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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-04-1010.
NEOPLASIA
From the Department of Molecular Oncology and the
Department of Hematology/Oncology, Research Institute for Radiation
Biology and Medicine, Hiroshima University, Hiroshima,
Japan.
Somatically acquired point mutations of AML1/RUNX1 gene
have been recently identified in rare cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Moreover, germ line mutations
of AML1 were found in an autosomal dominant disease, familial platelet
disorder with predisposition to AML (FPD/AML), suggesting that AML1
mutants, as well as AML1 chimeras, contribute to the transformation of
hematopoietic progenitors. In this report, we showed that AML1 point
mutations were found in 6 (46%) of 13 MDS patients among atomic bomb
(A-bomb) survivors in Hiroshima. Unlike acute or chronic leukemia
patients among A-bomb survivors, MDS patients exposed relatively
low-dose radiation and developed the disease after a long latency
period. AML1 mutations also were found in 5 (38%) of 13 therapy-related AML/MDS patients who were treated with alkylating
agents with or without local radiation therapy. In contrast, frequency
of AML1 mutation in sporadic MDS patients was 2.7% (2 of 74). Among
AML1 mutations identified in this study, truncated-type mutants lost
DNA binding potential and trans-activation activity. All
missense mutations with one exception (Gly42Arg) lacked DNA
binding ability and down-regulated the trans-activation
potential of wild-type AML1 in a dominant-negative fashion. The
Gly42Arg mutation that was shared by 2 patients bound DNA even more
avidly than wild-type AML1 and enhanced the
trans-activation potential of normal AML1. These results
suggest that AML1 point mutations are related to low-dose radiation or
alkylating agents and play a role distinct from that of leukemogenic
chimeras as a result of chromosomal translocations caused by sublethal
radiation or topoisomerase II inhibitors.
(Blood. 2003;101:673-680) Genes encoding transcription factors that play
critical roles in hematopoiesis are frequently involved in the genetic
alterations in leukemia and myelodysplastic syndrome (MDS). A good
example is AML1/RUNX1/CBFA2, which encodes a component of
the AML1-core binding factor In addition to chromosomal translocations, recent studies have
identified several somatically acquired point mutations of the
AML1 gene that occurred exclusively within the DNA binding runt homology domain (RHD) in sporadic AML/MDS
patients.8,9 Because of the low frequencies of point
mutations (0%-7.1%) and the divergent effects of these mutants on the
trans-activation activity of the AML1-CBF The AML1 gene also was reported as a target of gene
alteration by ionizing radiation (IR) and anticancer drugs in
experimental systems.17,18 Moreover, human leukemias
associated with AML1 gene translocations after anticancer
therapy or low-dose radiation have been reported,19,20
although the role of radiation in these patients is
controversial.21 In this report, we tested the frequency
of point mutations in the AML1 gene in MDS patients among
atomic bomb (A-bomb) survivors in Hiroshima, as well as in
therapy-related MDS/AML. We found unexpectedly high frequencies of
AML1 mutations in these patients, suggesting that radiation and anticancer drugs contribute to the development of MDS/AML through
mutations of the AML1 gene.
Patients' materials
Polymerase chain reaction-single-strand conformation
polymorphism (PCR-SSCP)
Identification of AML1 mutations PCR products that showed abnormal bands were subcloned into a pCR2.1 vector (Invitrogen, Carlsbad, CA), and 8 independent clones were sequenced in both directions using BigDye Terminator Cycle sequencing kit (Perkin-Elmer) and analyzed on ABI Prism 310 Genetic Analyzer (Perkin-Elmer). To confirm mutations, PCR products from cDNA were also sequenced. First-strand cDNA was synthesized using total RNA and random hexamers with SuperScript II reverse transcriptase (Gibco). The cDNA products were amplified with the following primers: 5'-GCAGGGTCCTAACTCAATCG-3'/5'-GCTCGGAAAAGGACAAGCTC-3', and subcloned PCR products were sequenced as described above.Cell culture and transfection The cell lines Cos-7 and HeLa were cultured in Dulbecco modified Eagle medium (Gibco), and U937 in RPMI 1640 (Gibco) supplemented with 10% fetal calf serum, 2 mM glutamine at 37°C in a humidified atmosphere with 5% CO2. Cos-7 cells were transfected using SuperFect (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For reporter assay, HeLa cells were transfected by the calcium phosphate precipitation method25 and U937 cells by Effectene (Qiagen).Plasmid constructions The entire coding regions of wild-type AML1 or CBF generated
by PCR using Pfu polymerase (Stratagene, La Jolla, CA), were subcloned into pcDNA3.1 expression vector (Invitrogen). PCR-generated fragments encoding AML1 or AML1 mutants with an N-terminus FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) epitope were also subcloned into pcDNA3.1 vector. The integrity of the amplified sequence was
confirmed by DNA sequencing. A reporter plasmid containing a macrophage
colony-stimulating factor receptor (M-CSFR) promoter (pM-CSF-R-luc)26 was kindly provided by Dr D. Zhang (Beth
Israel Hospital and Harvard Medical School).
