Concurrent epigenetic silencing of wnt/β-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia

Abstract

Background The Wnt/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/β-catenin pathway inhibitor genes – CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 – in the cell lines EHEB and MEC-1 as well as patient samples. Methods Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models. Results For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect. Conclusions The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

Somatic mutation as a mechanism of Wnt/β-catenin pathway activation in CLL

“…Extensive Wnt pathway dysregulation has been previously noted in CLL, and is linked in part to changes in DNA methylation. [11][12][13]30 LEF1 overexpression has been reported in the premalignant form of CLL, monoclonal B-cell lymphocytosis, 8 and is thought to drive CLL. Because expression of Wnt pathway members appears to be crucial for CLL survival and our data demonstrate that altered gene expression is indistinguishable between samples with and without mutations, we reason that somatic mutation is another layer of regulation affecting CLL function.…”

Genetic lesions associated with chronic lymphocytic leukemia chemo-refractoriness

“…39 The role of the Wnt pathway in CLL has been explored in several papers. [40][41][42][43] Gene expression analysis showed that several members of the Wnt family, as well as the transcription factor LEF1, are overexpressed in CLL cells compared with peripheral blood B cells from healthy donors. 40,41 On the other hand, it was reported that Wnt/b-catenin inhibitor genes were hypermethilated.…”

Molecular pathogenesis of CLL and its evolution

“…Interestingly, the assessment of the methylation status of specific CpG pairs in ZAP70 has been proposed for clinical prognostic purposes, as they are associated with ZAP70 expression, activity and clinical outcome [75][76][77]. Also, several works have described altered regulation of Wnt signaling through aberrant methylation of different genes of this CLL-relevant pathway [78][79][80][81].…”