Pediatr Blood Cancer 2011;56:349–352

The Dual Role of HLXB9 in Leukemia

Stuart Ferguson, PhD, Hannah E. Gautrey, MS, and Gordon Strathdee, PhD

Background. The HLXB9 gene encodes a homeodomain con-taining transcription factor which has been implicated in thedevelopment of both solid and hematological malignancies. Inleukemia it is one of the two fused genes, along with ETV6, in a recur-rent translocation frequently observed in infant AML. Procedure.Here we investigate the role of epigenetic inactivation of the HLXB9gene in leukemia. Quantitative DNA methylation analysis was per-formed using the COBRA assay, and qRT-PCR was used to assess theeffects of methylation on expression in hematological cell lines andprimary ALL samples. Results. Hypermethylation of the HLXB9 gene

was found to be a frequent event in childhood ALL, occurring in 33%of cases. However, it was rarely or never observed in other types ofleukemia, including AML, CML, and CLL, with the exception of adultALL, in which 39% of cases were hypermethylated. Furthermore,hypermethylation of HLXB9 results in loss of expression in hemato-logical cell lines and primary ALL samples. Conclusion. These resultssuggest that HLXB9 may have a dual role in childhood leukemia, asan oncogene in infant AML but as a tumor suppressor in childhoodALL. Pediatr Blood Cancer 2011;56:349–352. © 2010Wiley-Liss, Inc.

Key words: acute lymphocytic leukemia; DNA methylation; epigenetics; HLXB9; homeobox

INTRODUCTION

HLXB9, also known as MNX1, is a homeodomain containingtranscription factor which maps to the distal end of the long armof chromosome 7. Although little is known of its normal function,it has been found to be involved in several important pathologies.Firstly, mutation or deletion of HLXB9 was found to be the primarycause of Currarino syndrome, a rare genetic syndrome leading tosacral agenesis and the presence of a pre-sacral mass or teratoma[1,2]. Shortly afterwards HLXB9 was also reported to be involved ina recurrent translocation, specifically found in infant acute myeloidleukemia (AML) cases in which the HLXB9 gene is frequentlyfused to the ETV6 gene on chromosome 12 [3]. Interestingly, theHLXB9 mutations that were associated with the development ofteratomas as part of the Currarino syndrome appear to be eitherdeletions or loss of function mutations, suggesting a potential tumorsuppressor role [2,4]. In agreement with this, loss of HLXB9 hasalso been associated with progression in breast cancer and coloncancer [5,6]. In contrast, in infant AML, the translocations involvingHLXB9 have been shown to be associated with ectopic expressionof WT HLXB9, suggesting an oncogenic role [7]. Similarly, a raret (6;7) translocation found in an adult AML patient was found tolead to over-expression of HLXB9 [8]. Thus, both inactivation andover-expression of HLXB9 may play a role in tumor development.

Altered DNA methylation is a key feature in the development ofall types of cancer. In particular, hypermethylation of promoter-associated CpG islands is a frequent mechanism leading to theinactivation of genes during tumor development [9]. For example,the clustered HOX genes, which like HLXB9 are homeodomain-containing transcription factors, are known to be frequent targetsfor methylation-based inactivation, both in the development of solidtumors [10–14] and also leukemia [15–17]. Thus, to determine thepotential role of DNA methylation of the HLXB9 gene in con-trolling its expression in leukemia we determined the methylationstatus of its promoter region in samples from all common types ofchildhood and adult leukemia. This analysis determined that whilemethylation of HLXB9 was rarely or never seen in most types ofleukemia, including childhood AML where it has an apparent onco-genic role, hypermethylation of HLXB9 was commonly seen inchildhood acute lymphoid leukemia (ALL), as well as in adult ALL.Expression analysis determined that in both cell lines and in primary

ALL samples hypermethylation of the gene was associated withloss of expression. These results suggest that the role of HLXB9 inchildhood leukemia may be much more widespread than previouslythought and in addition to its oncogenic role in infant AML, it mayalso play a tumor suppressor role in ALL.