Immunoprecipitations and Western blot analysis Cos-7 cells were transfected using Superfect (Qiagen) with 5 µg of pcDNA3.1-CBF and 5 µg FLAG-tagged AML1 or AML1 mutants expression plasmid. After 24 hours, the cells were lysed in the lysis
buffer (20 mM Tris[tris(hydroxymethyl)aminomethane]-HCl, pH
7.5; 150 mM NaCl; 1% Nonidet P40; 1 mM phenylmethlsulfonyl fluoride
(PMSF); 1 µg/mL leupeptin). The lysates were sonicated and then
incubated with protein G (Pharmacia Biotech) to block nonspecific
binding proteins. A portion of each lysate was removed for immunoblot
analysis. A 20-µL volume of a 50% slurry of anti-FLAG M2 beads
(Sigma, St Louis, MO) was added to the lysates, incubated for 4 hours
at 4°C, and washed 3 times with lysis buffer. FLAG beads were blocked
in phosphate-buffered saline (PBS) containing 1% bovine serum albumin
prior to addition to the lysates. For Western blot, the lysates or the
supernatants from immune complex beads after boiling in Laemmli buffer
were separated by 15% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis gel and transferred to Hybond ECL (Amersham
Pharmacia Biotech). The membrane was blocked in 5% nonfat milk in
PBS-T (0.1% Tween-20) and hybridized sequentially with primary
antibodies and a horseradish peroxidase-conjugated secondary antibody
(Amersham Pharmacia Biotech). The primary antibodies used in this study
were anti-M2 antibody (Sigma), anti-AML1 polyclonal antibody (Oncogene
Research Products, Boston MA), and anti-CBF polyclonal antibody
(Oncogene Research Products). Bound antibodies were detected by
enhanced chemiluminescence (ECL) using a Western blotting kit (Amersham
Pharmacia Biotech).
Electrophoretic mobility shift assay Nuclear extracts from Cos-7 cells, which were transiently transfected with the corresponding expression plasmid, were prepared as described previously.27 Protein concentrations were determined with Bradford reagents (Bio-Rad, Hercules, CA). Electrophoretic mobility shift assay (EMSA) was performed as previously described.28 DNA binding reactions were prepared in a buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 1 mM MgCl2, 0.1 mM ethylene glycol-bis (-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA),
0.4 mM dithiothreitol, 40 mM KCl, and 60 µg of salmon sperm DNA per
milliliter. Annealed oligomers containing the AML1 binding site were
labeled with  -[32P]dATP (deoxyadenosine triphosphate;
Amersham Pharmacia Biotech) in a standard Klenow reaction mixture. For
competition studies, 100 ng unlabeled, annealed oligomers containing
the wild-type (TGTGGT) or mutated (TGTTAG) AML binding site was added
into the DNA binding reaction mixtures. For supershift analyses, 1 µg
of AML1 antibody was used.
Transcriptional assay HeLa cells were transiently transfected by calcium phosphate precipitates containing the luciferase reporter plasmid pM-CSF-R-luc, pcDNA3.1-CBF, and pcDNA3.1-FLAG-tagged AML1 or AML1 mutants
expression plasmid. The total amount of plasmid for each transfection
was adjusted to 9.25 µg by adding empty expression plasmid. Cells were harvested and lysed 48 hours after transfection, and luciferase assays using a luminometer Fluroskan Ascent (Labsystems, Helsinki, Finland) were performed by Dual luciferase assay systems (Promega, Madison, WI) according to the manufacturer's instructions. Results of
the reporter assays represented the average values for relative luciferase activity generated from 3 independent experiments that were
normalized using the activity of pRL-tk (Promega) as an internal control. To analyze functional competitions between wild-type AML1 and
mutants, U937 cells were transfected with pM-CSF-R-luc, wild-type AML1,
and AML1 mutants expression plasmid using Effectene (Qiagen). Empty
plasmid was added to bring the total amount of DNA per transfection to
2.8 µg. Luciferase assays were performed as described above.
