Imaging, Diagnosis, Prognosis

Inactivation of HOXA by Hypermethylation in Myeloid and Lymphoid Malignancy is Frequent and Associated with Poor Prognosis Gordon Strathdee,1Tessa L. Holyoake,2 Alyson Sim,1Anton Parker,4 David G. Oscier,4 Junia V. Melo, 5 Stefan Meyer,6,7 Tim Eden,7 Anne M. Dickinson,8 Joanne C. Mountford,2 Heather G. Jorgensen,2 Richard Soutar, 3 and Robert Brown1

Abstract Purpose: The HOX genes comprise a large family of homeodomain-containing transcription factors, present in four separate clusters, which are key regulators of , hematopoietic differentiation, and leukemogenesis. We aimed to study the role of DNA methyl- ation as an inducer of HOX silencing in leukemia. Experimental Design: Three hundred and seventy-eight samples of myeloid and lymphoid leukemia were quantitatively analyzed (by COBRA analysis and pyrosequencing of bisulfite- modified DNA) for methylation of eight HOXA and HOXB cluster genes. The biological significance of the methylation identified was studied by expression analysis and through re-expression of HOXA5 in a chronic myeloid leukemia (CML) blast crisis cell line model. Results: Here, we identify frequent hypermethylation and gene inactivation of HOXA and HOXB cluster genes in leukemia. In particular, hypermethylation of HOXA4 and HOXA5 was frequently observed (26-79%) in all types of leukemias studied. HOXA6 hypermethylation was predomi- nantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. methylation exhibited clear correla- tions with important clinical variables, most notably in CML, in which hypermethylation of both HOXA5 (P = 0.00002) and HOXA4 (P = 0.006) was strongly correlated with progression to blast crisis. Furthermore, re-expression of HOXA5 in CML blast crisis cells resulted in the induc- tion of markers of granulocytic differentiation. Conclusion: We propose that in addition to the oncogenic role of some HOX family members, other HOX genes are frequent targets for gene inactivation and normally play suppressor roles in leukemia development.

The human HOX genes are a large gene family, consisting of transgenic mouse models have implicated several family 39 members, which are located in four gene clusters (HOXA- members in the control of normal hematopoiesis (2). HOXD). They each encode homeodomain-containing tran- For example, overexpression of Hoxa5 in CD34+ progenitor scription factors which are known to be key regulators of cells induced increased levels of granulocytic/monocytic differ- embryonic development (1). The HOX genes are also expressed entiation and inhibited erythroid/megakaryocytic differentiation in adult cells, in which they play important roles in the control (3). In addition, inhibition of Hoxa5, using antisense oligonu- of (1). Expression of many of the HOX cleotides, inhibited granulocytic/monocytic differentiation but genes, primarily from the HOXA and HOXB clusters, has been increased erythroid/megakaryocytic differentiation (4), suggest- identified in CD34+ hematopoietic progenitor cells, and ing that Hoxa5 is a key regulator of myeloid differentiation.

Authors’ Affiliations: 1Centre for Oncology and Applied Pharmacology, Cancer LRF UK Biobank) at Newcastle (A.M. Dickinson). S. Meyer is a CRUK Clinician Research U.K., Beatson Laboratories and 2Division of Cancer Sciences and Scientist. T. Eden is supported by theTeenage CancerTrust. Molecular Pathology, University of Glasgow, 3Department of Haematology, The costs of publication of this article were defrayed in part by the payment of Western Infirmary, Glasgow, United Kingdom; 4Department of Haematology, Royal page charges. This article must therefore be hereby marked advertisement in Bournemouth Hospital, Bournemouth, United Kingdom; 5Department of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Haematology, Imperial College London, Hammersmith Hospital, London, United Note: Supplementary data for this article are available at Clinical Cancer Research Kingdom; 6Stem Cell and Leukaemia Proteomics Laboratory, Paterson Institute of Online (http://clincancerres.aacrjournals.org/). Cancer Research and 7Paediatric and Adolescent Oncology Unit, University of Current address for G. Strathdee: Life Knowledge Park, Institute of Human Manchester and Christie Hospital NHS Trust, Manchester, United Kingdom; and Genetics, Newcastle University, Newcastle-upon-Tyne, NE13BZ, United Kingdom. 8Haematological Sciences, School of Clinical and Laboratory Sciences, The Requests for reprints: Gordon Strathdee, Life Knowledge Park, Institute Medical School, Newcastle-upon-Tyne, United Kingdom of Human Genetics, Newcastle University, Central Parkway, Newcastle-upon-Tyne Received 4/17/07; revised 6/12/07; accepted 6/26/07. NE1 3BZ, United Kingdom. Phone: 44-191-241-8829; Fax: 44-191-241-8810; Grant support: Cancer Research U.K. programme grant (R. Brown), and by E-mail: [email protected]. Leukaemia Research Fund U.K. grants (T.L. Holyoake, J.V. Melo, A. Parker, and F 2007 American Association for Cancer Research. D. Oscier), and the Biobank for Haematological Malignancies (affiliated to the doi:10.1158/1078-0432.CCR-07-0919

