Hypermethylation of the alternative AWT1 promoter in hematological malignancies is a highly specific marker for acute myeloid leukemias despite high expression levels
© Guillaumet-Adkins et al.; licensee BioMed Central Ltd. 2014
Received: 15 November 2013
Accepted: 19 December 2013
Published: 9 January 2014
Wilms tumor 1 (WT1) is over-expressed in numerous cancers with respect to normal cells, and has either a tumor suppressor or oncogenic role depending on cellular context. This gene is associated with numerous alternatively spliced transcripts, which initiate from two different unique first exons within the WT1 and the alternative (A)WT1 promoter intervals. Within the hematological system, WT1 expression is restricted to CD34+/CD38- cells and is undetectable after differentiation. Detectable expression of this gene is an excellent marker for minimal residual disease in acute myeloid leukemia (AML), but the underlying epigenetic alterations are unknown.
To determine the changes in the underlying epigenetic landscape responsible for this expression, we characterized expression, DNA methylation and histone modification profiles in 28 hematological cancer cell lines and confirmed the methylation signature in 356 cytogenetically well-characterized primary hematological malignancies.
Despite high expression of WT1 and AWT1 transcripts in AML-derived cell lines, we observe robust hypermethylation of the AWT1 promoter and an epigenetic switch from a permissive to repressive chromatin structure between normal cells and AML cell lines. Subsequent methylation analysis in our primary leukemia and lymphoma cohort revealed that the epigenetic signature identified in cell lines is specific to myeloid-lineage malignancies, irrespective of underlying mutational status or translocation. In addition to being a highly specific marker for AML diagnosis (positive predictive value 100%; sensitivity 86.1%; negative predictive value 89.4%), we show that AWT1 hypermethylation also discriminates patients that relapse from those achieving complete remission after hematopoietic stem cell transplantation, with similar efficiency to WT1 expression profiling.
We describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. In addition AWT1 hypermethylation is ideally suited to monitor the recurrence of disease during remission in patients undergoing allogeneic stem cell transfer.
KeywordsWT1 Methylation Epigenetics Leukemia Imprinting
Overexpression of the Wilms’ tumor 1 gene (WT1) is implicated in the prognosis of leukemia with high expression predicting disease progression in acute myeloid leukemia (AML), as well as being intensively studied as a potential molecular marker for minimal residual disease (MRD) and treatment response. In the normal scenario, WT1 expression is restricted to CD34+ cells in the bone marrow and absent in peripheral blood cells; therefore expression of this zinc-finger transcription factor is limited to early progenitors of the blood system, suggesting that this gene plays a critical role in controlling proliferation and/or differentiation in hematopoietic stem cells .
Expression profiling of WT1 is becoming almost universal in characterizing AML. Recently, raised WT1 levels at diagnosis and post-induction are associated with poorer outcomes, whereas patients with very low WT1 level post-intensification had excellent outcomes . Despite aberrant expression of WT1 being a useful marker for diagnosis and monitoring MRD in AML the underlying epigenetic alterations associated with this expression are unknown . Using a panel of 28 hematological cancer cell lines and more than 350 primary samples we identify AML-specific AWT1 promoter hypermethylation that is present irrespective of underlying translocation/oncogenic fusion protein or mutations, which is accompanied by a concomitant epigenetic switch at the level of post-translational histone tail modifications. Finally we show that this robust AWT1 epigenetic signature is an excellent marker for discriminating AML from non-diseased peripheral blood and that this hypermethylation signature can accurately track disease progression, differentiating patients who relapse from those achieving complete remission after allogeneic hematopoietic stem cell transplantation (SCT).
