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Originally published as JCO Early Release 10.1200/JCO.2005.02.196 on April 4 2005

Journal of Clinical Oncology, Vol 23, No 17 (June 10), 2005: pp. 3877-3885
© 2005 American Society of Clinical Oncology.
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TWIST2 Demonstrates Differential Methylation in Immunoglobulin Variable Heavy Chain Mutated and Unmutated Chronic Lymphocytic Leukemia

Aparna Raval, David M. Lucas, Jennifer J. Matkovic, Kristi L. Bennett, Sandya Liyanarachchi, Donn C. Young, Laura Rassenti, Thomas J. Kipps, Michael R. Grever, John C. Byrd, Christoph Plass

From the Division of Human Cancer Genetics, Division of Hematology-Oncology, Department of Medicine, and Department of Biostatistics, The Ohio State University, Columbus, OH; and Division of Hematology/Oncology, Department of Medicine, University of California, San Diego, CA

Address reprint requests to John C. Byrd, MD, Division of Hematology-Oncology, Starling Loving Hall, Room 302, The Ohio State University, Columbus, OH 43210; e-mail: byrd-3{at}medctr.osu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
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 (VH) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig VH 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 VH show TWIST2 methylation, while only 16% of patient samples with unmutated Ig VH 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 VH 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
CLL is one of the most common types of adult leukemia in the United States.1 The clonal B-cell of CLL has a low proliferation rate and disrupted apoptosis, leading to accumulation of cells and disruption of normal immune function.2 The clinical course of CLL is highly variable, and survival from the time of diagnosis of CLL can range from months to decades. Adverse features that associate with short time from diagnosis to development of symptomatic disease, reduced progression-free survival following therapy, and inferior survival include select chromosomal deletions such as del(17p13.1)3 and del(11q22.33),4 mutations of p53,5 and absence of somatic mutations in immunoglobulin variable heavy chain (Ig VH) genes.6-8 Chromosomal aberrations are less common at diagnosis, but develop disproportionately in VH unmutated CLL.5,9 VH mutational status has been shown to segregate patients with poor outcome, but testing for this parameter currently is not clinically available. Attempts have been made to develop surrogate markers, for example, ZAP-70 and CD38 expression. Although ZAP-70 expression appears to be a stronger predictor of the need for early therapy than does Ig VH mutation status, determination of ZAP-70 does not obviate the value of Ig VH mutation status in predicting clinical outcome.10 As such, identifying genetic features that explain the difference in biology in patients of different Ig VH mutational status is a high priority.

One such parameter that could explain these differences is the inappropriate epigenetic regulation of key transcriptional regulatory or antiapoptotic proteins. Various studies have characterized tumor suppressor genes that are silenced in cancer due to epigenetic modifications such as altered DNA methylation.11,12 Regions rich in CpG dinucleotides, so-called CpG islands, are commonly present in the 5' upstream region of genes. DNA methylation of CpG islands results in the condensation of the chromatin structure and downregulation of transcription.13 Aberrant DNA methylation has been detected in genes involved in cellular pathways including apoptosis, cell cycle, DNA repair, and adherence.14,15 Until recently, the relevance of epigenetic gene silencing in CLL has been uncertain. We recently have reported the global analysis of aberrant promoter DNA methylation in CLL utilizing a technique called restriction landmark genome scanning (RLGS).16,17 This preliminary study identified a large number of genes that are methylated in CLL in a non-random manner,18 indicating that DNA methylation plays an important role in the biology of CLL. To date, no studies in CLL have identified specific epigenetic events associated with Ig VH mutational status.