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Inappropriate expression of HOX genes has also been leukemia from Glasgow Royal Infirmary, Royal Bournemouth Hospital, implicated in the development of hematopoietic malignancies Newcastle Royal Victoria Infirmary, Glasgow Western Infirmary, (5). The HOXA9 gene is one of the targets (along with NUP98) Hammersmith Hospital (London), Royal Liverpool University Hospital in the acute myeloid leukemia (AML)–associated (Liverpool, United Kingdom), and Royal Manchester Children’s Hospital. Peripheral blood samples were also obtained from healthy translocation t(7;11)(p15;p15) (refs. 6, 7), and HOXA9 over- volunteers. Ethical approval was obtained for all samples collected. expression has been shown to induce AML in mouse models Both childhood ALL and AML samples were obtained from diagnostic HOX ¶ (8). Similarly, several other genes located in the 5 end of bone marrow aspirates and all samples were determined to have >95% clusters (paralogues 9-13) have also been identified as partners blasts by morphologic assessment of bone marrow aspirate films. All with NUP98 in leukemia-associated translocations (9). Over- CML samples were derived from peripheral blood. All chronic phase expression of four other family members (Hoxa7, Hoxa10, CML samples consisted of leukocytes derived from peripheral blood Hoxb3, and Hoxb8) have all been correlated with defective from patients undergoing leukapheresis. These samples were taken at hematopoiesis and myeloproliferative disease/AML in mouse diagnosis from patients with very high white cell counts and will contain models (5). In addition, the key regulator of HOX gene >95% BCR/ABL-positive cells. The majority of blast crisis samples were expression, MLL, is involved in multiple AML and acute obtained by the isolation of leukocytes directly from peripheral blood lymphoblastic leukemia (ALL)–associated translocations (10) samples, although a small number were also isolated from leukaphe- resis. The blast crisis samples all had between 80% and 99% blasts and and simultaneous overexpression of multiple HOX genes, essentially no identifiable normal cells. CLL samples were derived from identified by microarray analysis, has been correlated with peripheral blood mononuclear cells obtained by Ficoll gradient specific subtypes of AML (11) and ALL (12). centrifugation. This should result in at least 80% (and usually >90%) Alterations in the patterns of DNA methylation are critical malignant cells in all samples. Adult AML samples were obtained either in the development of all types of cancer (13). In particular, from bone marrow or peripheral blood samples (22 peripheral blood transcriptional repression due to hypermethylation of promoter- samples and 61 bone marrow samples). Blast counts were available from associated CpG islands has been shown to inactivate many genes 68 of 83 AML patients. Only one sample had a count <50% and the whose down-regulation is known to be important in tumor overwhelming majority had z80% blasts (54 of 68 samples). No development, both in hematologic malignancies and differences were detected between the methylation frequencies of in solid tumors (14, 15). We have recently shown that hyper- samples derived from peripheral blood or bone marrow. methylation of the HOXA4 gene is associated with transcrip- For the CML chronic phase series, patients at high risk (for progression to blast crisis) were defined as those having one or more tional repression in chronic lymphocytic leukemia (CLL; of the following: high Hasford/Sokal score (19, 20), incomplete ref. 16) and correlates with high-risk of disease. In addition, response to imatinib (21), or the presence of deletions on the short HOXA5 we have also identified as one of a small group of genes arm of chromosome 9 (22). Incomplete response to imatinib was whose cell type–specific expression is controlled by DNA defined as less than complete cytogenetic response by 12 months after methylation in normal cells (17) and have shown increased initiation of therapy, loss of complete cytogenetic response, or levels of DNA methylation and consequent gene inactivation development of imatinib resistance. For AML, patients were separated in a small number of AML samples (18). into good prognosis [t(8;21), t(15;17) and inv(16)] or intermediate The above findings suggested that DNA methylation may prognosis (normal karyotype; ref. 23), based on cytogenetic analysis. have an important role in aberrant control of HOX gene For CLL, the immunoglobulin variable heavy chain mutational status expression during the development of leukemia. Thus, in was known for all cases. Tissue culture and cells. The LAMA84 CML cell line was maintained contrast to the oncogenic role identified for some HOX genes, HOXA9 in RPMI with 2 mmol/L of glutamine and 10% FCS in 95% air/5% CO2 most notably , other family members may function as at 37jC. suppressors of the malignant phenotype. In this study, we have COBRA analysis. COBRA analysis was done largely as described analyzed the methylation status of eight HOXA and B cluster before (24). One microgram of genomic DNA was modified with genes in a large number of chronic and acute, myeloid, and sodium bisulfite using the CpGenome modification kit (Chemicon lymphoid malignancies using quantitative methylation analy- International) as per the manufacturer’s instructions. All samples were sis. These studies have identified frequent hypermethylation of resuspended in 40 AL of Tris-EDTA and 1 AL of this was used for HOX genes, in particular within the HOXA cluster, in all types subsequent PCR reactions. The samples were amplified in 25 AL of leukemia studied. The patterns of methylation identified in volumes containing 1 manufacturer’s buffer, 1 unit of FastStart taq specific types of leukemia were nonrandom and exhibited a polymerase (Roche), 1 to 4 mmol/L of MgCl2, 10 mmol/L of deoxy- nucleotide triphosphates, and 75 ng of each primer. PCR was done number of clear correlations with the clinical characteristics of with one cycle of 95jC for 6 min, 35 cycles of 95jC for 30 s, 58jC the patients. Most strikingly, in chronic myeloid leukemia to 63jC for 30 s, and 72jC for 30 s, followed by one cycle of 72jC HOXA4 HOXA5 (CML), methylation of both and was strongly for 5 min. All PCR reactions were carried out on a PTC-225 DNA associated with progression to blast crisis. Furthermore, re- engine tetrad (MJ Research). Following amplification, the PCR pro- expression of HOXA5 in blast crisis CML cells resulted in the ducts were digested with the appropriate restriction enzymes, specific induction of markers of granulocytic differentiation. These for the methylated sequence after sodium bisulfite modification. results suggest that inactivation of HOX genes, particularly Digested PCR products were separated on 1.5% or 2% agarose gels HOXA4 and HOXA5, plays an important role in the develop- and visualized by ethidium bromide staining on GeneSnap gel ment of leukemia. documentation system (Syngene). Quantitation of methylation levels was carried out by measurement of band intensities using the GeneTools system (Syngene) and methylation levels in a particular sample were calculated correcting for the smaller size (and thus lower Materials and Methods ethidium bromide binding capacity) of the methylated band. In vitro – methylated DNA (Chemicon International) was diluted into DNA Patient samples. DNA was isolated from peripheral blood or bone extracted from normal peripheral blood to produce standards (100%, marrow samples obtained from patients with clinically diagnosed 66%, 33%, and 0%) of known methylation status for all COBRA assays

