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Imaging, Diagnosis, Prognosis |
Authors' Affiliations: 1 Centre for Oncology and Applied Pharmacology, Cancer Research U.K., Beatson Laboratories and 2 Division of Cancer Sciences and Molecular Pathology, University of Glasgow, 3 Department of Haematology, Western Infirmary, Glasgow, United Kingdom; 4 Department of Haematology, Royal Bournemouth Hospital, Bournemouth, United Kingdom; 5 Department of Haematology, Imperial College London, Hammersmith Hospital, London, United Kingdom; 6 Stem Cell and Leukaemia Proteomics Laboratory, Paterson Institute of Cancer Research and 7 Paediatric and Adolescent Oncology Unit, University of Manchester and Christie Hospital NHS Trust, Manchester, United Kingdom; and 8 Haematological Sciences, School of Clinical and Laboratory Sciences, The Medical School, Newcastle-upon-Tyne, United Kingdom
Requests for reprints: Gordon Strathdee, Life Knowledge Park, Institute of Human Genetics, Newcastle University, Central Parkway, Newcastle-upon-Tyne NE1 3BZ, United Kingdom. Phone: 44-191-241-8829; Fax: 44-191-241-8810; E-mail: G.R.Strathdee{at}newcastle.ac.uk.
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Abstract |
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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 predominantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. HOX gene methylation exhibited clear correlations 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 induction 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.
Inappropriate expression of HOX genes has also been implicated in the development of hematopoietic malignancies (5). The HOXA9 gene is one of the targets (along with NUP98) in the acute myeloid leukemia (AML)–associated chromosome translocation t(7;11)(p15;p15) (refs. 6, 7), and HOXA9 overexpression has been shown to induce AML in mouse models (8). Similarly, several other HOX genes located in the 5' end of clusters (paralogues 9-13) have also been identified as partners with NUP98 in leukemia-associated translocations (9). Overexpression of four other family members (Hoxa7, Hoxa10, Hoxb3, and Hoxb8) have all been correlated with defective hematopoiesis and myeloproliferative disease/AML in mouse models (5). In addition, the key regulator of HOX gene expression, MLL, is involved in multiple AML and acute lymphoblastic leukemia (ALL)–associated translocations (10) and simultaneous overexpression of multiple HOX genes, identified by microarray analysis, has been correlated with specific subtypes of AML (11) and ALL (12).
Alterations in the patterns of DNA methylation are critical in the development of all types of cancer (13). In particular, transcriptional repression due to hypermethylation of promoter-associated CpG islands has been shown to inactivate many genes whose down-regulation is known to be important in tumor development, both in hematologic malignancies and in solid tumors (14, 15). We have recently shown that hypermethylation of the HOXA4 gene is associated with transcriptional repression in chronic lymphocytic leukemia (CLL; ref. 16) and correlates with high-risk of disease. In addition, we have also identified HOXA5 as one of a small group of genes whose cell type–specific expression is controlled by DNA methylation in normal cells (17) and have shown increased levels of DNA methylation and consequent gene inactivation in a small number of AML samples (18).
The above findings suggested that DNA methylation may have an important role in aberrant control of HOX gene expression during the development of leukemia. Thus, in contrast to the oncogenic role identified for some HOX genes, most notably HOXA9, other family members may function as suppressors of the malignant phenotype. In this study, we have analyzed the methylation status of eight HOXA and B cluster genes in a large number of chronic and acute, myeloid, and lymphoid malignancies using quantitative methylation analysis. These studies have identified frequent hypermethylation of HOX genes, in particular within the HOXA cluster, in all types of leukemia studied. The patterns of methylation identified in specific types of leukemia were nonrandom and exhibited a number of clear correlations with the clinical characteristics of the patients. Most strikingly, in chronic myeloid leukemia (CML), methylation of both HOXA4 and HOXA5 was strongly associated with progression to blast crisis. Furthermore, re-expression of HOXA5 in blast crisis CML cells resulted in the induction of markers of granulocytic differentiation. These results suggest that inactivation of HOX genes, particularly HOXA4 and HOXA5, plays an important role in the development of leukemia.