Abundant biallelic expression of the WT1/AWT1 transcripts result in nuclear retained proteins in myeloid derived leukemic cell lines
qRT-PCR was performed on a wide range of leukemia and lymphoma cell lines to evaluate the transcription levels of the WT1, AWT1 and the non-coding antisense transcript WT1-AS. In all myeloid origin cell lines evaluated the WT1 and AWT1 transcripts were readily detectable, with high correlation (Pearson correlation, r = 0.90), indicating that they are likely to be co-regulated. The WT1-AS transcript was also expressed in most of the myeloid cell lines (Figure 1B). In contrast, the majority of B-, T- cell leukemic cell lines and lymphomas did not express these transcripts, with the exception of MOLT4 and NALM-20, the later being a cell line with a t(9;22) translocation and biphenotypic B-cell and myeloid cell characteristics . The transcripts within the WT1 locus have been described to be expressed from the paternal allele in an imprinted manner [6, 7]. The over-expression of these transcripts in the cell lines allowed us to determine allelic expression. Using primer pairs designed to distinguish individual isoforms (i.e. the forward primer was in the unique first exon of each transcript) and incorporate SNPs identified in the UCSC sequence browser (GRC27/hg19- dbSNP 135) into the final RT-PCR product, we observe that WT1, AWT1 and WT1-AS are biallelically expressed in all heterozygous cell lines (Figure 1C). Finally, western blotting using an antibody directed against the C-terminal of the WT1 protein revealed that the abundant RNA levels for WT1 and AWT1 isoforms are translated into nuclear retained proteins in the myeloid derived cell lines KG1A, NB4 and K562, consistent with their transcription factor function (Figure 1D).
AWT1 promoter hypermethylation despite over-expression in AML
The promoter region for WT1 is located within a CpG island of 2.4 kb that is unmethylated in all normal cell types as revealed by the WGBS (chr11: 32454875 32457311 = CD34+ 3% methylated; leukocytes 5% methylated). Approximately 4 kb downstream in intron 1, a second promoter region is embedded within an unmethylated CpG island of 560 bp (chr11: 32452145-32452708 = CD34+ 24% methylated; leukocytes 15% methylated), giving rise to the AWT1 and WT1-AS isoforms (Figure 2A). Adjacent to these promoters is a region of proposed allele specific DNA methylation, the WT1-ARR, which has been implicated in regulating expression of these transcripts (chr11: 32454083-32454765 = CD34+ 33% methylated; leukocytes 18% methylated). Our analysis utilizing standard bisulphite PCR and subcloning of normal leukocyte samples heterozygous for the SNP rs11031781 revealed that this region is mosaically methylated on both alleles (Figure 2B), consistent with a lack of imprinted methylation despite normal leukocytes having methylation on the paternal allele at the H19 loci (Additional file 1: Figure S1A).
Methylation analysis of the entire panel of leukemia and lymphoma cell lines, determined by pyrosequencing, revealed that in myeloid derived cancer cell lines the WT1 CpG island promoter is unmethylated, except for the HL-60 cell line that was completely hypermethylated, whereas the AWT1 CpG island promoter was methylated in all cell lines evaluated. The WT1-ARR region showed varying amounts of methylation (Figure 2C). In B- and T-cell derived leukemic cell lines the WT1 promoter was frequently hypermethylated as was the AWT1 promoter. In lymphoma cell lines, the entire locus was fully methylated (Additional file 1: Figure S1B).
The AWT1 methylation detected by bisulphite analysis is 5mC and not 5hmC
Recently 5-hydroxymethylation (5hmC), an oxidized derivative of 5-methylcytosine (5mC) has been suggested to regulate gene expression and is moderately enriched within CpG rich promoters and actively transcribed gene bodies . Unfortunately the deamination of unmethylated cytosines that occurs during conventional bisulfite reactions does not distinguish between 5mC and 5hmC, therefore the bisulphite resistant cytosines we observed in the AML cell lines may reflect enrichment of 5hmC and not 5mC. In order to differentiate these methylation states, we performed DNA immunoprecipitation utilizing antibodies that can discriminate 5hmC from 5mC in the myeloid cell lines, K562, KG1A and NB4. Using this technique we show that the methylation present at the AWT1 promoter is 5mC (Figure 2D). We confirm these observations using a second method utilizing a highly specific 5hmC glucosyltransferase that tags a glucose moiety to 5hmC but not to 5mC, rendering h5mC refractory to digestion with the Glucosyl-5hmC Sensitive Restriction Endonucleases (GSRE) enzyme (Msp I-CCGG) (data not shown).
