Epigenetic inactivation of Notch-Hes pathway in human B-cell acute lymphoblastic leukemia

Abstract

The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA)/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL). We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2'-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL.

Figure 1. Methylation status of Notch pathway genes in leukemia cell lines and normal peripheral blood cells.

A. Methylation profile of Notch pathway genes in T cell and B cell leukemia cell lines and normal peripheral blood cells. Bisulfite pyrosequencing was performed to determine the methylation status of Notch pathway genes in 8 T cell and 7 B cell leukemia cell lines and 10 normal peripheral blood cells. Green: methylation density<15%; Yellow: methylation density between 15–29.9%; Pink: methylation density between 30–59.9%, Red box: methylation density >60%. Methylation density >15% was used as the cut off to determine a sample as methylated. Methylation frequency is the percentage of methylated cell lines versus the total number of lines studied for each gene. Position fr. TSS: distance of pyrosequencing sites (bp) away from transcription start site (TSS). B. Methylation frequencies of Notch1, Notch2, Notch3, JAG1, DLL1, DLL2, DLL4, Hes2, Hes4, Hes5 and Hes6 genes in leukemia cell lines as detected by pyrosequencing for T cell and B cell lines respectively. *, p<0.05. C&D. Methylation densities of Notch1, Notch2, Notch3, JAG1, DLL1, DLL2, DLL4, Hes2, Hes4, Hes5 and Hes6 genes in T and B cell lines respectively. Pyrosequencing data was used to determine methylation density.

Figure 2. Methylation status of Notch3, JAG1, Hes2, Hes4 and Hes5 genes in normal CD19⁺ B cells, bone marrows from patients with B-ALL and T-ALL.

A & B: Methylation characteristics of Notch3, JAG1, Hes2, Hes4 and Hes5 genes in normal CD19⁺ B cells, B-ALL and T-ALL as shown by table (A) and figure (B). *, p<0.05. C&D. Methylation levels of Notch3, JAG1, Hes2, Hes4 and Hes5 genes in primary B-ALL and T-ALL. Pyrosequencing was performed to determine methylation density. N represents the number of cases in each group.

Figure 3. Expression and histone modification analysis of Hes5 genes in leukemia cell lines and normal hematopoietic lineages.

A. Expression levels of Notch1, Notch2, Notch3, JAG1, Hes1 and Hes5 in normal whole bone marrow (BM), CD34+BM, peripheral blood (PB), CD19⁺ B cells (PB-B) and CD3+ T cells (PB-T). The relative gene expression was determined by real-time PCR assays and normalized to GAPDH. B. Hes5 mRNA expression levels in leukemia cell lines by RT-PCR. Hes5 methylation levels from Figure 1A are shown under each cell lines. C. left. Hes5 expression is significantly lower in Hes5 methylated cell lines when compared with unmethylated cell lines. C. right. Hes5 mRNA expression inversely correlated to methylation level. The solid line represents the regression of the degree of methylation on the Hes5 expression level. D. ChIP assay of the Hes5 CpG islands. Chromatin DNA was immunoprecipitated with antibodies against the indicated histone modifications. Immunoprecipitated DNA was amplified by real-time PCR. Percent input was determined as the amount of immunoprecipitated DNA relative to input DNA. Experiments were performed in duplicate. Bars, SD.

Figure 4. Restoration of Hes5 expression by treatment with 5-aza-2′-deoxycytidine (DAC) and suberoylanilide hydroxamic acid (SAHA) in leukemia cell lines.

A. Hes5 expression and the effect of demethylating treatment. The leukemia cells were either untreated (C), or treated with DAC only, SAHA only or both (D+S) as described in material and methods. Quantitative RT-PCR was used to measure Hes5 mRNA expression. B. Effect of epigenetic modulation on Hes5 gene methylation. Pyrosequencing was performed to determine methylation level. C. DAC and SAHA treatment increase acetylated histone H3 as detected by ChIP-real time PCR. D. Promoter hypermethylation silences expression of Hes5. Top. Diagram of the human Hes5 promoter region studied. CpG sites are indicated by short vertical bars. Arrows point to transcription start site (TSS). The region for bisulfite sequencing, ChIP PCR and promoter activity assay are indicated. Left bottom: Methylation analysis of Hes5 gene promoter region by bisulfite sequencing. Each row of circles represents the sequence of an individual clone. Open circles, unmethylated CpG sites; filled circles, methylated CpG sites. Right bottom: Promoter activity of the Hes5 CpG islands. The relative luciferase activities of the unmethylated and methylated pGL3-Hes5 constructs in 293T cells.

Figure 5. Distinct expression pattern of Hes5 in primary B cell leukemia compared to T-ALL and their response to 5aza-dC (DAC) treatment.

A. Relative Hes5 mRNA expression in pre-treatment bone marrows from patients with T cell acute lymphoblastic leukemia (T-ALL) and B-ALL, as measured by quantitative RT-PCR, normalized to GAPDH. B. Inverse correlation between Hes5 mRNA expression and Hes5 methylation levels in pre-treatment patients, as measured by pyrosequencing. The solid line represents the regression of the degree of methylation on the Hes5 expression level. C. Methylation levels of Hes5 and LINE in B-ALL patient at different time points (days 1–35) after DAC treatment, as measured by pyrosequencing. D. Bisulfite sequencing map of Hes5 gene from a B-ALL patient at days 1 and 30 of DAC treatment.

Figure 6. Hes5 inhibits proliferation and induces apoptosis in B cells but not in T cells.

A. REH, RS4;11 and T-ALL1 cell lines were transduced with lentivirus expressing Hes5 or empty vector. Cell numbers were measured on day using trypan blue exclusion assay. B. Cell cycle distributions measured 2 days after lentivirus infection using propidium iodide (PI) staining to measure DNA content. The percentage of cells in sub-G1 (<2N DNA) is presented. C. Analysis of apoptosis 3 days after lentivirus infection using flow cytometry, PI staining and annexinV staining.