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(except for analysis of HOXA5 in which in vitro –methylated DNA was vector (Clontech). This vector contains an IRES sequence to allow the diluted into genomic DNA extracted from the SVCT cell line, which independent expression of HOXA5 and GFP from the same vector to is known to be unmethylated at the HOXA5 loci; ref. 18). The primers allow identification of transfected cells. used are listed in Supplementary Table S1. Transient transfections and fluorescence-activated cell sorting. Tran- Pyrosequencing. The samples were amplified in 50-AL volumes sient transfections of the LAMA84 CML cell line were carried out containing manufacturer’s buffer, 2 AL of bisulfite-modified DNA, using the Nucleofector device (program T16; Amaxa Biosystems). Cells 6 2 units of FastStart taq polymerase, 3 mmol/L of MgCl2, 50 mmol/L of (2 10 ) were transfected with 2 Ag of plasmid DNA using deoxynucleotide triphosphates, and 0.2 Amol/L of each primer. PCR Nucleofector kit V (Amaxa Biosystems), as per the manufacturer’s was done with one cycle of 95jC for 6 min, 45 cycles of 95jC for protocol. Twenty-four hours after transfection, cells were sorted for 30 s, 58jC for 30 s, and 72jC for 30 s, followed by one cycle of 72jC positive GFP expression using a FACSVantage cell sorter (Becton for 5 min. All PCR reactions were carried out on a PTC-225 DNA engine Dickinson) and returned to culture. Following sorting, the cell tetrad (MJ Research). Following amplification, sequencing was done populations were >90% GFP-positive. GFP expression at various time using a PSQ 96MA pyrosequencer (Biotage AB) as per the manufac- points after sorting was assessed using a FACSCalibur system (Becton turer’s protocol. The primers used for the initial PCR were identical to Dickinson). Total RNA was extracted from the HOXA5/GFP and the those used for COBRA analysis (Supplementary Table S1), with the control GFP-alone transfected cells at 3 and 5 days posttransfection addition of a 5¶ biotin label on the reverse primer. The sequencing and used for quantitative RT-PCR as described below. primer was 5¶-GATGAGTTTTGTTTTTAG-3¶. Quantitative reverse transcription-PCR. Total RNA was isolated Results from leukemia samples as described above. RNA (2-5 Ag) was then used for cDNA synthesis using the SuperScript first-strand cDNA Hypermethylation of HOXA4 and HOXA5 correlates with blast synthesis kit (Invitrogen) as per the manufacturer’s protocol. Quanti- tative reverse transcription-PCR (RT-PCR) analysis was done in 20-AL crisis in CML. We have previously identified frequent meth- HOXA4 volumes containing 1 master mix(DyNAmo SYBR green qPCR kit; ylation of (16) in CLL and hypermethylation of Finnzymes), 75 ng of each primer, and 1 AL of cDNA. PCR was done HOXA5 in a small series of patients with AML (18), suggesting with one cycle of 94jC for 15 min, followed by cycles of 94jC for that genes in the central portion of the HOX clusters may be 30 s, 55jCto61jC for 30 s and 72jC for 30 s, with plate reads carried targets for aberrant methylation in leukemia. To investigate this out at 77jCto87jC at the end of each cycle. Each of the PCR assays possibility in CML, we performed methylation analysis of were run in triplicate. Glyceraldehyde-3-phosphate dehydrogenase was HOXA and B cluster genes (HOXA4, A5, A6, A7, B4, B5, B6, used as a control for normalizing relative expression levels in the and B7) in 44 samples of chronic phase CML. The analysis was different samples. Reactions were carried out on an Opticon 2 DNA done using the COBRA assay to assess methylation levels in the engine (MJ Research). Levels of A6 (normalized to glyceraldehyde-3- proximal promoter/first exon of each gene (examples in Fig. 1). phosphate dehydrogenase) were calculated using software provided by the manufacturer. Forward primer: 5¶-GGTTTA CCCTTGGATGCAGC Multiple restriction digests were used to ensure at least 4 (and and reverse 5¶-GGCGGTTCTGGAACCAGATC. up to 11) separate CpG sites were analyzed for each loci. Also, HOXA5 expression vector. The complete HOXA5 cDNA sequence known methylation standards of 100%, 66%, 33%, and 0% was obtained from Open Biosystems. The cDNA was then subcloned methylation were assessed to ensure appropriate quantitation into the pIRES2-enhanced green fluorescent (GFP) expression of all assays. No methylation was detected in normal peripheral

