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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2452-2460
ABL1 Methylation Is a Distinct Molecular Event Associated With
Clonal Evolution of Chronic Myeloid Leukemia
By
Fotis A. Asimakopoulos,
Pesach J. Shteper,
Svetlana Krichevsky,
Eitan Fibach,
Aaron Polliack,
Eliezer Rachmilewitz,
Yinon Ben-Neriah, and
Dina Ben-Yehuda
From the Department of Hematology, Hadassah University Hospital, and
Lautenberg Center for Immunology, Hebrew University-Hadassah Medical
School, Jerusalem, Israel.
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ABSTRACT |
Methylation of the proximal promoter of the ABL1 oncogene is
a common epigenetic alteration associated with clinical progression of
chronic myeloid leukemia (CML). In this study we queried whether both
the Ph'-associated and normal ABL1 alleles undergo
methylation; what may be the proportion of hematopoietic progenitors
bearing methylated ABL1 promoters in chronic versus acute phase
disease; whether methylation affects the promoter uniformly or in
patches with discrete clinical relevance; and, finally, whether
methylation of ABL1 reflects a generalized process or is
gene-specific. To address these issues, we adapted the techniques of
methylation-specific PCR and bisulfite-sequencing to study the
regulatory regions of ABL1 and other genes with a role in DNA
repair or genotoxic stress response. In cell lines established from CML
blast crisis, which only carry a single ABL1 allele nested
within the BCR-ABL fusion gene, ABL1 promoters
were universally methylated. By contrast, in clinical samples from
patients at advanced stages of disease, both methylated and
unmethylated promoter alleles were detectable. To distinguish between
allele-specific methylation and a mixed cell population pattern, we
studied the methylation status of ABL1 in colonies derived from
single hematopoietic progenitors. Our results showed that both
methylated and unmethylated promoter alleles coexisted in the same
colony. Furthermore, ABL1 methylation was noted in the vast
majority of colonies from blast crisis, but not chronic-phase CML. Both
cell lines and clinical samples from acute-phase CML showed nearly
uniform hypermethylation along the promoter region. Finally, we showed
that ABL1 methylation does not reflect a generalized process
and may be unique among DNA repair/genotoxic stress response genes. Our
data suggest that specific methylation of the Ph'-associated
ABL1 allele accompanies clonal evolution in CML.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
myeloproliferative disorder accounting for approximately 25% of all
leukemia cases. Original pioneering work showed that the disease
results from clonal expansion of a hematopoietic stem
cell.1-3 The initiating molecular event is a reciprocal
translocation, t(9;22), cytogenetically detectable by the presence of
the Philadelphia chromosome (Ph').4,5 This
translocation results in fusion of the ABL1 gene, located on
chromosome 9, to the BCR gene on chromosome 22 with formation of the BCR/ABL hybrid gene.6,7 The product
of the ABL1 gene is a tyrosine kinase which, like other enzymes
of this type, has been postulated to play a role in cellular growth
control and response to DNA damage.8,9 Exactly how
alteration of this gene is responsible for leukemogenesis is unknown,
but the end result is an expansion of the stem cell pool with
overproduction of mature myeloid cells with essentially normal
morphology and function. After a period of 3 to 5 years of relative
quiescence ("chronic phase") characterized by functional
maturation of the hematopoietic progenitors, the disease progresses to
an accelerated and subsequent blastic phase that is virtually
indistinguishable from acute leukemia. In these late stages of the
disease, collectively termed "acute-phase CML," a myeloid or
lymphoid descendent of the originally affected stem cell loses its
capacity for terminal differentiation. The early molecular events
underlying disease progression to blast crisis remain largely obscure;
however, a number of late cytogenetic and molecular abnormalities have
been described in subsets of patients with established blastic
transformation.7,10,11
Clinical diagnosis of CML is usually established in the chronic phase,
with its onset, whether several days or years before detection,
remaining elusive. At this stage, there is a window of opportunity for
permanent cure after bone marrow transplantation (BMT); however, BMT is
usually no longer effective after the disease has
evolved.12 Therefore, monitoring the rate of clinical
progression is of prime importance in the management of the disease.
