ABSTRACT
B-cell chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells indicating disruption of apoptosis. Restriction Landmark Genome Scanning was done to identify novel target genes silenced by CpG island methylation in CLL. Secreted frizzled-related protein 4 (SFRP4), a negative regulator of the Wnt signaling pathway, was found to be frequently methylated in CLL samples. Wnt signaling has been shown to control normal apoptotic behavior and is required for normal B-cell development whereas aberrant activation of this pathway has been observed in CLL. We show aberrant DNA methylation and silencing of SFRP4, as well as of additional SFRP family members, in primary CLL samples. Induction of their expression in a dose-dependent manner following treatment with a demethylating agent, 5-aza-2'-deoxycytidine, was shown. Of the five SFRP family members studied in detail, SFRP1 was hypermethylated and down-regulated in all CLL patient samples studied, suggesting that this epigenetic event is a critical step during leukemogenesis. Our results suggest that silencing of SFRPs by CpG island methylation is one possible mechanism contributing to aberrant activation of Wnt signaling pathway in CLL.
Subject(s)
DNA Methylation , Gene Silencing , Intercellular Signaling Peptides and Proteins/biosynthesis , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Membrane Proteins/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Transformation, Neoplastic , CpG Islands , Decitabine , Dose-Response Relationship, Drug , Gene Expression Profiling , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/physiopathology , Membrane Proteins/genetics , Membrane Proteins/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Signal Transduction , Wnt Proteins/physiologyABSTRACT
PURPOSE: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V(H)) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V(H) mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells. MATERIALS AND METHODS: TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells. RESULTS: Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V(H) show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V(H) were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression. CONCLUSION: TWIST2 is differentially methylated in CLL cells relative to Ig V(H) mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.
Subject(s)
Azacitidine/analogs & derivatives , DNA Methylation , Gene Expression Regulation, Leukemic , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Repressor Proteins/genetics , Somatic Hypermutation, Immunoglobulin , Transcription Factors/genetics , Antimetabolites, Antineoplastic/therapeutic use , Azacitidine/therapeutic use , B-Lymphocytes/pathology , DNA Modification Methylases/antagonists & inhibitors , Decitabine , Gene Expression Regulation, Neoplastic , Helix-Loop-Helix Motifs , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Prognosis , Promoter Regions, Genetic , Treatment Outcome , Twist-Related Protein 1ABSTRACT
The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.