In the present report we study promoter methylation of TWIST2 (also called DERMO1), one of the genes reported in our previous global methylation analysis in CLL.18 TWIST2 is located on chromosome 2 and is a basic helix-loop-helix (bHLH) transcription factor.19 While TWIST2 function has not been characterized in lymphoid cells, expression of TWIST2 in other cell types has been shown to downregulate p14arf thus antagonizing p53-induced apoptosis.20 We demonstrate that TWIST2 promoter methylation results in gene silencing that can be relieved by treatment with the hypomethylating agent decitabine. Most interestingly, methylation of TWIST2 occurs significantly more frequently in the mutated Ig VH subtypes of CLL patients, while the majority of patients with unmutated Ig VH do not show TWIST2 promoter methylation. As we know that p53 dysfunction is a common phenomenon in patients with unmutated Ig VH, the expression of TWIST2 in Ig VH unmutated samples would explain an additional mechanism that results in p53 dysfunction in this subtype.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
Patient Selection and Sample Collection
Blood was obtained from patients with B-cell CLL through the CLL Research Consortium Tissue Bank, and from CLL patients and healthy volunteers at Ohio State University (Columbus, OH) after obtaining informed consent using an institutional review board–approved protocol. All patients examined in this series had immunophenotypically defined CLL as outlined by the modified 96 National Cancer Institute criteria.21 The CLL cell line WaC3CD5 (described previously)22 and RAJI cell line were incubated (37°C and 5% CO2) in RPMI 1640 supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from Invitrogen, Carlsbad, CA). Where indicated, WaC3CD5 cells were treated with decitabine, or 5-aza-2'-deoxycytidine (Sigma-Aldrich, St Louis, MO) at 0.01, 0.1, 0.5, 1.0, 2.0, or 5.0 µmol/L for up to 48 hours. RAJI cells were treated with 0.5 µM decitabine for 3, 6, 9, and 12 days.

Ig VH Gene Analysis
Analysis was performed by the CLL Research Consortium Tissue Bank as previously described.23 Somatic mutations were identified by comparison with the most homologous germ line Ig VH gene. Sequences with less than 98% homology to germ line were considered mutated.

Southern Blot Hybridization
Restriction enzymes were obtained from New England Biolabs (Beverly, MA). DNA was digested with EcoRV or with EcoRV followed by AscI. For each sample, 3.5 µg DNA was separated on a 0.8% agarose gel and transferred by vacuum to a nylon membrane (Zeta Probe, Biorad, Hercules, CA). Probes were prepared by purifying BsaI-digested restriction fragments from the AscI-EcoRV plasmid clone A2G15. Fragments were labeled with [{alpha}-32P]dCTP using the Prime-It II kit (Stratagene, La Jolla, CA), hybridized overnight, and exposed for 72 hours on a Storm Phosphorimager (Amersham Biosciences, Piscataway, NJ). ImageQuant software (Amersham Biosciences) was used for quantifying intensities of the fragments.

Bisulfite Genomic Sequencing and Combined Bisulfite Restriction Analysis
One microgram of genomic DNA from CLL samples and normal controls were treated with sodium bisulfite according to published protocols24 with modifications. Primers were designed to allow amplification of both methylated as well as unmethylated DNA. The TWIST2 forward primer was 5'-AAGGGGGAGGTAAAATTGAAA-3' and the reverse primer was 5'-CTAAACTAAATTACTAAATAATTATC-3'. The polymerase chain reaction (PCR) was carried out in a 50 µL reaction using 1-µl bisulfite-treated DNA, 60 picomole of each primer, 1.25 mmol/L of each dNTP, 2.5 units of Platinum Taq polymerase (Invitrogen), and 5 µL 10x PCR buffer. Reaction conditions had an initial denaturation step for 10 minutes at 95°C, 35 cycles of 96°C x 30 seconds, 52°C x 30 seconds, 72°C x 30 seconds, and a final extension step at 72°C x 10 minutes. For bisulfite sequencing, the PCR product was purified from a 1.5% agarose gel using the Qiagen Gel Extraction kit (Qiagen, Valencia, CA) according to manufacturer's protocol and subcloned using the TOPO TA-Cloning kit (Invitrogen). Five to 10 clones for each sample were sequenced. For combined bisulfite restriction analysis (COBRA), the bisulfite-treated DNA was PCR-amplified. The PCR product was digested by BstU I restriction enzyme and separated on an 8% polyacrylamide gel.