Identifying and understanding the genetic and molecular mechanisms
crucial for disease evolution to the blastic phase will not only aid in determining the proper timing for BMT, but also provide greater insight
into the process of malignant transformation.
The ABL1 gene is expressed in all normal human cells. This gene
has 2 alternative first exons, Ia and Ib, which are differentially transcribed from their own promoters13,14
(Fig 1). The proximal promoter (Pa) and a
distal promoter (Pb) are 175 kb apart and direct the synthesis of 2 mRNA species of 6 and 7 kb, respectively. In approximately 90% of
Ph' translocations the proximal promoter, Pa, is nested within
the BCR/ABL transcriptional unit.15 In normal cells, both ABL1 promoters are active in gene expression and the activity of the Pa promoter appears to be unaffected by the Pb
promoter.
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| Fig 1.
The position of the Ia CpG island within the
BCR-ABL fusion gene is shown. The normal ABL1
proto-oncogene (top) comprises 2 alternative promoters (Pb and Pa) each
adjacent to exons Ib and Ia, respectively. Ninety percent of CML
breakpoints occur in the long intron separating exons Ib and Ia. After
the translocation event, the Ia promoter and its associated CpG island
become nested within the fusion gene.
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Our laboratory first reported that the CpG island associated with the
proximal promoter of ABL1 undergoes methylation upon clinical
progression of CML and that this epigenetic alteration may account for
the loss of ABL1 expression in cell lines established from
patients in the blastic phase of CML.16 Using a
methylation-sensitive restriction endonuclease approach, we showed that
peripheral blood and/or bone marrow samples from patients in the
chronic phase of CML generally lacked methylation at this locus,
whereas cell lines and clinical samples from blastic-phase CML had
invariably undergone methylation. Patients in the accelerated phase of
CML showed intermediate patterns of methylation but they also
progressed to patterns of dense methylation upon evolution to blast
crisis. We consolidated and expanded these observations in a large
study of ABL1 methylation patterns in 99 CML patients at
various stages of disease.17 This work demonstrated that
partial methylation patterns were also observed in patients with
chronic-phase disease of long duration (more than 24 months).
Intriguingly, treatment with interferon- resulted in reversal of
methylation regardless of cytogenetic response; these effects were not
observed with hydroxyurea treatment. Reports from other laboratories
subsequently confirmed these observations.18,19
These data raise a number of important questions concerning the precise
role of ABL1 promoter methylation in CML progression. Methylation may be restricted to ABL1 or represent a widespread phenomenon. Recently, reversal of the methylation status of imprinted genes was reported in blastic-phase CML, raising the possibility that
maintenance of stable methylation patterns may cease upon disease
evolution.20 Earlier reports on differential methylation of
the calcitonin and BCR loci in acute-phase CML have also
provided hints to this scenario.21-23 Additionally, a
quantitative appreciation of the size of the clone bearing methylated
ABL1 promoter alleles would be central to the investigation of
the role of ABL1 methylation in CML. Furthermore, knowledge of
the number of promoter alleles in each cell undergoing methylation in
acute-phase CML has been lacking.
To address these questions we adapted the technique of
methylation-specific polymerase chain reaction
(MSP)24 and bisulfite sequencing to study the
promoter regions of ABL1 and a number of other genes. Our
results show that methylation is a highly specific process that is
likely to be restricted to the ABL1 allele nested within the
Ph' fusion gene. Moreover, it appears that in blastic-phase CML,
in contrast to earlier phases of the disease, the vast majority of
clonogenic progenitors carry methylated ABL1 promoter alleles.
Therefore, we put forward the suggestion that specific methylation of
the ABL1 promoter is the mechanism disrupting the balance of
BCR-ABL to ABL protein that has been postulated to underlie CML
progression.25 Reversal of ABL1 hypermethylation may offer new avenues for therapeutic intervention in this common leukemia.
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MATERIALS AND METHODS |
Cell lines and patient samples.