AWT1 hypermethylation is associated with a switch from permissive to repressed chromatin state
The methylation profiles in cell lines reflect the epigenetic state in primary AML at diagnosis
Methylation analysis in other primary hematological malignancies
We extended our methylation analysis to other forms of hematological cancers. Similar to AML, chronic myeloid leukemia cases with BCR-ABL translocations had unmethylated WT1 promoters and AWT1 hypermethylation, however, polycythaemia vera and essential thrombocythaemia samples had methylation profiles indistinguishable from normal leukocyte controls. In samples classified with B-cell t(9:22) BCR/ABL, t(4:11) MLL/AFF1, t(12:21) TEL/AML1 and T-cell lymphoblastic leukemias or follicular and Burkitt lymphomas we identified very variable methylation at the promoters, which was often significantly different from normal controls but not reaching the defined 40% methylation cut-off (Figure 4A, C).
AWT1 hypermethylation discriminates AML patients who relapse from those achieving complete remission after SCT
Recent studies have illustrated the power of methylation profiling to identify important cancer-related genes associated with the pathogenesis and classification of hematological cancers. For example DBC1 hypermethylation is a prognostic marker for AML with normal karyotype . In addition large-scale epigenetic studies have demonstrated that aberrant methylation is a hallmark of specific leukemia sub-types, often associated with known translocations or genetic mutations. This is best highlighted by the AML hypermethylation signature associated with IDH1/IDH2 mutations, which disrupt TET2 function . In our current study we observe extreme hypermethylation of the AWT1 promoter, an epigenetic mark generally associated with robust gene silencing, in myeloid-derived hematological cancer cell lines and primary samples. These results are somewhat surprising as high expression levels of the various WT1 transcripts are frequently observed in AML [17, 18], a scenario that is normally mutually exclusive with abundant promoter methylation.
Loss-of-function mutations in TET2, an enzyme that converts 5mC to 5hmC have been observed in ~8% of AML cases . Interrogation of the mutation status of TET2 in the Human Cancer Genome database revealed that Jurkat was the only cell in our panel that had pathological changes (p.Q831H). This suggests that absence of demethylation by mutated TET2 is not associated with the observed AWT1 hypermethylation.
Previous studies in gliomas and breast tumors have also revealed, contrary to expectation, a high frequency of WT1 expressing samples with promoter (CpG island 188) hypermethylation [20, 21]. Interestingly one study assessed the affect of 5-aza-deoxycytidine on WT1 expression and observed that despite decreases in methylation there was not a concomitant change in WT1 expression . Interestingly exposing the KG1A cell line to Decitabine resulted in drastic (>50%) demethylation of the AWT1 promoter but only a modest doubling in expression levels (Additional file 4: Figure S2). Despite being a specific cancer marker, the tumor-specific methylation at this promoter interval appears to be inconsequential to gene transcription and that other mechanism regulate expression. This implies that when interpreting DNA methylation patterns both cellular context and the genomic position of the CpG sites must be taken into consideration.
Our results in cell lines imply that the WT1 and AWT1 isoforms are co-regulated, suggesting they share cis-acting regulatory elements. Recently an enhancer region located ~1 kb past the shared 3′UTR has been identified and shown to bind the myeloid-specific transcription factor GATA-2 , with abundant expression of GATA-2 and WT1 in AML being associated with bad prognosis . An expression analysis in the 26 of the hematological cancer cell lines and 53 primary AML samples revealed GATA-2 and WT1/AWT1 co-expressed in 96% cases (51/56) consistent with this mechanism being a regulator of expression at the locus (Additional file 5: Figure S3). Computational analysis of the ENCODE dataset reveals that the GATA-2 site is enriched for H3K27ac, a histone modification frequently associated with enhancer elements . This chromatin state may therefore be permissive to chromatin looping between this enhancer and the WT1 promoter region. Interrogation of the Chromatin Interaction Analysis Paired–End Tags (ChIA-PET) dataset in K562 cells suggests there is a strong physical association between these two genomic elements facilitated by POLII (Additional file 5: Figure S3).
