Downregulation of death-associated protein kinase 1 (DAPK1) in chronic lymphocytic leukemia

Abstract

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.

Figure 1

DAPK1 Promoter Methylation in CLL Samples (A) Schematic representation of the DAPK1 gene showing the location of the CpG island (black bar) and the four bisulfite reaction amplicons (A1–A4). The arrow indicates the predicted transcription start site. The lower panel shows a graphical display of quantitative DNA methylation data for the DAPK1 promoter region. Each square represents a single CpG or a group of two or three CpGs analyzed, and each row represents a sample. Methylation frequencies extend from light yellow (0%) to dark blue (100%). Gray indicates unavailable data. Samples included seven CD19+ selected control B cells, four control normal PBMCs, seven CD19+ selected CLL cells, and PBMCs from 62 sporadic CLL samples, Raji and Jurkat cells. Asterisks indicate CLL samples that showed less than 11% methylation. (B) Plot of average percentage methylation in regions A1–A4, in controls (seven CD19+ selected B cells and four PBL samples; total = 11), CD19+ selected CLL cells (n = 7), unselected CLL cells (n = 62), and Raji and Jurkat cell lines.

Figure 2

Epigenetic Silencing of DAPK1 in CLL (A) pGL3 luciferase constructs ligated with different DAPK1 5′ upstream inserts (nos. 1–4) were transfected into Jurkat cells, and reporter activity was studied 48 hr after transfection. pGL3 vector without insert was used as a negative control. Error bars indicate standard deviation (SD). (B) Quantitative DAPK1 expression in normal B cells and seven CD19+ selected CLL samples. The expression in CLL samples is shown relative to the expression in B cells (defined as 1.0). Error bars indicate SD. (C) Box plot of DAPK1 expression in 50 CLL samples as measured by semiquantitative RT-PCR and compared to its expression in six normal B cell samples. The distribution is significantly different (p < 0.01). (D) Luciferase assays in 293T cells with either methylated or unmethylated DAPK1 construct no. 2. Error bars indicate SD. (E) DAPK1 expression in Raji cells treated with 0.5 μM decitabine. RT-PCR was performed for DAPK1 and GAPDH on untreated and treated cell lines. Jurkat cells were used as a positive control. (F) Bisulfite sequences in BS1 region (c.1-1509–c.1-1262) for untreated, 6 and 12 days decitabine-treated (0.5 μM) Raji cells. Each row represents a clone. The open circles indicate unmethylated CpG, and closed circles indicate methylated CpG dinucleotides. The overall methylation frequency is given in percentage. Error bars indicate SD.

Figure 3

DAPK1 Regulates Apoptosis in Lymphoid Cells (A) DAPK1 expression in Jurkat cells stably transfected with either vector alone, DAPK1 siRNA-A, or DAPK1 siRNA-C as measured by western blot. Tubulin expression served as a control. (B) Percent live cells were measured in Jurkat cells stably transfected with vector alone or DAPK1 siRNA-C, treated with activating-Fas antibody (100 ng/ml). After 16 hr, cells were harvested and suspended in binding buffer with annexin V-FITC and propidium iodide, followed by flow cytometry to assess cell death. Error bars indicate SD. (C) p53 expression in cells treated with either no, 50 ng/ml, or 100 ng/ml Fas-activating antibody.

Figure 4

DAPK1 Expression in CLL Family 4532 (A) Pedigree of CLL family no. 4532. Open circle and square represent unaffected female and male, respectively, while closed circle and square represent affected female and male. (B) Sequencing of SNP c.114A > G from genomic DNA and cDNA of two unaffected (IV-3 and IV-4) and two affected (III-3 and III-4) individuals. (C) RT-PCR on RNA isolated from monochromosomal hybrid clones with either WT or the CLL chromosome 9 from fibroblast cells of individual III-4 is shown. RPL4 expression was used as an internal control. Error bars indicate SD. (D) DAPK1 protein expression from WT and CLL alleles in monochromosomal hybrids. Jurkat and WaC3CD5 cells were used as positive controls, and NIH3T3 cells were used as the negative control to show specificity to human DAPK1. (E and F) The RT-PCR product amplifying SNP c.1510A > G in unaffected (IV-3; E) and affected (III-4; F) fibroblast cell lines was cloned, and individual clones were genotyped. Shown is the percentage of A and G clones in IV-3 and III-4, and n is the number of clones studied. The difference in allelic expression of DAPK1 in III-4 and IV-3 was statistically significant (p < 0.01).

Figure 5

An A to G Change at c.1-6531 bp Regulates DAPK1 Expression in CLL Family (A) A 357 bp PCR product with SNP c.1-6531A > G was ligated upstream to DAPK1 promoter (c.1-2215–c.1-1151) luciferase construct with either A (DAP-A) or G (DAP-G) as SNP. (B) The DAPK1 promoter alone (no. 1), DAP-A, or DAP-G constructs were transfected into Jurkat cells, and the luciferase activity was measured. Error bars indicate SD. (C) Nuclear extracts from Jurkat cells were analyzed by EMSA assay using WT or CLL oligo. Five specific bands (I–V) are marked. (D) Ten- or fifty-fold molar excess concentrations of cold oligos were used for competition assays. An unlabeled Oct-1 oligo was used as a negative control. (E) EMSA assays were performed using WT, WT mutant, CLL, and CLL mutant oligos. In mutant oligos, the adjacent bases to the c.1-6531 SNP were mutated. (F) For the supershift assay, antibodies against HOXB7, USF2, and MSX2 were added to the Jurkat nuclear extract, and gel shift was studied using the CLL oligo.

Figure 6

Downregulation of DAPK1 Expression by HOXB7 (A and B) Fibroblast cell lines from affected (III-4) and unaffected (IV-4) family members were transfected with 60 nM of HOXB7 siRNA or scrambled siRNA, and HOXB7 expression was studied at different time points by semiquantitative SYBR green RT-PCR. HOXB7 expression in untreated cells was set as 1.0. Error bars indicate SD. (C and D) DAPK1 expression was studied in the fibroblast cell lines transfected with HOXB7 siRNA or scrambled siRNA for different time points by quantitative SYBR green RT-PCR. DAPK1 expression in untreated cells was set as 1.0. Error bars indicate SD. (E and F) Allele-specific expression of DAPK1 in an affected (E) and unaffected (F) family member was studied in fibroblasts from III-4 and IV-4, before and after transfection of HOXB7 siRNA. RT-PCR products including SNP c.1510A>G were cloned and genotyped. Shown is the percentage of the number of clones from the two alleles, and (n) indicates number of clones studied.

Figure 7

DAPK1 Expression and Promoter Methylation in CLL Cells of Family No. 4532 (A) RT-PCR for DAPK1 with GAPDH as an internal control on RNA extracted from blood cells of no. 4532 family members III-1, III-2, III-3, III-4 and IV-5 and from selected CD19+ normal B-cells from 3 healthy volunteers. Error bars indicate SD. (B) DNA methylation analysis in CLL cells from CD19+ selected control B-cells, total PBMCs from one unaffected family member (IV-1) and two affected individuals (III-1 and III-4). Bisulfite treated DNA was amplified for the BS1 and BS2 regions. Each row represents a clone. The open circles indicate unmethylated and closed circles indicate methylated CpGs.