Identification and characterization of genes deregulated by DNA methylation in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is one of the most common leukemias in adults in the Western world characterized by the accumulation of mature-appearing B-cells in the peripheral blood, lymphoid tissues and bone marrow. The use of novel inhibitors targeting BCR- and PI3K-signaling in CLL as well as the application of CD20-antibodies in the clinic have led to improved outcome for the patients. Nevertheless, CLL remains an incurable disease and in order to develop new treatment strategies, there is still a strong need to elucidate the pathomechanism of CLL further. CLL is amongst other aspects a disease of the epigenome, displaying genome-wide hypomethylation while specific regions are hypermethylated, resulting in the deregulation of tumor suppressors, non-coding RNAs and oncogenes. Interestingly, DNA methylation in CLL patients was shown to be relatively stable over time suggesting that genes which are deregulated by this DNA modification are affected permanently, independent of disease stage and course. In this project, it was therefore aimed to identify and characterize genes deregulated in CLL by DNA methylation, which are likely involved in the pathomechanism of the disease. To this end, expression and methylation array profiling was conducted in CD19+ sorted CLL cells and B-cells of healthy donors. Out of the 1,866 genes identified as differentially expressed between cancer cells and their non-malignant counterpart, 33 showed significantly differentially methylated regions (DMR) that displayed negative correlation with gene expression. Technical validation using mass spectrometry based quantification of methylation confirmed the presence of DMRs in 17 of these genes including the transcription and stem cell factor KLF4. KLF4, of which repression and hypermethylation could be confirmed in a larger patient subset, was previously shown to play an important role in B-cell differentiation and maturation and further to act as tumor suppressor in other B-cell malignancies including classical- and non-Hodgkin lymphoma. Moreover, KLF4 was shown to be a downstream target of NOTCH1 which is recurrently mutated and constitutively active in CLL. In fact, treatment of six different lymphoblastoid and leukemia cell lines and of primary peripheral blood mononuclear cells (PBMCs) from eight CLL patients with γ-secretase inhibitor resulted in the inhibition of NOTCH1 activity and in the re-expression of KLF4 on RNA and protein level, suggesting transcriptional regulation of KLF4 by NOTCH1. Furthermore, expression of KLF4 was correlated with levels of BCL-2 family members BAK, BAX and BCL-2 as well as the cell cycle regulator CCND1 in primary CLL and B-cells. Although, overexpression of KLF4 in three leukemia cell lines did not induce apoptosis in these cells, it resulted in the deregulation of a number of genes involved in prominent signaling pathways which are highly relevant for the pathogenesis of CLL, including BCR- and PI3K-signaling. In summary, these findings suggest the repression of KLF4 by DNA promoter methylation and NOTCH signaling in CLL. Overexpression of KLF4 results in the deregulation of genes involved in signaling pathways important for B-cells which indicates that KLF4 is in part involved in keeping the CLL cells in a highly activated state.