METHODS

Cell Culture

All cell lines were maintained in RPMI with 2 mM glutamineand 10% fetal calf serum in 95% air/5% CO2 at 37◦C.

Patient Samples

DNA was isolated from peripheral blood or bone marrow sam-ples obtained from patients with clinically diagnosed leukemia fromNewcastle Royal Victoria Infirmary, Royal Manchester Children’sHospital, Glasgow Royal Infirmary, and Royal Bournemouth Hos-pital. Peripheral blood samples were also obtained from healthyvolunteers. Ethical approval for all samples collected had beenobtained.

Methylation Analysis of the HLXB9 Promoter Region

COBRA analysis was performed largely as described before[18]. Two hundred nanograms of genomic DNA was modified withsodium bisulfite using the MethylampTM One-Step DNA Modifi-cation Kit (Epigentek, Brooklyn, NY) as per the manufacturer’sinstructions. All samples were resuspended in 15 �l of TE and

Crucible Laboratory, Institute for Ageing and Health, NewcastleUniversity, Newcastle upon Tyne, UK

Grant sponsor: Tyneside Leukaemia Research Association; Grantsponsor: Biotechnology and Biological Sciences Research Council.

Conflict of interest: Nothing to declare.

Stuart Ferguson and Hannah E. Gautrey contributed equally to this work.

*Correspondence to: Gordon Strathdee, Crucible Laboratory, Institutefor Ageing and Health, Newcastle University, Newcastle upon TyneNE1 3BZ, UK. E-mail: g.r.strathdee@newcastle.ac.uk

Received 12 January 2010; Accepted 6 May 2010

© 2010 Wiley-Liss, Inc.DOI 10.1002/pbc.22679Published online 10 November 2010 in Wiley Online Library(wileyonlinelibrary.com).

350 Ferguson et al.

1 �l of this was used for subsequent PCR reactions. The sam-ples were amplified in 25 �l volumes containing 1× manufacturer’sbuffer, 1 U of FastStart taq polymerase (Roche, Lewes, UK), 2.5 mMMgCl2, 10 mM dNTPs, and 75 ng of each primer. PCR was per-formed with 1 cycle of 95◦C for 6 min, 35 cycles of 95◦C for30 sec, 61◦C for 30 sec, and 72◦C for 30 sec, followed by 1 cycleof 72◦C for 5 min. Following amplification, the PCR products weredigested with the appropriate restriction enzymes (BsiEI, BstUI,HhaI; New England Biolabs, Hitchin, UK), specific for the methy-lated sequence after sodium bisulfite modification. Digested PCRproducts were separated on a 2–3% agarose gels and visualized byethidium bromide staining. In vitro methylated (IVM) DNA (Mil-lipore, Watford, UK) was diluted into DNA extracted from normalperipheral blood to produce standards (100%, 66%, 33%, and 0%) ofknown methylation status for all COBRA assays. Primers used wereF 5′-gggtagaggttttaggttgagtt, reverse 5′-attctttacrctacaaccctcaaac.

HLXB9 Expression Analysis

cDNA synthesis was carried using the SuperScriptTM III FirstStrand Synthesis System (Invitrogen, Paisley, UK) as per manu-facturer’s protocol. Quantitative RT-PCR analysis was performedin 10 �l volumes containing 1× master mix (SYBR® GreenJumpStartTM Taq ReadyMix kit; Sigma, Dorset, UK), 37.5 ng ofeach primer, and 0.5 �l cDNA. PCR was performed with 1 cycleof 94◦C for 15 min, followed by cycles of 94◦C for 30 sec, 55(�2-microglobulin) or 60◦C (HLXB9) for 30 sec, and 72◦C for30 sec, with plate reads carried out at 80◦C (�2-microglobulin)or 87◦C (HLXB9) at the end of each cycle. Each of the PCRassays was run in triplicate. �2-microglobulin was used as a controlfor normalizing relative expression levels in the different sam-ples. Reactions were carried out on a TaqMan 7900HT (AppliedBiosystems, Warrington, UK). Primer sequences were forwardprimer 5′-aagatgcccgacttcaactc and reverse 5′-gacaggtacttgttgagcttgfor HLXB9 and forward primer 5′-gcattcagacttgtctttcagc and reverse5′-atgcggcatcttcaaacctc for �2-microglobulin.