Mutations in WT1 have been reported in 6-10% of AML cases, 20% of biphenotypic leukemias and sporadic T-cell acute lymphoblastic leukemias (T-ALL) [25–27]. This is a similar frequency to that found in sporadic Wilm’s tumors [28, 29]. In AML and T-ALL cases with heterozygous mutations, expression analyses have demonstrated biallelic expression of both the mutated and wild-type WT1 alleles consistent with our observations that the WT1 and AWT1 transcripts are not subject to genomic imprinting [27, 30]. Interestingly, direct sequencing of all exons of the WT1 gene in our panel of cell lines identified only one mutation in addition to known polymorphisms, an A > G nonsense mutation in exon 7 (p.R369*) of the non-expressing U937 cell line.
Despite the plethora of research on the role of WT1 in leukemia, it remains unclear if the abundant WT1 levels represent ectopic expression during carcinogenesis or simply reflects the arrested differentiation during early hematopoiesis, since most AML have an immature phenotype. However, the hypermethylation observed at this domain is associated with the tumorgenic changes since methylation at the WT1 and AWT1 CpG islands is never observed in any normal hematopoietic cell type or CD34+ stem cells.
The discovery of novel molecular markers that are highly specific and sensitive will improve strategies for both detecting and classifying tumor types, but also in the management of cancer by monitoring a tumors response to novel therapies. Here we show that using a cut-off of >40% methylation at the AWT1 promoter, the PPV for our assay in AML is 100%, indicating that the analysis can correctly detect and classifies a healthy individual. The sensitivity and NPV are 86.1% and 89.4% respectively, making this an ideal marker to correctly classify AML compared to controls. It must be noted, however, that when assessing all hematological malignancies together, hypermethylation of the AWT1 promoter it does not perform well as a diagnostic factor for unspecified leukemias/lymphomas (sensitivity 47%; specificity 100%; PPV 100%; NPV 43.6%) (Additional file 2: Table S3). Therefore quantitating AWT1 hypermethyaltion is ideally suited to monitor MRD in which the diagnosis of AML has already been performed, and the recurrence of the disease during remission needs to be accurately monitored as revealed by our analysis of patients undergoing allogeneic SCT.
In summary, differences in methylation patterns among tumors may be correlated with clinical features of patients and can serve as markers in cancer classification. Here we describe a methylation signature of the AWT1 promoter CpG island that is a promising marker for classifying myeloid-derived leukemias. When compared to the results of other well-characterized methylation markers in additional forms of cancer (i.e. MGMT in glioblastomas and GSTP1 in prostate cancers) [31, 32] the outcomes are extremely encouraging and further work on larger cohorts, maybe in combination with other leukemia-specific methylation markers, are required to confirm if our observations can be useful in a clinical setting. It remains to be determined what mechanisms are responsible for the correlated up-regulation of WT1 and AWT1 expression, with DNA methylation, micro-RNA, enhancer activation or even utilization of an unidentified enhancing long non-coding RNA all possibly having an influence .
Patient samples and cell lines
DNA was extracted from frozen peripheral blood and/or bone marrow samples from 356 patients at diagnosis and 102 control peripheral blood samples. For 9 individuals, we also had samples from multiple time points following SCT. Full clinical classification of the cohort can be found in Additional file 2: Table S1 and Additional file 3: Table S2. In addition, DNA and RNA were isolated from 28 derived from various hematological malignancies along with CD15+ (neutrophils and monocytes), CD19+ (B -cells) and CD3+ (T-lymphocyte) cell fractions. The cell lines were purchased directly from the ATCC or DSMZ cell line repositories and were grown in DMEM media with 10% heat inactivated fetal calf serum supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin. In addition all cell lines were routinely tested to ensure they were free of mycoplasm contamination. These studies were approved by the ethical committee of Bellvitge Institute for Biomedical Research/CEIC (PR159/08) and all patients gave informed consent.