Quantitative COBRA
For 100% methylated control, DNA from normal blood cells was treated with SssI in presence of S-adenosyl methionine (New England Biolabs) and purified using the Qiagen Gel Extraction kit. This methylated DNA was mixed with untreated DNA from normal blood cells at different ratios to obtain 100%, 50%, 25%, 10%, 5%, 1%, and 0% methylated DNA standards. One microgram of each standard DNA was treated with bisulfite as explained above. COBRA was performed on patient samples and standard DNA. Digested fragments were visualized on a 2.0% agarose gel and transferred by vacuum to a nylon membrane (Zeta Probes). The reverse primer used for PCR was end-labeled in the presence of [{gamma}-32P] ATP by T4 polynucleotide kinase (Gibco, Carlsbad, CA) according to the manufacturer's instructions, hybridized overnight, and exposed for 72 hours on a Storm Phosphorimager (Ambersham Biosciences). The ratio between the intensities of undigested DNA and total DNA was plotted against the percentage of in vitro methylated DNA to create a standard curve, and percent methylation in Ig VH mutated and unmutated patient samples was interpolated from these values.

Reverse Transcription PCR
Total RNA from patient samples, as well as untreated and decitabine-treated WaC3CD5 cells, were isolated using TRIzol (Invitrogen) and further purified by RNeasy Mini Kit (Qiagen). Three micrograms of total RNA was used for reverse transcription using SUPERSCRIPT First-Strand Synthesis kit (Invitrogen). The primers used were: TWIST2 forward primer 5'-GCCGCCAGGTACATAGACTT -3', reverse primer 5'-CCCCAAACATAAGACCCAGA-3'; GPI forward primer 5'-GACCCCCAGTTCCAGAAGCTG-3', reverse primer 5'-GCATCACGTCCTCCGTCACC-3'; GAPDH forward primer 5'-CCCCTTCATTGACCTCAACTACAT-3', reverse primer 5'-CGCTCCTGGAAGATGGTGA-3'. Each PCR was performed in 50-µL final volume containing 1-µL cDNA from above, 1x PCR buffer, 1.5 mM MgCl2, 10 pmol of each primer, 200 µM of each dNTP, 5% DMSO and 2.5 U Platinum Taq DNA polymerase (Invitrogen). Reaction conditions had an initial denaturation step of 10 minutes at 95°C, 35 cycles of 96°C x 30 seconds, 58°C (TWIST2) x 30 seconds or 60°C x 30 seconds (GPI and GAPDH), 72°C x 30 seconds, and a final extension step at 72°C x 10 minutes.

Statistics
Standard curves to calculate percent DNA methylation were fit using exponential curves with all r-square values in excess of 0.95. Comparisons of patient groups based on percent methylation were performed using the Wilcoxon rank-sum test. P values ≤ .05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
TWIST2 Methylation by Southern Blot
In a previous study, we used RLGS on 10 CLL patient samples and showed that TWIST2 was methylated in two of 10 CLL patient samples.18 To further confirm that TWIST2 was aberrantly methylated in these samples at the restriction landmark site AscI, we performed Southern blotting using genomic DNA from 10 CLL samples, CD19+ selected normal control B-cells, and normal peripheral blood, digested with EcoRV alone or in combination with AscI. Figure 1A shows the TWIST2 gene structure and location of the AscI-EcoRV fragment (4.3 kb) detected by RLGS. The probe was designed to detect a 4.3 kb, AscI-EcoRV fragment if the AscI site is unmethylated and a 6.5 kb EcoRV-EcoRV fragment if the AscI site is methylated. DNA from normal peripheral-blood cells and CD19+ B cells showed only a 4.3 kb fragment, indicating that the AscI site is not methylated in these cells (Fig 1B). The presence of both 4.3 and 6.5 kb fragments in patients 3 and 5 confirmed the RLGS data that indicated partial methylation of TWIST2 in these samples. In addition, low-level methylation was observed in patients 7, 8, 9, and 10. Overall, this result is consistent with our earlier report that the AscI site within TWIST2 is methylated in CLL.