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Materials and Methods |
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Both childhood ALL and AML samples were obtained from diagnostic bone marrow aspirates and all samples were determined to have >95% blasts by morphologic assessment of bone marrow aspirate films. All CML samples were derived from peripheral blood. All chronic phase CML samples consisted of leukocytes derived from peripheral blood from patients undergoing leukapheresis. These samples were taken at diagnosis from patients with very high white cell counts and will contain >95% BCR/ABL-positive cells. The majority of blast crisis samples were obtained by the isolation of leukocytes directly from peripheral blood samples, although a small number were also isolated from leukapheresis. The blast crisis samples all had between 80% and 99% blasts and essentially no identifiable normal cells. CLL samples were derived from peripheral blood mononuclear cells obtained by Ficoll gradient centrifugation. This should result in at least 80% (and usually >90%) malignant cells in all samples. Adult AML samples were obtained either from bone marrow or peripheral blood samples (22 peripheral blood samples and 61 bone marrow samples). Blast counts were available from 68 of 83 AML patients. Only one sample had a count <50% and the overwhelming majority had 
80% blasts (54 of 68 samples). No differences were detected between the methylation frequencies of samples derived from peripheral blood or bone marrow.
For the CML chronic phase series, patients at high risk (for progression to blast crisis) were defined as those having one or more of the following: high Hasford/Sokal score (19, 20), incomplete response to imatinib (21), or the presence of deletions on the short arm of chromosome 9 (22). Incomplete response to imatinib was defined as less than complete cytogenetic response by 12 months after initiation of therapy, loss of complete cytogenetic response, or development of imatinib resistance. For AML, patients were separated into good prognosis [t(8;21), t(15;17) and inv(16)] or intermediate prognosis (normal karyotype; ref. 23), based on cytogenetic analysis. For CLL, the immunoglobulin variable heavy chain mutational status was known for all cases.
Tissue culture and cells. The LAMA84 CML cell line was maintained in RPMI with 2 mmol/L of glutamine and 10% FCS in 95% air/5% CO2 at 37°C.
COBRA analysis. COBRA analysis was done largely as described before (24). One microgram of genomic DNA was modified with sodium bisulfite using the CpGenome modification kit (Chemicon International) as per the manufacturer's instructions. All samples were resuspended in 40 µL of Tris-EDTA and 1 µL of this was used for subsequent PCR reactions. The samples were amplified in 25 µL volumes containing 1x manufacturer's buffer, 1 unit of FastStart taq polymerase (Roche), 1 to 4 mmol/L of MgCl2, 10 mmol/L of deoxynucleotide triphosphates, and 75 ng of each primer. PCR was done with one cycle of 95°C for 6 min, 35 cycles of 95°C for 30 s, 58°C to 63°C for 30 s, and 72°C for 30 s, followed by one cycle of 72°C for 5 min. All PCR reactions were carried out on a PTC-225 DNA engine tetrad (MJ Research). Following amplification, the PCR products were digested with the appropriate restriction enzymes, specific for the methylated sequence after sodium bisulfite modification. Digested PCR products were separated on 1.5% or 2% agarose gels and visualized by ethidium bromide staining on GeneSnap gel 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 ethidium bromide binding capacity) of the methylated band. In vitro–methylated DNA (Chemicon International) was diluted into DNA extracted from normal peripheral blood to produce standards (100%, 66%, 33%, and 0%) of known methylation status for all COBRA assays (except for analysis of HOXA5 in which in vitro–methylated DNA was diluted into genomic DNA extracted from the SVCT cell line, which is known to be unmethylated at the HOXA5 loci; ref. 18). The primers used are listed in Supplementary Table S1.