Cell lines BV173, KBM5, EM2, and K562 are Philadelphia-positive CML
cell lines established from patients in blast crisis (see ref 16 and
references therein). The first 3 cell lines contain duplicates of a
Ph' chromosome but no normal homologue of chromosome 9; K562
contains 3 Ph' chromosomes as well as a normal homologue of
chromosome 9. The patient population used in this work has been
previously studied in our laboratory using the conventional methylation-sensitive enzyme methodology.17
Cell isolation and DNA extraction from clinical samples.
Mononuclear layers of peripheral blood or bone marrow cells were
isolated on a Ficoll-Hypaque density gradient (Pharmacia Biotech,
Upsala, Sweden). Genomic DNA was subsequently extracted using standard organic extraction procedures.
Bisulfite modification of DNA.
Genomic DNA was modified by sodium bisulfite as follows: 5 µg
DNA/reaction was denatured at 95°C for 15 minutes. Samples were subsequently chilled on ice for 2 minutes and 2N freshly prepared NaOH
was added to a final concentration of 0.5N in a total volume of 30 µL. After a 15-minute incubation at 37°C, 450 µL of freshly prepared bisulfite reagent (see below) was added, and the mixtures were
overlaid with mineral oil and allowed to incubate at 55°C for 4 hours. After incubation, DNA was extracted using the Wizard DNA
Clean-up kit (Promega, Madison, WI). Purified DNA was
recovered in water and 2N NaOH added to a final concentration of 0.3N;
samples were allowed to incubate for 15 minutes at 37°C. On
conclusion of this step, 1 to 1.5 µg of lambda DNA (NEB, Beverly,
MA) was added and the DNA was ethanol precipitated
overnight at  80°C. DNA was subsequently recovered by
centrifugation and dissolved in sterile water in preparation for
polymerase chain reaction (PCR). The bisulfite reagent was prepared as
follows: 1.9 g of sodium bisulfite (Sigma, St Louis, MO)
was slowly dissolved in sterile water on a gentle rocker; the total
volume was 4 mL, which also contained 0.7 mL of a 2N NaOH solution and
0.5 mL of a saturated solution of hydroquinone (Sigma) in water.
Single-step methylation-specific PCR (ABL1, MSH2,
MSH6 loci).
For the ABL1, MSH2, and MSH6 loci, a single
step methylation-specific PCR approach was used. Eight hundred
nanograms of genomic DNA modified as described above was included in
100 µL PCR reaction volumes containing buffer to 1X final
concentration (Tris-HCl 10 mmol/L pH 9, KCl 50 mmol/L,
MgCl2 1.5 mmol/L, Triton X100 0.1%, bovine serum albumin
[BSA] 0.2 mg/mL), primers (see below), and 1 U Taq polymerase. dNTP
concentrations were calibrated for each PCR reaction as a way to
achieve gentle adjustments of the magnesium concentration.
Table 1 provides primer sequences for each
locus as well as the concentration of dNTPs and cycling parameters used in each PCR reaction. As a positive control, DNA from normal donor buffy coat samples was in vitro methylated at CpG sites using CpG
methylase (SssI methylase) (NEB) as per the manufacturer's recommendations.
Two-step methylation-specific PCR (ATM and MLH1
loci).
For ATM and MLH1, a 2-step PCR approach was used.
Two-step PCR significantly boosted sensitivity and specificity of
single-step PCR and was particularly useful for islands where CpG sites
were relatively sparse. In the first round of PCR, most of the CpG island in each case was amplified from modified genomic DNA using primers that were insensitive to methylation, ie, did not include any
CpG sites. In the second round, a small amount of template from the
first reaction was used in PCR together with primers specific to
selected CpG sites. Table 1 provides the sequences for each set of
primers as well as cycling parameters for each reaction.
ABL1 sequencing of bisulfite-modified DNA.
In an attempt to amplify the ABL1 CpG island, we originally
designed primers flanking a 519-bp fragment corresponding to the sense
strand (after bisulfite modification, opposite strands are no longer
complementary and primers can be designed to amplify either strand).
However, we were unable to amplify the sense strand from samples
containing heavily methylated DNA, whereas any unmethylated DNA
component was readily amplifiable. We speculate that this may be due to
the complex conformation of heavily methylated molecules. The
ABL1 promoter is very rich in guanine residues that could easily form hard-to-resolve secondary structures with cytosine residues
remaining after bisulfite modification of a heavily methylated sample.