Fig. 1. COBRA analysis of HOX gene methylation in leukemia. Examples of COBRA analysis, with the specific HOX gene and disease type indicated below each example. For each example, the first lane is normal peripheral blood (PBL) and the final lane is 100% in vitro ^methylated(IVM) control. Positions of unmethylated (undigested) and methylated (digested) bands; and presence (+) or absence (-)of hypermethylation is indicated below each sample.

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Table 1. Hypermethylation frequencies of HOX genes in leukemia

Chronic phase CML, Blast crisis-CML, AML, CLL, Childhood ALL, Childhood AML, n =44(%) n =23(%) n =83(%) n =151(%) n =48(%) n =28(%) HOXA4 59 91 64 38 69 54 HOXA5 34 87 59 59 77 39 HOXA6 000341914 HOXA7 0000137 HOXB4 0000217 HOXB5 000000 HOXB6 0000134 HOXB7 0 0 0 0 26 11

NOTE: Number (n) of samples analyzed for each gene at which methylation was detected in the first 20 samples (except for HOXA6 in CLL, where n = 61). For loci in which no methylation was detected in the first 20 samples, no further samples were analyzed (except HOXB5 in childhood ALL, where n = 48, and in blast crisis CML, where n = 23 for all loci).

blood for any of the loci, except the HOXA5 gene. As previously HOXA5 gene (34%) and the neighboring HOXA4 gene (59%) reported (18), HOXA5 exhibited methylation of 50% of alleles were found to be frequently hypermethylated in CML chronic in normal peripheral blood and, to account for this, hyper- phase samples (Fig. 1; Table 1). In contrast, no methylation was methylation of HOXA5 was defined as >80% methylation at detected at the other HOXA and HOXB cluster genes analyzed. the majority of CpG sites tested. Using this approach, both the The presence of methylation of HOXA5 in normal cells means that accurate quantitation of the methylation levels in leukemia samples is crucial to definitively identify samples with Table 2. Correlations between HOX gene increased methylation levels. Thus, to confirm that the accuracy hypermethylation and patient outcome of the methylation levels identified at the HOXA5 gene by COBRA analysis, all 44 CML chronic phase samples were c Leukemia Hypermethylated % Methylated* P further analyzed by pyrosequencing. The accuracy of this gene(s) method in quantitating DNA methylation levels has previously CML HoxA5 been shown (25, 26). This analysis produced essentially Blast crisis vs. 87% (20/23) 0.00002 identical results to the COBRA analysis, confirming the Chronic phase 33% (15/45) High risk for 57% (8/14) 0.003 hypermethylation of all samples identified as hypermethylated progressionb vs. by COBRA analysis and lower levels of methylation in all Low risk 6% (1/16) samples without hypermethylation (as determined by COBRA for progression analysis; Supplementary Table S2). Furthermore, the strong HoxA4 Blast crisis vs. 91% (21/23) 0.006 agreement of the COBRA analysis with the pyrosequencing data Chronic phase 60% (27/45) also shows the accuracy of quantitation using the COBRA High risk for 86% (12/14) 0.02 method. progression vs. CML is a triphasic disorder, normally presenting in the Low risk 44% (7/16) chronic phase of the disease, in which differentiation is for progression HoxA4 and relatively normal and finally progressing to blast crisis, in A5 comethylation which normal differentiation is arrested (27). As HOX genes are Poor response 83% (5/6) 0.001 key regulators of hematopoietic differentiation, we hypothe- to imatinib vs. sized that alterations in HOX gene methylation may be Complete response 10% (2/21) to imatinib associated with progression to blast crisis. Samples from AML HoxA4 and patients with the chronic phase of the disease were separated A5 comethylation into high- and low-risk (of transformation to blast crisis) Normal karyotype vs. 69% (22/32) 0.0002 x groups based on known prognostic markers (as defined in ‘‘Favorable’’ cytogenetics 14% (3/21) HOXA4 CLL HoxA4 Materials and Methods) and compared with and IgVh unmutated vs. 57% (25/44) 0.003 HOXA5 methylation status. Hypermethylation of either gene IgVh mutated 30% (32/107) individually or co-hypermethylation of both genes were clearly associated with the high-risk group of patients (Table 2), NOTE: IgVh, immunoglobulin variable heavy chain. suggesting that methylation of HOXA4 and HOXA5 is *Percentage of samples methylated at the indicated gene (no. of associated with an increased risk of progression to blast crisis. methylated samples/total no. of samples). c To further assess the potential link between HOXA4 and P values determined using the Fisher exact test. HOXA5 bRisk for progression (from chronic phase to blast crisis) in CML as methylation and CML blast crisis, 23 blast crisis defined in Materials and Methods. samples were analyzed for hypermethylation as above. This x‘‘Favorable cytogenetics’’ in AML samples include t(8;21), analysis identified exceptionally high levels of hypermethyla- t(15;17), and inv(16). tion of both genes in blast crisis samples (91% for HOXA4 and 87% for HOXA5) and both HOXA5 and HOXA4 exhibited

