Genome-Wide Methylation Arrays Reveal Distinct Methylation Profiles in Prognostic Subsets of Chronic Lymphocytic Leukemia.

Aberrant DNA methylation has been shown to play a strong role in tumorogenesis, where genome-wide hypomethylation and regional hypermethylation of tumor suppressor gene (TGS) promoters are characteristic hallmarks of many cancers. In chronic lymphocytic leukemia (CLL), the epigenetic mechanism of gene regulation has thus far received limited attention, although promoter methylation and transcriptional silencing has been shown for certain individual genes, for example, DAPK1, ZAP70 and PEG10. To date, only the ‘Restriction Landmark Genomic Scanning' technique has been performed to assess the genome-wide methylation status in CLL. However, this technique spans only 3000 CpG islands and does not give a full coverage of the genome.

Here, we analyzed the global methylation profiles in CLL by applying high-resolution genome-wide methylation arrays from Illumina that cover 28,000 CpG sites, spanning 14,000 genes. Specifically, 23 CLL samples belonging to the immunoglobulin heavy-chain variable (IGHV) mutated (favorable prognostic) and IGHV unmutated/IGHV3-21 (poor-prognostic) subsets were analysed. The raw data was processed using the BeadStudio software followed by bioinformatic analysis where the arcsin transformed data was used in a moderated t-test to find differentially methylated genes. Only genes with a large absolute difference between the groups were included for further analysis. Methylation-specific PCR (MSP-PCR) and realtime-PCR (RQ-PCR) were performed on a selection of genes to confirm the array data. Additionally, bi-sulfite sequencing was employed on selected genes to confirm the degree of methylation. Moreover, CLL samples were treated with the DNA methyl transferase inhibitor 5-aza-2'-deoxycytidine combined with and without the histone deacetylase inhibitor (HDAC) trichostatin A to induce re-expression of selected methylated genes

Overall, we observed significant differences in methylation patterns between the CLL subgroups. Specifically, we identified TSGs that were preferentially methylated in the IGHV unmutated (7 genes, e.g. VHL, ABI3) and IGHV3-21(1 gene, SLC22A18) subgroups. We also identified 10 unmethylated and hence potentially expressed genes shown to be involved in activation of proliferative pathways such as the NFkB pathway (e.g. ADORA3), and the MAP/ERK kinase pathway (e.g. FABP7) in the IGHV unmutated and IGHV3-21 subgroups. In contrast, these latter genes were silenced by methylation in IGHV mutated patients. The methylation status was verified for 4 genes (BCL10, PRF1, ADORA3 and IGSF4) by MSP-PCR and the expression status of 7 genes (BCL10, PRF1, ADORA3, IGSF4, NGFR, ABI3 and VHL) was confirmed using RQ-PCR. Furthermore, bi-sulfite sequencing confirmed the degree of methylation for 2 methylated TSGs (VHL and ABI3) in unmutated CLL samples. Finally, the significance of DNA methylation in regulating gene promoters was shown by re-inducing 3 methylated TSGs ( VHL, ABI3 and IGSF4) in IGHV unmutated samples using the methyl-inhibitor 5-aza-2'-deoxycytidine.

Taken together, our data for the first time reveals differences in global methylation profiles between prognostic subsets of CLL, which may unfold important epigenetic silencing mechanisms involved in CLL pathogenesis. Specific inhibition of expression of unmethylated genes involved in facilitating tumorogenesis and re-expression of methylated tumor suppressor genes within the poor-prognostic CLL subgroups may represent potential new drug therapy targets.

No relevant conflicts of interest to declare.