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  		Fig 1. TWIST2 gene structure and DNA methylation. (A) Schematic shows location of EcoRV-AscI fragment, probe used for Southern blotting and bisulfite polymerase chain reaction (BS-PCR) primers; (B) Southern Blot shows percentage methylation in each sample; (C) bisulfite sequencing of patient samples. Each row represents a clone. The open circles indicate unmethylated CpG, and closed circles indicate methylated CpG. Pt, patient.

 
Bisulfite Genomic Sequencing of TWIST2
Numerous reports have demonstrated the involvement of promoter methylation in the regulation of gene expression in cancer. Thus, we next examined methylation within the 5' upstream regulatory region of TWIST2 with bisulfite genomic sequencing in CLL patient samples. DNA from normal blood and B cells and from patient samples 3, 4, and 5 were treated with sodium bisulfite. This treatment causes conversion of unmethylated cytosines to uracil, while methylated cytosines remain unchanged. PCR amplification of these treated DNAs converts uracil to thymine, and subsequent sequencing of the PCR products then allows determination of the methylation status of the original DNA at each CpG site. Figure 1A shows both the location of the CpG island within the TWIST2 gene and the location of the primer pairs (bisulfite-PCR) designed to amplify the -193bp to -52bp region of the promoter. The resulting PCR product was cloned, and eight to 10 clones of each sample were sequenced. Figure 1C shows the methylation status of 13 CpG sites present between -193bp to -52bp. Patient samples 3 and 5 showed a high level of methylation in CpG sites 5 to 13, while normal blood cells and CD19+ B cells showed no or very low methylation in that region. As previously reported by RLGS and as shown in Figure 1A, in patient No. 4 the TWIST2 promoter was not methylated. These results indicate that TWIST2 is methylated in the 5' upstream regulatory region in a subset of CLL samples but not in normal B cells.

TWIST2 Methylation Regulates Its Expression
It is possible that the methylation observed could be restricted to T cells or granulocytes in the patient samples. To further demonstrate that TWIST2 is methylated in CLL cells, we studied promoter methylation in seven CLL samples that had undergone positive selection using magnetic beads conjugated to anti-CD19 (MACS, Miltenyi Biotec, Auburn, CA). The resulting cells are at least 95% CD19+ (data not shown). Methylation of DNA from these samples was then analyzed by COBRA assay. The TWIST2 PCR product is 141 bp long and has three methylation-sensitive BstUI sites. If the original DNA is methylated, BstUI digestion produces multiple smaller fragments, visualized by agarose gel electrophoresis. We observed that four of seven CD19+ selected CLL samples had a high level of methylation (patient samples 2, 3, 4, and 7), while three samples showed reduced levels (Fig 2A). Normal B cells and the CLL cell line WaC3CD5 were used as negative and positive controls, respectively. These results establish the fact that the 5' upstream regulatory region of TWIST2 is methylated in a subset of CLL samples.



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  		Fig 2. TWIST2 methylation and expression in selected chronic lymphocytic leukemia (CLL) samples. (A) BstUI restriction digestion of selected CLL cells results in undigested and digested fragments that indicate unmethylated and methylated DNA, respectively. The normal B cells and WaC3CD5 were used as negative and positive controls, respectively. (B) Reverse transcription polymerase chain reaction was performed for TWIST2 and GAPDH.