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Hypermethylation of HOXA cluster genes is common in both myeloid and lymphoid malignancies. To further assess the role of methylation in the aberrant control of HOX in leukemia, we extended the above analysis to determine the frequency of HOX gene methylation in samples of CLL, AML, childhood ALL, and childhood AML. This analysis was carried out using COBRA assays as described for the CML samples above. Methylation analysis of the eight HOXA and B cluster genes was carried out initially on a panel of 80 samples, consisting of 20 samples each of the four types of leukemia. The use of 20 samples from each leukemia type yielded a >95% chance of identifying genes methylated with a frequency of z15%. In leukemia types in which a particular gene was identified as methylated in the original panel, the analysis was expanded to larger sample sets (see Table 1) to more clearly define the level of hypermethylation and to allow Fig. 2. Methylation of HOXA5 and HOXA4 in blast crisis CML patient samples. a comparison of methylation status to clinical characteristics. Examples of COBRA analysis of the blast crisis samples for HOXA5 (top)and HOXA4 (bottom).Top, all examples are of myeloid blast crisis (HOXA5). Bot tom, As in the CML samples, this analysis identified frequent both types of blast crisis samples are included (HOXA4); first lane, normal hypermethylation of both the HOXA4 and HOXA5 genes in peripheral blood (PBL); final lane, 100% in vitro ^methylated(IVM) control. all types of leukemia analyzed. The relative frequencies of Positions of unmethylated (undigested) and methylated (digested) bands; presence (+) or absence (-)of hypermethylation is indicated below each sample. hypermethylation were, however, disease-specific, with HOXA4 hypermethylation levels being greater than HOXA5 in myeloid malignancies, whereas the opposite was true for highly significant increases in the frequency of hypermethyla- lymphoid malignancies (Table 1). Furthermore, hypermethy- HOXA6 tion in the blast crisis phase of the disease (P = 0.00002 for lation of the gene was frequently identified in both HOXA5 and P = 0.006 for HOXA4). Notably, for HOXA5,a adult CLL and childhood ALL, but was rare in the myeloid HOX known regulator of myeloid differentiation (3, 4), hyper- malignancies (Table 1). The other five genes analyzed methylation was invariably observed in myeloid blast crisis exhibited no evidence of methylation in adult leukemia. In samples (15 of 15 samples), suggesting a specific role for loss of contrast, all the HOX genes, except HOXB5, exhibited HOXA5 in progression to myeloid blast crisis. hypermethylation in the childhood ALL and AML samples In order to further validate these results, a second, (Table 1). In addition, although no clear comethylation of independent DNA sequence within the CpG islands of both HOX genes was seen in adult leukemias, there were very HOXA4 and HOXA5 was also analyzed by COBRA (examples strong correlations between methylation of different HOX in Fig. 2), and the methylation levels at the HOXA5 gene were genes in the childhood samples, even between HOX genes again confirmed using pyrosequencing analysis (Supplemen- from different clusters (Table 3). tary Table S2). Both the additional COBRA analysis and the Hypermethylation of gene promoter–associated CpG islands pyrosequencing data confirmed the results of the original has been shown to lead to inactivation of gene expression (28). analysis for all samples, confirming the exceptionally high rate We have previously shown that hypermethylation of both of HOXA4 and HOXA5 hypermethylation in CML blast crisis HOXA4 (16) and HOXA5 (18) is associated with the loss of samples. Hypermethylation of the other HOX genes was also expression in primary leukemia samples. To confirm that assessed in the blast crisis samples; however, as in the chronic promoter hypermethylation of the other frequently hyper- phase samples, no significant methylation was seen for any of methylated HOX gene, HOXA6, was associated with the loss the other loci (Table 1), showing that hypermethylation is of expression, quantitative RT-PCR analysis was used to assess specifically targeted to the HOXA4 and HOXA5 loci. expression in primary CLL samples. As shown in Fig. 3,