RESULTS

The HLXB9 Promoter Is Frequently Hypermethylatedin ALL

Hypermethylation of the HLXB9 gene was assessed in childhoodALL samples using the COBRA assay, which enables quantita-tive DNA methylation analysis. Digestion with multiple restrictionenzymes allowed methylation to be assessed at multiple CpG sitesacross the proximal promoter region, from −233 to +67, relative tothe transcriptional start site. Samples were regarded as hypermethy-lated when 50% or more of the sites analyzed exhibited methylationlevels of 50% or greater. This analysis identified frequent hyper-methylation of the HLXB9 gene in childhood ALL samples (33%,15/46 samples, examples in Fig. 1A).

Translocations involving HLXB9 in AML seem to be almostexclusively limited to infant cases. However, no correlation wasseen between patient age and HLXB9 methylation in childhoodALL suggesting that, unlike the AML-associated translocation ofHLXB9, hypermethylation of HLXB9 is a frequent event in ALLregardless of age of onset.

Fig. 1. Methylation of the HLXB9 promoter is common in ALL.Examples of COBRA analysis of the HLXB9 gene in (A) childhoodALL samples and (B) adult ALL samples. The positions of bands rep-resenting methylated or unmethylated DNA are indicated by arrows.As indicated below the gels, samples were defined as methylated (+) ifmethylation levels of 50% or greater were observed. Samples with lessor no methylation were defined as unmethylated (−). 100%, 66%, and33% represent controls with 100%, 66%, and 33% IVM DNA mixedwith PBL DNA. IVM, in vitro methylated DNA; PBL, peripheral bloodleukocyte.

HLXB9 Is Not Frequently Hypermethylated in OtherTypes of Leukemia

To determine if HLXB9 methylation was a childhood ALL-specific phenomenon or whether it was seen in across all typesof leukemia, the methylation status of the gene was also studiedin CLL, CML, AML (childhood and adult), and adult ALL. Asshown in Figure 2 in contrast to the frequent methylation seen inALL samples the HLXB9 gene was never (adult AML, CML, andCLL) or rarely (childhood AML) methylated in most other types ofleukemia. In contrast to this though, hypermethylation was a com-mon event in adult ALL, with a frequency very similar to that seen inchildhood ALL (39%, 28/72 samples, examples in Fig. 1B). Thus,hypermethylation of HLXB9 is essentially restricted to ALL.

Hypermethylation of the HLXB9 Promoter IsAssociated With Loss of Gene Expression

The role of HLXB9 in normal hematopoietic cells is notknown; however, other than those cases exhibiting an HLXB9-associated translocation, expression of HLXB9 has been reportedto be relatively rare in AML. Thus, to assess whether the observedhypermethylation of HLXB9 is likely to be physiologically relevantwe assessed the correlation between expression and methylation inhematopoietic cell lines and in primary ALL samples. Methylationanalysis (as described above) and quantitative RT-PCR analysis wascarried out on a panel of 14 leukemia/lymphoma cell lines, includ-ing myeloid, B cell, and T-cell-derived lines. Hypermethylation ofHLXB9 was found to be common in cell lines, with 7/14 cell linesexhibiting hypermethylation. This was invariably associated withloss of gene expression, with 7/7 unmethylated cell lines expressingHLXB9 compared with 0/7 methylated lines (P = 0.001, Mann–Whitney U-test; Fig. 3A). Interestingly, expression was observedin myeloid, B-cell, and T-cell-derived cell lines, indicating thatHLXB9 is likely to be expressed in multiple hematopoietic lineages.

