Differential DNA methylation patterns leading to differential gene expression: identifying p63 as a novel player in CLL
Two primary and interconnected epigenetic mechanisms are histone modifications and DNA methylation. Whereas DNA methylation is considered to be a very stable epigenetic modification, histone modifications are more labile. Levels of histone modifications are maintained by the balance between the activities of histone-modifying enzymes that add or remove specific modifications. As aberrant histone modification levels result from an imbalance in these modifying enzymes in diseased tissue, correcting the increased or decreased level of a particular enzyme could restore the natural equilibrium in the affected cells.
- We compared the global DNA methylation profile of subgroups of CLL patients with aggressive disease and found distinct DNA methylation patterns, leading to differential gene expression. Amongst the genes showing the greatest differences was TP63 which was found to be overexpressed (while in parallel hypomethylated) in CLL subset #8 (a subgroup with the highest risk of Richter’s transformation amongst all CLL) compared to all other subgroups analyzed.
- The TP53 gene family member TP63 (p63/p51/p40/TP73L/KET) is almost never mutated in cancer, yet alterations in its expression are known to critically affect tumorigenesis and chemoresistance (Melino, 2011). TP63 exercises transcriptional regulation of a large number of genes and microRNAs responsible for cell adhesion, apoptosis, proliferation, and differentiation.
- In CLL there are few studies with markedly different results. Humphries et al (2013) showed that TAp63 mRNA levels were higher in CLL with unmutated IGHV genes and emphasized the pro-apoptotic function of TP63. Moreover they showed that B-cell receptor (BCR) engagement leads to repression of TP63 mRNA expression in malignant B cells. Binsky et al (2010) demonstrated that stimulation of CD74 with its natural ligand, migration inhibitory factor (MIF), initiates a signaling cascade that results in upregulation of TP63, which directly regulates CLL survival. In addition, TP63 expression was found to elevate the expression of the integrin VLA-4, particularly during the advanced stage of the disease.
- So far we have analyzed TP63 expression and function in well characterized CLL subgroups, starting from stereotyped subsets #6 and #8. We found that subset #8 shows significantly higher TP63 expression compared to all other subgroups under study and that BcR stimulation induced TP63 upregulation in subset #8. Moreover we found that BcR stimulation in CLL subset #8 protect cells from apoptosis and siRNA experiments against TP63 confirmed that TP63 is a prosurvival factor in CLL.