Table 3. Comethylation of HOX genes in childhood leukemia

Meth A4 (48)* Meth A5(49) Meth A6 (13) Meth A7 (8) Meth B4 (12) Meth B6 (7) Meth B7 (15) Meth A4 NA Meth A5 39c (0.0012)b NA Meth A6 13 (0.0032) 13 (0.0065) NA Meth A7 8 (0.046) 7 (>0.05) 4 (0.031) NA Meth B4 9 (>0.05) 11 (>0.05) 5 (0.034) 5 (0.0024)NA Meth B6 5 (>0.05) 6 (>0.05) 3 (>0.05) 4 (0.0021) 4 (0.012) NA Meth B7 12 (>0.05) 14 (0.027) 6 (0.022) 6 (0.0007)10(3.20E-07) 4 (0.031) NA

*Number of samples hypermethylated at the above loci (out of a total of 72 childhood ALL and AML samples). cNumber of samples hypermethylated at both loci. bP value, determined by Fisher exact test. Values in boldface were significant after correction for multiple comparisons.

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differentiation in CML blast crisis cells, the HOXA5 gene was transiently re-expressed in the hypermethylated LAMA84 CML blast crisis cell line. The HOXA5 cDNA sequence was introduced into the pIRES2-enhanced GFP vector, which also provides the expression of GFP, to allow the detection of transfected cells. Cells were fluorescence-activated cell–sorted for GFP expression 24 h posttransfection and assayed at 5 days posttransfection for expression of a panel of markers of differentiation using quantitative RT-PCR (compared with cells transfected with a vector expressing GFP alone). At 5 days posttransfection, HOXA5-transfected cells exhibited clear increases in expression of several markers of myeloid differen- tiation, including CD13, C/EBPa, and C/EBPq (all associated