 
We next examined these same samples to determine whether TWIST2 promoter methylation regulates its expression. Reverse transcription (RT) -PCR data in Figure 2B shows that the four CLL patient samples (2, 3, 4, and 7) and WaC3CD5 cells in which promoter methylation was detected by COBRA did not express TWIST2. However, normal B cells and patient sample 1 expressed TWIST2 mRNA. Interestingly, of three samples that showed a very low level of methylation (samples 1, 5, and 6), two did not express the TWIST2 transcript. This indicates that although promoter methylation could be one of the mechanisms regulating TWIST2 expression in CLL, there are additional regulatory pathways involved in TWIST2 silencing. Taken together, our data indicates that DNA methylation from -193bp to -52bp regulates TWIST2 gene expression in a subset of CLL patients, and suggests that TWIST2 could play an important role in the biology of CLL.

Re-Expression of TWIST2 by Decitabine Treatment
As shown in Figure 2, the TWIST2 promoter is methylated in WaC3CD5 and these cells do not express TWIST2 transcript. Because our data suggests TWIST2 is at least partially regulated by promoter methylation, we hypothesized that an agent that reverses methylation could lead to re-expression of this gene. We therefore incubated WaC3CD5 cells with decitabine, which inhibits DNA methyltransferase 1 and 3b. We have previously shown depletion of DNMT1 enzyme in WaC3CD5 cell line with increasing doses of decitabine.18 As expected, there was no TWIST2 expression in untreated WaC3CD5 cells at baseline by RT-PCR. However, TWIST2 expression was induced with increasing concentrations of decitabine after 48 hours, while the expression of internal control glucose-6-phosphate isomerase was constant in all the samples (Fig 3A). Similarly, treatment of RAJI cell with decitabine resulted in induction of TWIST2 expression (Fig 3B). Bisulfite sequencing of untreated and 6 days of decitabine treatment was performed to study the effect of the drug on promoter methylation (Fig 3C). The TWIST2 promoter was found to be highly methylated in RAJI cells (97%) but treated cells showed a marked reduction in promoter methylation in three of five clone sequences (53%; P < .001). These results indicate that TWIST2 expression is silenced in these cell lines due to promoter methylation.



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  		Fig 3. Re-expression of TWIST2 after decitabine treatment. WaC3CD5 cells (A) and RAJI cells (B) were treated with decitabine as indicated. Reverse transcription polymerase chain reaction was performed for TWIST2, GPI, and GAPDH. Normal lung or B cells were used as positive controls. (C) Bisulfite sequencing was performed for untreated and 6-day decitabine treated (0.5 µM) RAJI cells.

 
TWIST2 Methylation Strongly Correlates With VH Mutational Status
Given the established role of TWIST2 in silencing p53 function in nonlymphoid cell lines20 and the high preponderance of p53 dysfunction in patients with VH unmutated CLL,20 we hypothesized that epigenetic regulation of this gene may vary based upon VH mutational status.5,9 We therefore performed COBRA analysis on 53 CLL patients with known VH mutational status. Out of 53 samples, 31 samples expressed unmutated Ig VH and 22 samples use mutated Ig VH genes. Interestingly, we found that TWIST2 was predominantly methylated in samples with mutated IgVH genes, a marker for favorable prognosis in CLL. We observed that 16 (72%) of 22 samples with mutated Ig VH genes showed TWIST2 methylation, while only five (16%) of 31 cases with unmutated Ig VH genes were methylated (P < .001). A representative COBRA assay for each subset is shown in Figure 4.



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  		Fig 4. TWIST2 methylation by combined bisulfite restriction analysis assay in two different chronic lymphocytic leukemia subsets: The undigested band (141 bp) represents unmethylated DNA while digested fragments indicate methylated DNA. (A) TWIST2 methylation in immunoglobulin variable heavy chain (Ig VH) unmutated samples and (B) methylation in Ig VH mutated samples.