Pyrosequencing. The samples were amplified in 50-µL volumes containing manufacturer's buffer, 2 µL of bisulfite-modified DNA, 2 units of FastStart taq polymerase, 3 mmol/L of MgCl2, 50 mmol/L of deoxynucleotide triphosphates, and 0.2 µmol/L of each primer. PCR was done with one cycle of 95°C for 6 min, 45 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by one cycle of 72°C for 5 min. All PCR reactions were carried out on a PTC-225 DNA engine tetrad (MJ Research). Following amplification, sequencing was done using a PSQ 96MA pyrosequencer (Biotage AB) as per the manufacturer's protocol. The primers used for the initial PCR were identical to those used for COBRA analysis (Supplementary Table S1), with the addition of a 5' biotin label on the reverse primer. The sequencing primer was 5'-GATGAGTTTTGTTTTTAG-3'.
Quantitative reverse transcription-PCR. Total RNA was isolated from leukemia samples as described above. RNA (2-5 µg) was then used for cDNA synthesis using the SuperScript first-strand cDNA synthesis kit (Invitrogen) as per the manufacturer's protocol. Quantitative reverse transcription-PCR (RT-PCR) analysis was done in 20-µL volumes containing 1x master mix (DyNAmo SYBR green qPCR kit; Finnzymes), 75 ng of each primer, and 1 µL of cDNA. PCR was done with one cycle of 94°C for 15 min, followed by cycles of 94°C for 30 s, 55°C to 61°C for 30 s and 72°C for 30 s, with plate reads carried out at 77°C to 87°C at the end of each cycle. Each of the PCR assays were run in triplicate. Glyceraldehyde-3-phosphate dehydrogenase was used as a control for normalizing relative expression levels in the different samples. Reactions were carried out on an Opticon 2 DNA engine (MJ Research). Levels of A6 (normalized to glyceraldehyde-3-phosphate dehydrogenase) were calculated using software provided by the manufacturer. Forward primer: 5'-GGTTTA CCCTTGGATGCAGC and reverse 5'-GGCGGTTCTGGAACCAGATC.
HOXA5 expression vector. The complete HOXA5 cDNA sequence was obtained from Open Biosystems. The cDNA was then subcloned into the pIRES2-enhanced green fluorescent protein (GFP) expression vector (Clontech). This vector contains an IRES sequence to allow the independent expression of HOXA5 and GFP from the same vector to allow identification of transfected cells.
Transient transfections and fluorescence-activated cell sorting. Transient transfections of the LAMA84 CML cell line were carried out using the Nucleofector device (program T16; Amaxa Biosystems). Cells (2 x 106) were transfected with 2 µg of plasmid DNA using Nucleofector kit V (Amaxa Biosystems), as per the manufacturer's protocol. Twenty-four hours after transfection, cells were sorted for positive GFP expression using a FACSVantage cell sorter (Becton Dickinson) and returned to culture. Following sorting, the cell populations were >90% GFP-positive. GFP expression at various time points after sorting was assessed using a FACSCalibur system (Becton Dickinson). Total RNA was extracted from the HOXA5/GFP and the control GFP-alone transfected cells at 3 and 5 days posttransfection and used for quantitative RT-PCR as described below.
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Results |
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CML is a triphasic disorder, normally presenting in the chronic phase of the disease, in which differentiation is relatively normal and finally progressing to blast crisis, in which normal differentiation is arrested (27). As HOX genes are key regulators of hematopoietic differentiation, we hypothesized that alterations in HOX gene methylation may be associated with progression to blast crisis. Samples from patients with the chronic phase of the disease were separated into high- and low-risk (of transformation to blast crisis) groups based on known prognostic markers (as defined in Materials and Methods) and compared with HOXA4 and HOXA5 methylation status. Hypermethylation of either gene individually or co-hypermethylation of both genes were clearly associated with the high-risk group of patients (Table 2 ), suggesting that methylation of HOXA4 and HOXA5 is associated with an increased risk of progression to blast crisis.