Fig. 3. HOXA6 hypermethylation correlates with loss of expression. Quantitative with differentiation down the granulocytic lineage), but no RT-PCR analysis for HOXA6 expression in methylated (right) and unmethylated detectable expression of CD11b (marker of terminal granulo- (left) CLL samples. HOXA6 expression levels correlated with the methylation status cytic differentiation) or CD14 (monocytic differentiation; of the gene (P = 0.0012, Mann-Whitney U test). ref. 29; Fig. 4). Therefore, the pattern of markers expressed following HOXA5 transfection is consistent with differentiation hypermethylation of HOXA6 was also found to be associated down the granulocytic (as opposed to the monocytic) lineage with loss of gene expression (P = 0.0012, Mann-Whitney U test). (29). Although clearly up-regulated, the expression levels of Hypermethylation of HOXA4 and HOXA5 is associated with these markers remained low and no clear evidence of AML patients with poorer prognosis. As shown above, hyper- morphologic differentiation was seen, consistent with the lack methylation of both HOXA4 and HOXA5 was associated with of CD11b expression. This is likely due to the transient nature disease progression in CML. In addition, we have previously of HOXA5 re-expression, as well as selective loss of HOXA5- shown that hypermethylation of HOXA4 was associated with positive cells from the population as shown by the decreased CLL patients with a poor prognosis (16). To determine if HOX levels of GFP-positive cells at day 5 in the HOXA5/GFP gene methylation was generally associated with patients with a transfectants (1.2% GFP-positive cells) versus the GFP alone poorer prognosis, the methylation data was also compared with transfectants (5.6% GFP-positive cells; Fig. 4B). Thus, re- clinical data for adult AML (for the childhood leukemias, the expression of HOXA5 in CML blast crisis cells resulted in sample numbers were insufficient to allow meaningful com- altered expression patterns consistent with the induction of parisons to be made). In AML, cytogenetic abnormalities are differentiation, and further implicating the loss of HOXA5 one of the most important prognostic indicators (23). expression in the arrest of normal differentiation seen in CML Comparison of HOXA4 and HOXA5 methylation with patient blast crisis. cytogenetics showed clear differences in frequency. Almost all samples analyzed in this study were either from good prognosis Discussion cytogenetic groups [t(8;21), t(15;17), and inv(16)] or exhibited a normal karyotype (associated with an intermediate prognosis; The HOX genes play crucial roles in the control of ref. 23). Although methylation of both HOXA4 (38%) and differentiation of adult hematopoietic cells (1). The results HOXA5 (43%) was observed in patients exhibiting cytogene- presented here show that several members of the HOX gene tics associated with a good prognosis, it was significantly family, particularly from the HOXA cluster, are frequently more frequent in patients with a normal karyotype who have inactivated by CpG island hypermethylation in leukemia. For a poorer prognosis [78% for both; P = 0.004 (HOXA4) and two of these genes, HOXA4 and HOXA5, hypermethylation was P = 0.018 (HOXA5)]. Furthermore, the clear majority of seen in all types of leukemias studied, including both myeloid patients with normal karyotype leukemias showed methylation and lymphoid malignancies. Methylation of both genes of both genes (69%), whereas this was rare in the good showed clear associations with clinical characteristics of the prognosis patients (14%, P = 0.0002; Table 2). different leukemia types. Of particular interest, methylation of Re-expression of HOXA5 in CML blast crisis cells results in the both HOXA5 (P = 0.00002) and HOXA4 (P = 0.006) were induction of differentiation. HOXA5 has previously been significantly more frequent in CML blast crisis (87% and 91%, shown to be an important regulator of normal myeloid respectively) than in the chronic phase of the disease (31% and differentiation and promotes differentiation down the mono- 59%, respectively). Hypermethylation of HOXA5, a known cytic/granulocytic pathway (3, 4). The strong correlation regulator of myeloid differentiation, was invariably observed in identified between hypermethylation of HOXA5 and CML myeloid blast crisis samples (15 of 15 samples hypermethy- blast crisis led to the hypothesis that loss of HOXA5 expression lated), suggesting that loss of HOXA5 expression may be crucial may play an important role in the arrest of normal differen- in the progression to myeloid blast crisis. Furthermore, hyper- tiation in this phase of the disease. Indeed, we have previously methylation of both genes was also significantly more frequent shown that treatment of a CML blast crisis cell line with a DNA in chronic phase patients at high risk of progression (as methylation inhibitor led to the re-expression of HOXA5 and determined by previously identified prognostic markers) than myeloid differentiation of the cells (18). A major drawback of in patients at low risk. These results suggested an important role this approach is that the expression of many other genes will be for the loss of HOXA4 and HOXA5 in the progression to blast affected by the DNA methylation inhibitor, and thus, to more crisis. Importantly, the observed changes in methylation are not directly assess the potential role of HOXA5 in the control of simply a reflection of an increased number of immature cells in