This hypothesis would predict that amplification of the opposite strand
should be readily achieved because the complementary cytosines should
transform to thymidine after bisulfite modification. Indeed, this was found to be the case and amplification of the antisense strand was subsequently used for analysis. Because
methylation of CpG sites is a symmetrical process, we were able to
infer methylation patterns for the sense strand by studying the
opposite strand.
DNA modified as described above was sequenced on a fluorescent
sequencer, either immediately after PCR or subsequent to a cloning step
using the T-vector kit (Invitrogen, Carlsbad, CA). Individual clones were screened for the correct insert by PCR before
sequencing. Primers and conditions for amplifying the ABL1 promoter antisense strand are given in Table 1.
Culture of colonies derived from peripheral blood hematopoietic
progenitors.
Mononuclear cells were aseptically isolated from peripheral blood and
cultured in semisolid medium as previously described.26
Methylation-specific PCR of colonies.
Individual colonies were harvested in 150 µL of phosphate-buffered
saline (PBS), spun at maximum speed for 5 minutes, and resuspended in
22.5 µL of water containing 1.5 µg of lambda DNA per colony.
Samples were heated at 95°C for 10 minutes and subsequently processed as per normal bisulfite modification protocol.
| |
RESULTS |
Methylation-specific PCR shows uniform methylation of the
Ph'-associated ABL1 allele in cell lines established from
blastic-phase CML.
Cell lines EM2, KBM5, BV173, and K562 were established from patients in
the blastic phase of CML. The first 3 lines contain Ph'
chromosomes but lack copies of normal chromosome 9. Therefore, the only
ABL1 alleles present are the ones contained within
BCR-ABL fusion genes. Methylation-specific PCR showed
complete and universal methylation of ABL1 promoters in each of
these cell lines; no unmethylated promoter alleles were detected. By
contrast, cell line K562 contains 3 Philadelphia chromosomes as well as
a normal chromosome 9 homologue. In K562, methylation-specific PCR
showed both methylated and unmethylated ABL1 sequences
(Fig 2), a result which indicates that
ABL1 methylation patterns in cell lines are likely to be
specific and not reflect the indiscriminate spread of methylation
during repeated passage. These observations suggest that methylation of
ABL1 in CML is restricted to the allele nested within the
Ph' fusion gene.
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| Fig 2.
Methylation-specific PCR shows ABL1 methylation
patterns in CML cell lines. NBC, normal donor buffy-coat DNA; SssI-NBC,
normal donor buffy-coat DNA treated in vitro with CpG methylase (SssI
methylase); EM2, DNA from CML cell line EM2 containing only a duplicate
of the Ph' chromosome and lacking a normal homologue of
chromosome 9; K562, DNA from cell line K562 which contains 3 copies of
the Ph' chromosome in addition to a chromosome 9 homologue; M,
primers specific for methylated DNA; U, primers specific for
unmethylated DNA;  x, x/HaeIII marker.
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Clinical samples from patients with acute-phase CML contain both
methylated and unmethylated ABL1 promoters.
In contrast to cell lines, samples from 15 patients in acute-phase CML
showed both methylated and unmethylated ABL1 in every case
(Fig 3). This could be accounted for by the
presence of a mixed cell population consisting of CML cells with
biallelic methylation of ABL1 promoters alongside residual
normal cells carrying only unmethylated ABL1. Alternatively,
this pattern may denote the coexistence of a methylated and an
unmethylated promoter allele in each CML cell.
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| Fig 3.
Methylation-specific PCR shows ABL1 methylation
patterns in clinical samples from acute-phase CML. NBC, normal donor
buffy-coat DNA; SssI-NBC, CpG methylase (SssI methylase)-treated normal
donor buffy-coat DNA; EM2, CML cell line EM2. Native DNA lane contains
DNA that was not modified by bisulfite. M, primers specific for
methylated DNA; U, primers specific for unmethylated DNA; x,
x/HaeIII marker.
