represents the DNA methylation status in CLL cell lines.

Figure 3. Bisulfite sequencing of DLEU7

DNA methylation of 25 CG dinucleotides was examined in a 5′ region of DLEU7 that spans part of a region spanning the predicted 5′UTR and part of the first exon; the location of this region with respect to the TSS is shown. The CG dinucleotides are shown as heavy black lines. The methylation status of 25 CG dinucleotides was determined from bisulfite-treated DNA of CLL patients with different CD38 expression levels and either M-CLL or unmutated U-CLL IgV_H mutational status, two CLL cell lines (MEC1 and MEC2) and CD19⁺ B cells from a healthy donor. Each row is the result from an individual clone across the 25 CG dinucleotides. Filled circles indicate methylated cytosine and open circles are unmethylated sites. CLL: Chronic lymphocytic leukemia; M-CLL: Mutated CLL; TSS: Transcription start site; U-CLL: Unmutated CLL.

Figure 4. Pharmacologic reactivation in CLL cell lines

The mRNA expression levels of eight genes were quantified using real-time RT-PCR in untreated WAC3CD5, MEC1 and MEC2 cell lines. These were also treated with the demethylating agent 5′-Aza, TSA or the combination of 5′-Aza and TSA. A housekeeping gene (HRPT1) was used as an internal control for all of the above genes, except for DLC-1 where GAPDH was used. The relative expression level of each gene was determined using 2^-ΔΔCt, where Ct is the cycle threshold for positivity. 5′-Aza: 5-aza-2′-deoxycytidine; TSA: Trichostatin A.