www.aacrjournals.org 5053 Clin Cancer Res 2007;13(17) September 1,2007 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis the CML samples, as neither CD34+ nor CD133+ selected development of human leukemia. In AML, hypermethylation normal hematopoietic cells exhibiting increased methylation of of both HOXA4 and HOXA5 was significantly more frequent either HOXA4 (data not shown) or HOXA5 (18). in patients with a normal karyotype than in patients exhibi- This possibility was further supported by results from gene ting karyotypic abnormalities associated with good prognosis reintroduction studies which showed that re-expression of (78% versus 38%, P = 0.0045 for HOXA4 and 78% versus 43%, HOXA5 in LAMA84 CML blast crisis cells induced the P = 0.018 for HOXA5). Indeed, hypermethylation of both expression of several markers associated, in particular, with HOXA4 and HOXA5 in the same sample was seen in the differentiation down the granulocytic lineage. These results majority of patients with a normal karyotype (69%) but was are similar to those previously reported for the re-expression of rare in cases with a good prognosis (14%). Interestingly, C/EBPa in CML blast crisis cells, which also resulted in the patients with normal karyotypes have also previously been induction of granulocytic differentiation (30). As C/EBPa was shown to exhibit overexpression of multiple HOXA cluster one of the genes induced following HOXA5 re-expression, it is genes, including HOXA4 and HOXA5, using microarray studies possible that this represents a critical target for HOXA5-induced (11). Although this may at first seem contradictory, our differentiation. However, because induction of expression of previous analysis showed that hypermethylation of HOXA5 these markers did not occur until 5 days after transfection, this was associated with the loss of protein expression in myeloid would suggest that neither C/EBPa nor indeed any of the other leukemia cell lines and primary samples, even in the presence induced genes, are likely to be direct transcriptional targets for of a HOXA5-containing transcript (18), which is derived from HOXA5, but are probably secondary to the activation or upstream of the HOXA5 promoter and is presumably non- repression of other genes. Clearly, it will be important to do coding. This would be consistent with a model in which up- expression studies to allow the identification of specific regulation of HOXA cluster expression, likely targeting the transcriptional targets of HOXA5 in these cells and the known oncogenic HOXA genes, HOXA7 and HOXA9 (31), particular pathways activated or repressed by HOXA5 reintro- would lead to a much higher selective pressure for the duction. inactivation of HOXA genes that functioned to induce In addition to the frequent targeting of HOXA4 and HOXA5 differentiation or inhibit leukemic cell growth (HOXA4 and in CML, hypermethylation of these genes was also frequently HOXA5). In agreement with this, recent studies of MLL-AF10– observed in AML, CLL, and childhood ALL and AML, suggesting dependent mouse models of leukemogenesis have shown that a general role for the inactivation for these HOX genes in the although the presence of the fusion protein is associated with up-regulation of a Hoxa5-containing transcript (32), transfor- mation by the fusion protein is more efficient in bone marrow from Hoxa5-deficient mice, indicating that Hoxa5 antagonizes MLL-AF10–dependent transformation (33). Interestingly the same authors also showed that transformation by the CALM- AF10 fusion protein, which is associated with a more specific up-regulation of Hoxa5, was suppressed in the absence of Hoxa5 (33), indicating that the roles of the HOX are likely to be context-dependent. However, the high frequency of HOXA4 and HOXA5 hypermethylation observed in these studies suggests that their predominant role will be inhibitory to leukemogenesis. Concordant methylation of genes has been described in both hematologic and solid malignancies, and involves a nonran- dom distribution of methylation in which a subset of samples exhibit much higher levels of CpG island hypermethylation, or the so-called CpG island methylator phenotype (34). However, despite their close physical proximity, there is no clear evidence for comethylation of the HOX genes in adult leukemias. In contrast to the adult leukemias, hypermethylation of HOX genes exhibited very clear positive correlations in childhood leukemia (Table 3). Furthermore, methylation of HOX genes was also considerably more widespread in the childhood sam- ples than in the adult samples analyzed in this study (Table 1), although analysis of adult ALL samples will be required to confirm whether this is restricted to childhood leukemia. As other reports have suggested that CpG island methylation is, Fig. 4. Re-expression of HOXA5 in LAMA84 CML cells induces differentiation. A, quantitative RT-PCR analysis for five known markers of myeloid differentiation in if anything, less frequent in childhood (versus adult) leukemia RNA samples extracted from LAMA84 cells 5 d after transfection with either a (35), the high levels of HOX loci methylation suggests that vector expressing HOXA5 and GFP (HOXA5) or GFP alone (GFP). Expression of HOX CD13, C/EBPa,andC/EBPq were clearly increased in HOXA5-transfected cells tumor suppressor activity may be more widespread in (19.4-, 4.1-, and 19.6-fold, respectively), whereas CD11band CD14 were not genes in early life (resulting in selective pressure for inactivation expressed at detectable levels. B, GFP expression in cells transfected with either a of multiple HOX loci), but is restricted to a small subset vector expressing HOXA5 and GFP (HOXA5) or GFP alone (GFP) was assessed HOX by fluorescence-activated cell sorting analysis 5 d after initial transfection. The of genes in the adult. Alternatively, differences in percentage of cells positive for GFP expression is shown (bottom right quadrant). the mechanisms of gene regulation in early versus adult

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hematopoiesis may make HOX loci more susceptible to drugs. The identification of such targets for epigenetic therapies hypermethylation during childhood leukemogenesis. will be an important tool to allow appropriate monitoring and Overall, these results suggest an important role for DNA scheduling of epigenetic therapies, as well as potentially methylation, and consequent loss of expression, of multiple identifying patient populations most likely to benefit from HOX genes in both myeloid and lymphoid leukemia. these novel therapies. Furthermore, these studies identify a novel role for HOX genes (in particular, HOXA4 and HOXA5) in leukemia development as tumor suppressor genes, unlike the oncogenic role played by Acknowledgments more 5¶ HOXA cluster genes. Given the current interest in the We thank Prof. Richard E. Clark for providing CML samples and relevant clinical use of epigenetic therapies (directed against DNA methylation information, all hematologists from the United Kingdom who contributed to the or associated histone modifications) it will be crucial to collections of samples used for the analysis described here, and the LRF/CLWP for determine if HOX genes represent important targets for such access to banked samples.

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Gordon Strathdee, Tessa L. Holyoake, Alyson Sim, et al.

Clin Cancer Res 2007;13:5048-5055.

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