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Colonies derived from single hematopoietic progenitors in acute-phase
CML contain both methylated and unmethylated ABL1 promoters.
To distinguish between mixed-cell population pattern and
allele-specific methylation, we examined the methylation status of individual colonies derived from hematopoietic progenitors. Peripheral blood mononuclear cells from patients at chronic phase and patients in
acute-phase CML were seeded in methylcellulose-containing semisolid cultures in the presence of 10% (vol/vol) conditioned medium from cultures of the human bladder carcinoma cell line 5637. This
conditioned medium contains hematopoietic growth factors that promote
the growth of myeloid colonies.26 After a 14-day
incubation, colonies were harvested, and their DNA extracted and
subjected to modification by the bisulfite treatment protocol (see
Materials and Methods). The results indicated that DNA of colonies from
patients in chronic-phase CML generally lacked methylation at the
ABL1 promoter region, whereas colonies from patients with
acute-phase disease always contained a methylated allele alongside an
unmethylated allele (Fig 4).
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| Fig 4.
ABL1 methylation-specific PCR on individual
colonies. Each colony was split in half and used for
methylation-specific PCR employing primers specific for methylated DNA
(M) or for unmethylated DNA (U).
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The vast majority of clonogenic cells in acute-phase CML contain
methylated ABL1 promoters.
The study of single-progenitor-derived colonies also gave insight into
the size of the clone-bearing methylated ABL1 promoters at
different phases of disease. In all patients in blastic phase studied,
nearly all of clonogenic cells contained methylated ABL1 promoters (Table 2). By contrast, the vast
majority of patients in the chronic phase of the disease demonstrated
absence of ABL1 methylation in all colonies studied. A patient
recently diagnosed to be in chronic-phase CML (LR) showed ABL1
promoter methylation in 6 of 12 colonies studied. Follow-up has been
very short in this case, but notably this patient has maintained
persistently high peripheral blood eosinophil counts and has shown
resistance to hydroxyurea treatment. In accelerated-phase disease,
patient DK demonstrated intermediate "methylated" clone size (2 of 10 colonies studied bore methylated ABL1). A second patient
with a clinical picture of accelerated disease at the time of sample collection (patient AF) had undergone complete methylation of the
ABL1 promoter in all peripheral blood progenitors; this patient very promptly progressed to frank blast crisis. These findings suggest that methylation of ABL1 is tightly linked to
clonal evolution of CML.
Sequencing of bisulfite-modified DNA shows methylation patterns in
the ABL1 promoter region in acute-phase CML.
Traditional assays for detection of methylation using
methylation-sensitive restriction enzymes allow a limited appreciation of the extent of methylation in each case because only those CpG sites
that happen to be part of the recognition sequence of one or the other
of these enzymes can be studied. However, sequencing of DNA after
bisulfite modification has the power to reveal the methylation status
of every CpG site within the region of interest. To determine the
extent of methylation of the ABL1 promoter in acute-phase CML,
we amplified by PCR and subsequently sequenced the ABL1
promoter-associated CpG island from cell lines KBM5, BV173, and EM2 as
well as 7 patients in both the accelerated and blastic phases of CML
(Fig 5). Because we anticipated mixed
methylation patterns in the clinical samples (see above), PCR fragments
were cloned before sequencing and several clones containing full-length inserts from each patient were studied. Our results showed that in all
cell lines, all CpG sites within the ABL1 promoter without exception had undergone methylation importantly, all non-CpG cytosines had undergone conversion to thymidine after bisulfite treatment. DNA
from clinical samples was also densely hypermethylated along the entire
length of the CpG island. In most clones, occasional CpG sites were
unmethylated in the context of a densely methylated CpG island, but
they were not generally constant among patients. Clones
containing unmethylated ABL1 alleles were uniformly free of
methylation along the entire length of the CpG island in all cases
(Fig 5).
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| Fig 5.
Methylation topology of the ABL1 promoter region.