 
Quantitation of COBRA Assay and Clinical Correlation
Given the association of TWIST2 methylation with CLL cells that use mutated Ig VH genes, we next sought to develop a quantitative assay to measure percentage methylation in patient samples. Development of such an assay would allow serial assessment of TWIST2 expression during disease progression or treatment with a methylation-targeted therapy (ie, decitabine). Such a technique would also be valuable for following other genes that are differentially methylated in CLL as part of future clinical trials. For this, we combined two techniques, COBRA and Southern blotting. The standard DNA and 10 CLL samples were bisulfite-treated followed by COBRA and southern blotting (Fig 5A). As explained in Materials and Methods, the standard curve was used to calculate the percentage methylation in the 10 CLL patient samples (Fig 5A). Using this method, we next quantified DNA methylation at the BstUI sites in all 53 CLL samples. For this, each Southern blot was prepared with both patient samples and the in vitro methylated DNA standards. Thus, a standard curve was plotted for each blot and percentage methylation was calculated. TWIST2 promoter methylation in samples with unmutated versus mutated Ig VH genes was compared using the Wilcoxon rank sum test (Fig 5B). The difference in methylation between the two sets of samples was found to be statistically significant (P < .001). This finding was also supported by bisulfite sequencing data on two samples from each subset. Samples with mutated Ig VH genes had significant methylation between -193bp to -52 bp of the TWIST2 promoter, relative to samples with unmutated genes (Fig 5C). Normal CD19+ B cells and WaC3CD5 cells were used as negative and positive controls. This data demonstrates that TWIST2 is differentially methylated in CLL cells that use mutated Ig VH genes. In addition, we have established a reproducible assay to quantitatively measure changes in gene methylation in primary tumor samples.



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  		Fig 5. Quantitative methylation analysis. (A) Standard and chronic lymphocytic leukemia cell DNA was used for combined bisulfite restriction analysis and Southern blotting; (B) percentage methylation, between immunoglobulin variable heavy chain (Ig VH) unmutated and mutated samples, was compared by Wilcoxon rank-sum test; (C) bisulfite sequencing of normal B cells, WaC3CD5 cells, and two each of Ig VH mutated and unmutated patient samples.

 
TWIST2 Expression in CLL Samples Using Mutated Versus Unmutated IgVH Genes
In Figure 2, we showed that TWIST2 promoter methylation correlates with its expression in CD19+ selected CLL samples. If TWIST2 is more commonly methylated in samples with mutated Ig VH genes, it should be preferentially expressed in the samples with unmutated Ig VH genes. We examined TWIST2 expression in four samples with mutated Ig VH and five samples with unmutated Ig VH by RT-PCR (Fig 6). We observed that TWIST2 was silenced preferentially in the subtype with mutated Ig VH, while normal CD19+ B cells and four of five samples with unmutated Ig VH expressed the TWIST2 transcript. Lack of expression in patient No. 43 could be a result of either deletion or mutation of the gene in that patient, although there is not yet sufficient data to establish this.



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  		Fig 6. TWIST2 expression by reverse transcription polymerase chain reaction in normal B cells (lanes 3 and 4), immunoglobulin variable heavy chain (Ig VH) mutated (lanes 5 to 8) and Ig VH unmutated samples (lanes 9 to 13) are shown. Water and normal lung cDNA was used as negative and positive controls, respectively. GAPDH was used as the internal control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Authors' Disclosures of...
 REFERENCES
 
We have described, for the first time, a gene, TWIST2, in which methylation is highly associated with Ig VH mutational status in CLL samples. Identification of this gene was derived from a previous study utilizing RLGS, a technique developed to identify global genomic methylation. Following identification of this gene, we confirmed, using bisulfite sequencing, that spot loss on RLGS was not a consequence of deletion but rather promoter methylation. We demonstrated that TWIST2 is not methylated in normal B cells, and that its methylation associates with Ig VH gene mutational status in CLL patient samples. As the COBRA assay alone does not permit quantification of DNA methylation, we developed a combined Southern Blot-COBRA assay. This assay confirmed the significant overall difference of TWIST2 methylation in CLL cells that express mutated versus unmutated Ig VH. In addition, this new assay provides a method to assess the degree of specific gene methylation in serial tumor samples during disease progression or with treatment utilizing a DNA hypomethylating agent such as decitabine.