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In order to further validate these results, a second, independent DNA sequence within the CpG islands of both HOXA4 and HOXA5 was also analyzed by COBRA (examples in Fig. 2 ), and the methylation levels at the HOXA5 gene were again confirmed using pyrosequencing analysis (Supplementary Table S2). Both the additional COBRA analysis and the pyrosequencing data confirmed the results of the original analysis for all samples, confirming the exceptionally high rate of HOXA4 and HOXA5 hypermethylation in CML blast crisis samples. Hypermethylation of the other HOX genes was also assessed in the blast crisis samples; however, as in the chronic phase samples, no significant methylation was seen for any of the other loci (Table 1), showing that hypermethylation is specifically targeted to the HOXA4 and HOXA5 loci.
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15%. 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 a comparison of methylation status to clinical characteristics. As in the CML samples, this analysis identified frequent hypermethylation of both the HOXA4 and HOXA5 genes in all types of leukemia analyzed. The relative frequencies of hypermethylation were, however, disease-specific, with HOXA4 hypermethylation levels being greater than HOXA5 in myeloid malignancies, whereas the opposite was true for lymphoid malignancies (Table 1). Furthermore, hypermethylation of the HOXA6 gene was frequently identified in both adult CLL and childhood ALL, but was rare in the myeloid malignancies (Table 1). The other five HOX genes analyzed exhibited no evidence of methylation in adult leukemia. In contrast, all the HOX genes, except HOXB5, exhibited hypermethylation in the childhood ALL and AML samples (Table 1). In addition, although no clear comethylation of HOX genes was seen in adult leukemias, there were very strong correlations between methylation of different HOX genes in the childhood samples, even between HOX genes from different clusters (Table 3
).
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Re-expression of HOXA5 in CML blast crisis cells results in the induction of differentiation. HOXA5 has previously been shown to be an important regulator of normal myeloid differentiation and promotes differentiation down the monocytic/granulocytic pathway (3, 4). The strong correlation identified between hypermethylation of HOXA5 and CML blast crisis led to the hypothesis that loss of HOXA5 expression may play an important role in the arrest of normal differentiation in this phase of the disease. Indeed, we have previously shown that treatment of a CML blast crisis cell line with a DNA methylation inhibitor led to the re-expression of HOXA5 and myeloid differentiation of the cells (18). A major drawback of this approach is that the expression of many other genes will be affected by the DNA methylation inhibitor, and thus, to more directly assess the potential role of HOXA5 in the control of 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 differentiation, including CD13, C/EBP
, and C/EBP
(all associated with differentiation down the granulocytic lineage), but no detectable expression of CD11b (marker of terminal granulocytic differentiation) or CD14 (monocytic differentiation; ref. 29; Fig. 4
). Therefore, the pattern of markers expressed following HOXA5 transfection is consistent with differentiation down the granulocytic (as opposed to the monocytic) lineage (29). Although clearly up-regulated, the expression levels of these markers remained low and no clear evidence of morphologic differentiation was seen, consistent with the lack of CD11b expression. This is likely due to the transient nature of HOXA5 re-expression, as well as selective loss of HOXA5-positive cells from the population as shown by the decreased levels of GFP-positive cells at day 5 in the HOXA5/GFP transfectants (1.2% GFP-positive cells) versus the GFP alone transfectants (5.6% GFP-positive cells; Fig. 4B). Thus, re-expression of HOXA5 in CML blast crisis cells resulted in altered expression patterns consistent with the induction of differentiation, and further implicating the loss of HOXA5 expression in the arrest of normal differentiation seen in CML blast crisis.