The region amplified corresponds to coordinates 473 to +46 with
respect to the start of exon 1a or coordinates 37352 to 37871 of
GenBank sequence U07563.42 The relative position of dots on
the diagram corresponds to the relative position of CpG dinucleotides
along the island. Yellow dots indicate methylated CpG dinucleotides;
blue dots indicate unmethylated CpG dinucleotides. Amplification
products obtained from total peripheral blood and/or bone marrow
mononuclear cells were cloned and individual clones were sequenced on a
fluorescent sequencer. The designation "CML blast crisis cell
lines" refers to cell lines EM2, BV173, and KBM5. Patients 469 and
1457 were in accelerated-phase CML when samples were collected and
patient 424 was in blast crisis. For each patient, several clones of
methylated DNA are depicted alongside a representative clone of
unmethylated DNA.
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| Fig 6.
(A) Single-step methylation-specific PCR is shown for the
MSH6 locus. Lanes 1, 3, 4, 6, and 7: samples from patients in
acute-phase CML; lanes 2 and 5: samples from patients in chronic phase
CML; lane 8: normal donor buffy-coat DNA; lane 9: normal donor
buffy-coat DNA treated in vitro with CpG methylase (SssI methylase). M,
primers specific for methylated DNA; U, primers specific for
unmethylated DNA; x, x/HaeIII marker. (B) Two-step
methylation-specific PCR for the ATM promoter region. In the
first round, the CpG island is amplified from bisulfite-modified DNA
using primers that are insensitive to methylation (see Materials and
Methods). In the second round, primers specific for methylated DNA (M)
or unmethylated DNA (U) are used in PCR together with a small amount of
template from the first reaction (arrow). Lane 1: normal donor
buffy-coat DNA; lane 2: normal donor buffy-coat DNA treated in vitro
with CpG methylase (SssI methylase); lanes 3 through 6: samples from
patients in acute-phase CML. x, x/HaeIII marker.
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Both cell lines and clinical samples had previously been analyzed using
restriction enzyme-based methodology.17 These methods probed a solitary SacII site alongside 3 HpaII sites,
all clustered at the 3' end of the CpG island. All cell lines as
well as samples from 2 patients had shown methylation at all 4 enzyme
sites: bisulfite sequencing confirmed these results. The other 5 patients had shown lack of methylation at the HpaII sites while
the SacII site was methylated. Bisulfite sequencing confirmed
the methylation status of the SacII site in these patients;
however, it also demonstrated HpaII site methylation.
Therefore, HpaII analysis does not appear to be as sensitive as
SacII analysis for monitoring methylation in clinical samples.
Combined with the data obtained from study of progenitor-derived
colonies, the enhanced resolution of sodium bisulfite sequencing showed
that CML progression is characterized by an increase in the abundance
of fully methylated ABL1 promoters rather than by a linear
spread of methylation along the promoter region.
The ATM promoter remains free of methylation at all stages of
disease.
The promoter of the ataxia telangiectasia locus (ATM) was
examined by a modification of the classical methylation-specific PCR
protocol, "2-step MSP" (see Materials and Methods). In essence, this method is an adaptation of nested PCR protocols, where the first
amplification step encompasses most of the CpG island
using primers that are insensitive to methylation; ie, do not contain CpG dinucleotides and, therefore, equally amplify methylated and unmethylated sequences. It should be emphasized that primers are still
specific for bisulfite-modified DNA because thymidine is substituted
for all non-CpG cytosines. In the second PCR step, methylation-specific
primers are added to a small amount of template from the first step.
Using this approach, mononuclear cell samples from patients in
chronic-phase CML and patients in acute-phase CML as well as CML cell
lines (see above) were examined for methylation at 4 loci of the
ATM promoter region. The promoter was found to be free of
methylation in each of the 4 regions at all stages of disease
(Fig 6B, Table 3).
Promoters of mismatch repair genes MLH1, MSH2, and
MSH6 are unmethylated even in acute stages of CML.