While we have demonstrated differential methylation of TWIST2 in CLL cases with mutated Ig VH genes, the consequences of silencing this gene are uncertain. Given the unfavorable prognosis associated with VH unmutated status, we hypothesized that silencing of TWIST2 would diminish the biologic aggressiveness of this subtype of CLL. Indeed, several recent reports have demonstrated high-risk genetic features that can contribute to inactivating the p53 pathway through deletion or mutation of p53 and the related ATM gene also associate with poor prognosis.3,9,25-28 There are several studies characterizing the function of TWIST2, but its role in either normal B cells or in CLL cells is unknown. TWIST2 has significant homology and functional overlap with another bHLH factor, TWIST1, and both transcription factors inhibit differentiation of osteoblasts.29,30 TWIST2 also inhibits MyoD-mediated transactivation by acting as a co-repressor with histone deacetylase-1,19 and its overexpression has been implicated in disrupting p53 function.20 Transfection of mouse embryonal fibroblasts with either TWIST1 or TWIST2 causes inactivation of p53 function, in part via interference with p14ARF/MDM2 interaction.20 Overexpression of these genes resulted in abrogation of p21 or Bax induction following treatment with adriamycin,20 similar to that observed in primary CLL cells following ultraviolet irradiation when p53 or ATM mutations are present.8 Interestingly, mouse embryonal fibroblast cells transfected with TWIST1 or TWIST2 had diminished serum starvation- and drug-induced apoptosis relative to the vector control. Given the paucity of available data on TWIST2 function in B cells, our efforts are currently directed toward actively characterizing the role of TWIST2 in disrupting p53 function and apoptosis, both in CLL and related diseases.

In conclusion, we have described, for the first time, dichotomous methylation of specific gene (TWIST2) in different Ig VH mutational groups in patients with CLL. Using transformed B cells, we have demonstrated that regulation of expression of this bHLH transcription factor occurs in part through promoter methylation. Further studies are needed to investigate the imporantce of TWIST2 in normal B-cell development and to validate the association of this gene's methylation with VH mutational status and potentially ZAP-70 expression in a larger cohort of CLL patients. If these results are confirmed, the presence or absence of TWIST2 methylation might serve as an alternative mechanism for p53 dysfunction that mediates resistance to standard therapies utilized in CLL. In addition, as epigenetically targeted therapy with hypomethylating agents such as decitabine and 5-azacytidine is moving successfully forward in all types of leukemia including CLL, we have developed a semiquantitative method to serially follow promoter region methylation changes in primary tumor cells. Understanding the function of TWIST2 and other genes silenced selectively in subsets of CLL will be important in predicting which subset of patients might benefit from epigenetically targeted therapy.


   Authors' Disclosures of Potential Conflicts of Interest
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
REFERENCES
 
The authors indicated no potential conflicts of interest.


   Acknowledgment
 
We thank Yue-Zhong Wu, Linda Liu, Andrew Mone, and Elizabeth Angerman for technical assistance and critical reading of the manuscript. We also acknowledge the CLL Research Consortium for patient samples.


   NOTES
 
Supported in part by National Cancer Institute grants CA81534 to the CLL Research Consortium (L.R., T.K., M.R.G., J.C.B., D.L.), CA110496 (J.C.B., K.P., A.R., D.L.) CA93548 (C.P.), P30 CA16058 (C.P., L.J.R., T.J.K., J.C.B.), The Leukemia and Lymphoma Society of America (J.C.B., K.P.), The D. Warren Brown Foundation (J.C.B.). C.P. is a Leukemia and Lymphoma Society Scholar and J.C.B. is a Leukemia and Lymphoma Society Clinical Scholar. A.R is a Leukemia Society Fellow.

C.P. and J.C.B contributed equally to this article.

Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org.

Authors' disclosures of potential conflicts of interest are found at the end of this article.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Authors' Disclosures of...
 REFERENCES
 
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Submitted October 8, 2004; accepted January 28, 2005.




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