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Discussion |
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This possibility was further supported by results from gene reintroduction studies which showed that re-expression of HOXA5 in LAMA84 CML blast crisis cells induced the expression of several markers associated, in particular, with differentiation down the granulocytic lineage. These results are similar to those previously reported for the re-expression of C/EBP in CML blast crisis cells, which also resulted in the induction of granulocytic differentiation (30). As C/EBP was one of the genes induced following HOXA5 re-expression, it is possible that this represents a critical target for HOXA5-induced differentiation. However, because induction of expression of these markers did not occur until 5 days after transfection, this would suggest that neither C/EBP nor indeed any of the other induced genes, are likely to be direct transcriptional targets for HOXA5, but are probably secondary to the activation or repression of other genes. Clearly, it will be important to do expression studies to allow the identification of specific transcriptional targets of HOXA5 in these cells and the particular pathways activated or repressed by HOXA5 reintroduction.
In addition to the frequent targeting of HOXA4 and HOXA5 in CML, hypermethylation of these genes was also frequently observed in AML, CLL, and childhood ALL and AML, suggesting a general role for the inactivation for these HOX genes in the development of human leukemia. In AML, hypermethylation of both HOXA4 and HOXA5 was significantly more frequent in patients with a normal karyotype than in patients exhibiting karyotypic abnormalities associated with good prognosis (78% versus 38%, P = 0.0045 for HOXA4 and 78% versus 43%, P = 0.018 for HOXA5). Indeed, hypermethylation of both HOXA4 and HOXA5 in the same sample was seen in the majority of patients with a normal karyotype (69%) but was rare in cases with a good prognosis (14%). Interestingly, patients with normal karyotypes have also previously been shown to exhibit overexpression of multiple HOXA cluster genes, including HOXA4 and HOXA5, using microarray studies (11). Although this may at first seem contradictory, our previous analysis showed that hypermethylation of HOXA5 was associated with the loss of protein expression in myeloid leukemia cell lines and primary samples, even in the presence of a HOXA5-containing transcript (18), which is derived from upstream of the HOXA5 promoter and is presumably noncoding. This would be consistent with a model in which up-regulation of HOXA cluster expression, likely targeting the known oncogenic HOXA genes, HOXA7 and HOXA9 (31), would lead to a much higher selective pressure for the inactivation of HOXA genes that functioned to induce differentiation or inhibit leukemic cell growth (HOXA4 and HOXA5). In agreement with this, recent studies of MLL-AF10–dependent mouse models of leukemogenesis have shown that although the presence of the fusion protein is associated with up-regulation of a Hoxa5-containing transcript (32), transformation 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 proteins 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 nonrandom 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 samples 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, if anything, less frequent in childhood (versus adult) leukemia (35), the high levels of HOX loci methylation suggests that tumor suppressor activity may be more widespread in HOX genes in early life (resulting in selective pressure for inactivation of multiple HOX loci), but is restricted to a small subset of HOX genes in the adult. Alternatively, differences in the mechanisms of gene regulation in early versus adult hematopoiesis may make HOX loci more susceptible to hypermethylation during childhood leukemogenesis.
Overall, these results suggest an important role for DNA methylation, and consequent loss of expression, of multiple HOX genes in both myeloid and lymphoid leukemia. 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 more 5' HOXA cluster genes. Given the current interest in the use of epigenetic therapies (directed against DNA methylation or associated histone modifications) it will be crucial to determine if HOX genes represent important targets for such drugs. The identification of such targets for epigenetic therapies will be an important tool to allow appropriate monitoring and scheduling of epigenetic therapies, as well as potentially identifying patient populations most likely to benefit from these novel therapies.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Current address for G. Strathdee: Life Knowledge Park, Institute of Human Genetics, Newcastle University, Newcastle-upon-Tyne, NE1 3BZ, United Kingdom.
Received 4/17/07; revised 6/12/07; accepted 6/26/07.
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expression in a BCR-ABL+ cell line induces terminal granulocytic differentiation. J Biol Chem 2003;278:52651–9.This article has been cited by other articles:
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