Methylation-specific PCR was adapted to study the promoters of mismatch
repair genes MSH2, MSH6, and MLH1. For
MSH2 and MSH6, a classical, single-step MSP protocol
was used; to boost the specificity of detecting methylation of the
MLH1 promoter, 2-step MSP was developed for this gene. Our
panel of patients at chronic phase and patients in acute-phase CML as
well as CML cell lines was tested for methylation at 2 loci of the
MSH2 promoter, 2 loci of the MSH6 promoter, and 1 locus
of the MLH1 promoter. In all cases, all these loci were found
to be free of methylation in both chronic and acute stages of disease
(Fig 6A, Table 3).
| |
DISCUSSION |
The presence of a pathognomonic molecular abnormality in nearly all
cases as well as a rather invariable clinical course distinguish CML
from other myeloproliferative disorders. Despite significant advances
in the elucidation of the mechanisms responsible for disease initiation
and the molecular targets of the BCR-ABL tyrosine kinase, the events underlying transition to accelerated and blastic phases remain largely unknown.
We and others have shown that epigenetic alteration of the proximal
promoter of ABL1 appears to be specifically and consistently associated with progression of CML.16-19 Lack of
methylation in this region appears to be restricted to the chronic
phase whereas hypermethylation is observed when blast crisis has
evolved.16 Furthermore, long-standing chronic disease as
well as accelerated-phase disease are associated with intermediate
patterns of methylation, which can be reversed after treatment with
interferon-.17
Enzyme-based approaches to study methylation patterns in disease are
limited in many ways: Firstly, only a small proportion of CpG sites can
be studied and the possibility of patchy methylation cannot be
excluded. Secondly, the danger of false-positive results due to the
inability of restriction enzymes to digest is very real, given the fact
that many archival samples are rich in impurities that can inhibit
these enzymes. Thirdly, enzyme-based approaches result in the inability
to visualize unmethylated alleles because these are digested by the
enzyme and do not contribute to the subsequent PCR amplification. To
circumvent these problems, we adapted the techniques of
methylation-specific PCR and sequencing of sodium bisulfite-treated
DNA24 so as to study the promoter regions of ABL1
and other candidate genes.
We used the ability of methylation-specific PCR to reveal unmethylated
alleles to ask whether one or both ABL1 promoter alleles undergo methylation with disease progression. Whereas cell lines containing only Ph'-associated ABL1 alleles showed
uniform methylation, evidence of both methylated and unmethylated
promoter alleles was obtained from acute-phase CML clinical samples.
This may be attributable to the presence of a mixture of cell
populations: a leukemic population containing only methylated alleles
alongside a population of normal hematopoietic cells containing
unmethylated alleles; alternatively, it may indicate allele-specific
methylation. To distinguish between these possibilities, we studied
colonies derived from single hematopoietic progenitors grown in
semisolid medium. The results indicated that in acute-phase CML, each
and every progenitor carrying a methylated allele also carried an unmethylated allele. Thus, in acute-phase CML, methylation is likely to
be an allele-specific process. Given the evidence of specific
methylation of ABL1 in all 3 cell lines bearing only Ph'-associated ABL1 alleles, it would not be unreasonable
to assume that in clinical samples too the proximal ABL1
promoter nested within the Ph' fusion gene specifically undergoes
methylation in acute-phase CML. However, our data do not exclude the
possibility that in some patients the non-Philadelphia allele may
undergo methylation.
The study of colonies also enabled us to semi-quantitatively appreciate
the size of the methylated ABL1-bearing clone. This is very
difficult to infer from the study of uncloned populations where even a
few methylated ABL1 molecules can produce strongly positive
amplification results. Our data indicate that in blastic-phase CML, in
contrast to the chronic phase, the vast majority of clonogenic progenitors have undergone methylation. This observation indicates that
ABL1 promoter methylation is likely to be an inherent step in
the evolution of the disease.
Sequencing of bisulfite-modified DNA from cell lines and patients in
acute-phase CML was used to investigate the spatial degree of DNA
methylation. In both cell lines and patient samples, nearly all CpG
sites in the promoter region were methylated. Despite the fact that in
acute-phase CML methylation appears to be a fairly uniform process, it
is conceivable that study of methylation topology at earlier stages of
disease may show conserved patterns by which methylation spreads to
affect the whole CpG island.