To determine the epigenetic landscape of the WT1 region we analyzed publically available H3K4me3, H3K9me3 and H3K27me3 ChIP-seq datasets and whole genome bisulphite sequencing (WGBS) methylomes for CD34+ and three leukocytes. All data analyses were performed using in-house R-scripts. We subsequently targeted specific regions of interest using bisulphite PCR.
Approximately 50 ng of bisulphite converted DNA was used for each bisulphite PCR or pyrosequencing reaction (for primer sequences see Additional file 2: Table S4). To differentiate between 5′-hydroxymethylation (5hmC) and 5-methylcytosine (5mC) we utilized methylated DNA immunoprecipitations (meDIP) with a specific 5hmC or 5mC antibodies (Diagenode, Liege, Belgium) and the Quest 5hmC Glucosyltransferase Detection KitTM (Zymo).
For ChIP, 100 μg of chromatin was used for each immunoprecipitation reaction with Protein A Agarose/Salmon Sperm DNA and specific antibody raised against various post-translational histone H3 or H4 modifications (H3K4me2 Millipore 07–030; H3K4me3 Millipore 07–473; H3K9ac Cell Signalling 9671; H3K9me2 Millipore 07–441; H3K9me3 Abcam 8898; H4K20me3 Millipore 07–463; H3K27me3 Millipore 07–449). The levels of immunoprecipitated chromatin at specific regions were determined by qPCR using an Applied Biosystems 7900 Fast real-time PCR machine, using SYBR Green and IP levels were compared to Input and the results expressed as percentage of total input material.
The levels of various WT1 transcripts were assessed using qRT-PCR. All PCR amplifications were run in triplicate on a 7900 Fast real-time PCR machine (Applied Biosystems) following the manufacturers’ protocol. All primers were optimized using SYBR Green (Additional file 2: Table S4 for primer sequences) and melt curve analysis to ensure that amplicons were specific and free of primer-dimer products. Thermal cycle parameters included Taq polymerase activation at 95°C for 10 min for 1 cycle, repetitive denaturation at 95°C for 15 sec, and annealing at 60°C for 1 min for 40 cycles. All resulting triplicate cycle threshold (Ct) values had to be with 1 Ct of each other. The quantitative values for each triplicate were determined as a ratio with the level of β-Actin expression that was measured in the same sample, and then averaged to provide relative expression values. A cDNA mix of all cell lines and controls was used as an expression calibrator.
Western blot analysis
We extracted nuclear and cytoplasm protein fractions from cell lines for western blot using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific). SDS-PAGE gel electrophoresis and western blotting was performed as standard. To determine the cellular localization of proteins we incubated resulting membranes with primary antibodies against WT1 (Santa Cruz sc-192), β-Tubulin (Abcam ab6046) or Nucleolin (Santa Cruz sc-8031). Washed membranes were incubated with corresponding peroxidase-conjugated secondary antibody. The immunoreactive proteins were visualized using the ECL Western blotting detection kit (Amersham Biosciences).
Wilms tumour 1 gene
Acute myeloid leukemia
Minimal residual disease
Stem cell transplant
Reverse transcriptase polymerase chain reaction
Authors would like to thank Marta Kulis and Carles Arribas for help with pyrosequencing and we would like to thank all the patients and their families for participating in this project. This work was partially funded by European Community’s Seventh Framework Programme (FP7/2007-2013) under the HEALTH-F5-2011-282510 –BLUEPRINT (to M.E) and Spanish Ministerio de Educacion y Ciencia (BFU2011-27658 to D.M). D.M is a Ramon y Cajal research fellow and A.G.A was funded by a FPU studentship and was awarded an EMBO short-term fellowship to undertake part of this work.
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