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HLXB9 in Human Leukemia 351

Fig. 2. Methylation of HLXB9 is very rarely seen in other typesof leukemia. Examples of COBRA analysis in (A) chronic lympho-cytic leukemia (CLL), (B) chromic myeloid leukemia (CML), (C) adultAML, and (D) childhood AML, as indicated. The positions of bandsrepresenting methylated or unmethylated DNA are indicated by arrows.Hypermethylation of HLXB9 (defined as methylation levels of 50% orgreater) was not observed in CLL, CML, or adult AML (n = 20, 20, and33, respectively) and only rarely seen in childhood AML (e.g., samplesix in D is hypermethylated, indicated by *, overall 13% (3/23) sampleswere hypermethylated). 100%, 66%, and 33% represent controls with100%, 66%, and 33% IVM DNA mixed with PBL DNA. IVM, in vitromethylated DNA; PBL, peripheral blood leukocyte.

To confirm that methylation also correlated with expression in pri-mary samples a further 19 adult ALL samples, where sufficient RNAwas available, were also assessed for HLXB9 expression. This anal-ysis again confirmed the correlation between loss of expression andmethylation (Fig. 3B, P = 0.022, Mann–Whitney U-test). The lackof a perfect correlation as seen in the cell lines likely reflects theincreased heterogeneity of primary samples.

DISCUSSION

Several reports had previously suggested a potential role forHLXB9 in cancer, both in solid tumors and in infant AML. Loss offunction mutations are associated with the development of teratomasas part of the Currarino syndrome [2,4] and loss of expression hasbeen shown during progression of breast and colon cancer [5,6],whereas in infant AML it has been implicated as an oncogenedue to its frequent fusion to ETV6 and the ectopic expression ofWT HLXB9 in the HLXB9–ETV6 fusion cases [3,7]. Here thoughwe identify it as a frequent target for DNA methylation in child-hood ALL, suggesting that HLXB9 may have multiple roles in thedevelopment of childhood leukemia.

In contrast to the frequent hypermethylation of HLXB9 seenin childhood ALL it was never or rarely found to be hypermethy-lated in other types of leukemia (including childhood and adult

Fig. 3. Hypermethylation of the HLXB9 promoter leads to loss ofexpression. A: A panel of 14 cell lines was analyzed for HLXB9expression, using qRT-PCR (upper panel, normalized to expression of�2-microglobulin) and methylation by COBRA analysis (lower panel).This analysis identified loss of expression in all methylated cell lines,while expression of HLXB9 was retained in all unmethylated cell lines.B: Nineteen primary adult ALL samples were analyzed for HLXB9expression, using qRT-PCR (upper panel, normalized to expression of�2-microglobulin) and methylation by COBRA analysis (lower panel).This analysis identified a significant correlation between loss of HLXB9expression and methylation in the primary samples.

AML, CML, and CLL) with the exception of adult ALL wherehypermethylation frequency was very similar to childhood ALL.Thus, hypermethylation of the HLXB9 is largely an ALL-specificphenomenon.

The role of HLXB9 in normal hematopoietic cells is stillunknown. However, it has previously been implicated in normalhematopoietic differentiation and been shown to be expressed pre-dominantly in CD34+ cells [19]. Here we show that HLXB9 isexpressed in leukemia cell lines, derived from myeloid, B-cell,and T-cell leukemia, and in primary ALL samples, but that hyper-methylation of the gene results in loss of expression. Thus, onepotential explanation for the opposing roles of HLXB9 in ALL andAML would be that its normal function would be to drive lym-phoid differentiation and inhibit myeloid differentiation in CD34+

hematopoietic progenitor cells and thus loss of expression would

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352 Ferguson et al.

block lymphoid differentiation while over-expression would favora block in myeloid differentiation.

Thus, these data suggest that HLXB9 may play a dual role inthe development of leukemia, as a tumor suppressor gene in ALL,where it is frequently inactivated by epigenetic mechanisms, but asan oncogene in infant AML. Further functional studies will now berequired to define the role of HLXB9 both in normal hematopoiesisand in the development of both myeloid and lymphoid leukemia.

ACKNOWLEDGMENTS

The authors would like to thank Prof. A.M. Dickinson, Prof.A.G. Hall, Dr. S. Meyer, Prof. T.L. Holyoake, Prof. D.G. Oscier,and all clinicians involved in collection of clinical samples withoutwhich this study would not have been possible. This work was sup-ported by the Tyneside Leukaemia Research Association and theBiotechnology and Biological Sciences Research Council.

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