Furthermore, we addressed the degree of methylation of genes other than
ABL1. Among a large choice of genes to examine for methylation,
we chose to concentrate on the DNA mismatch repair genes MSH2,
MSH6, and MLH127-33 as well as the
ataxia-telangiectasia locus, ATM.34,35 Investigation of the
methylation pattern of mismatch repair genes might help to resolve the
current controversy concerning the type of mutator phenotype observed
in acute-phase CML36,37 because loss of MSH6
expression has been linked to mutator phenotype without microsatellite
instability,38 whereas loss of MSH2 expression is
associated with replication errors at these repeat
loci.28-31 The product of the ataxia-telangiectasia locus
(ATM) was found to constitutively interact with the
ABL1 tyrosine kinase and activate the latter upon
radiation-induced DNA damage.9,39 We reasoned that a
reciprocal relationship may exist in which methylation of at least one
member of the ABL1/ATM pair is required for CML evolution.
After a comprehensive search in samples from patients at all stages of
disease, we concluded that the promoters of mismatch repair genes as
well as ATM remain free of methylation even in acute stages of
disease. These findings, together with the previously reported absence
of methylation of the p16 and p15 genes in acute-phase
CML,40 suggest that ABL1, distinctly among DNA
repair and genotoxic stress-response genes, undergoes epigenetic
modification in this disorder.
Methylation of the ABL1 promoter appears to closely correlate
with CML progression: both the proportion of patients with methylated ABL1 and the numbers of progenitors bearing methylated
sequences in individual patients correlate with disease progression to
more aggressive forms. These data are compatible with 2 models
addressing the origins of ABL1 methylation. In the first,
methylation may be a stochastic event leading to clonal expansion: a
single CML progenitor may undergo methylation of ABL1 and/or
other critical gene promoters and acquire a growth advantage, with
subsequent clonal expansion and establishment of blastic
transformation, either directly or following accumulation of secondary
mutations. Alternatively, methylation may serve as a
molecular "clock," counting the time that has elapsed from the
initiating mutation, the generation of the BCR-ABL
fusion gene. In the latter model, methylation of the proximal promoter
of ABL1 may be an inevitable consequence of juxtaposition to
upstream Alu-rich BCR sequences, acting as "methylation
centers."41 Progenitors having survived long enough will
have undergone ABL1 methylation; they will also have had the
time to accumulate further mutations, thereby acquiring a more
aggressive phenotype that eventually would lead to the establishment of
blastic transformation.
ABL1 methylation appears to be an early and nearly universal
epigenetic alteration with effects integral to CML progression. BCR-ABL
and ABL appear to exert opposing and antagonistic actions at the level
of apoptosis, cell-cycle progression, and genetic instability.25 BCR-ABL is the malignant
counterpart, promoting cell-cycle progression and genetic instability
and inhibiting apoptosis, whereas the opposite roles have been
attributed to ABL. It is conceivable that a balance between the two,
maintaining the clinically benign chronic phase of CML, may be
dependent on continued transcription from the proximal ABL1
promoter. Silencing of the internal ABL1 promoter by
methylation may tip the balance in favor of BCR-ABL and provide the
ground for resistance to apoptosis and genomic instability that
characterize blastic transformation. Interferon therapy appears to
reestablish this balance leading to prolongation of the chronic
phase.17 In the future it will be fascinating to test this
hypothesis in animal models and provide new avenues for gene therapy of
this common and fatal form of leukemia.
| |
ACKNOWLEDGMENT |
The authors thank Aviva Simberger and Aliza Treves for valuable help
with colony assays, Dr Reuven Orr for provision of clinical samples,
and Dr Zahava Siegfried-Kluger for critical review of the manuscript.
| |
FOOTNOTES |
Submitted February 23, 1999; accepted May 25, 1999.
Supported by grants from the Golda Meir Fellowship Fund (to F.A.A.),
the Gabriella Rich Leukemia Fund, and the Office of the Chief Scientist
of the State of Israel (to D.B.-Y.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dina Ben-Yehuda, MD, Department of
Hematology, Hadassah University Hospital-Ein Karem, Jerusalem 91120, Israel; e-mail: dbyehuda{at}hadassah.org.il.
| |
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ABSTRACT
INTRODUCTION