High frequency of AML1 mutations in radiation-associated and therapy-related MDS/AML To investigate the AML1 mutations in radiation-associated and therapy-related MDS/AML, we analyzed exons 3 through 5 (corresponding to amino acid 1 to 177) of the AML1 gene by PCR-SSCP assay using genomic DNA extracted from mononuclear cells in the bone marrow of patients. Mutations were further confirmed by sequence analysis of RT-PCR products. AML1 mutations were found in only 2 (2.7%) of 74 sporadic MDS patients, in accordance with previous studies8-11 (Table 1). In contrast, AML1 mutations were identified in 6 (46%) of 13 MDS cases, who had been within 3 km of the hypocenter of the atomic bomb explosion in Hiroshima (see "Patients and methods"). The frequency of AML1 mutations in MDS patients among A-bomb survivors was significantly higher than that in sporadic MDS cases (P < .0001). The clinical findings of these patients are summarized in Table 2. Unlike acute and chronic leukemia patients among A-bomb survivors, only 3 (22%) of 14 MDS patients had been within 1 km of the hypocenter. Two cases had silent mutations (Pro157syn and Thr101syn), 3 cases had missense mutations (Gly42Arg, Asp171Asn, and Gly42Arg), and one case had a frame shift/nonsense mutation (Ser70fsTer93). Of sporadic MDS patients with AML1 mutations, one patient (case 7) had a past history of manufacturing a poison gas (mustard gas) during World War II.
Next, we analyzed the mutations of therapy-related MDS/AML cases.
Missense mutations were found in 2 (33%) of 6 cases (Table 3). A refractory anemia with excess of
blasts in transformation (RAEBt) patient (case 9) after
chemotherapy with mainly alkylating agents and a long period of
high-dose radiation therapy for astrocytoma had a missense mutation
(Arg177Gln), and the other patient (case 10, Gly138_Arg139insGlyGly), who had been exposed to the A-bomb at
a point 4.0 kilometers from the hypocenter, developed AML M0 after
intensive multidrug chemotherapy and radiation therapy for B-cell
lymphoma.
Finally, of the 7 patients who developed AML after myeloproliferative
disorders (MPD) or other hematologic diseases, 3 patients (43%) had
missense or nonsense mutations (Table 4).
These 3 patients were treated with busulfan, while none of the other 4 patients received this alkylating agent. Case 11, who developed AML
after myelofibrosis, had been treated with radiation therapy for
splenomegaly. Cases 11 and 12 had had no mutations and chromosomal
abnormalities in the chronic phase of MPD. No AML1 mutations were found
in 47 MPD and 20 CML patients in the chronic phase.
The AML1 mutations identified in our study are summarized in
Figure 1. We observed virtually equal
intensities of normal and shifted bands in the PCR-SSCP analysis of all
the samples with AML1 mutations and obtained comparable frequencies of
normal and mutated results in the sequence analysis. In addition, germ
line genomic DNA sequences were examined in specimens from nonleukemic organs from 6 cases (cases 4, 5, 6, 9, 10, and 12) and found to be
normal (data not shown), suggesting that mutations of the
AML1 gene were monoallelic at the somatic level. Mutations
of the CEBPA gene, which encodes the transcription factor
CCAAT/enhancer binding protein
Abilities of AML1 mutants to bind DNA and to heterodimerize
with CBF . To analyze the DNA binding ability of AML1 mutants, a
radiolabeled oligonucleotide probe containing the consensus binding
sequence for AML1 and nuclear extracts from Cos-7 cells transfected
with AML1 mutants were used in electromobility shift analysis (EMSA). A
DNA/protein complex was detected when using nuclear extract from a
transfectant expressing FLAG-tagged wild-type AML1 (Figure 2A, lane
2) that was not observed when using an
extract from the mock transfectant (lane 1). This complex was
supershifted with a specific serum against AML1 (lane 3) and was
competed for by the nonradiolabeled oligonucleotide containing AML1
binding site (lane 4), but was not competed for by those containing a
mutated AML1 binding site (lane 5), indicating that the complex
contains AML1. The complex was not detected using extracts from the
transfectants of AML1 mutants that occurred in RHD (lanes 6-8 and
10-13), while the intensity of complex was enhanced using an extract
from the transfectant expressing the Gly42Arg mutant (lane 9),
indicating that all the AML1 mutants except Gly42Arg lack
DNA-binding potential.
To test whether AML1 mutants are able to interact with CBF Transcriptional potential of AML1 mutants To investigate the transcriptional activities of the AML1 mutants, reporter experiments were performed using the promoter of M-CSFR, which is known to be transcriptionally regulated by AML1.26 When wild-type AML1 and CBF were cotransfected
in HeLa cells, the promoter activity was induced 8-fold compared to
transfection with CBF alone (Figure 3A, lanes 1 and
2). In contrast, none of the nonsilent
mutants except Gly42Arg induced significant trans-activation (lanes 3-10), in accordance with their DNA binding abilities (Figure 2A). To examine whether AML1 mutants act as dominant negative inhibitors of wild-type AML1, we performed the same reporter assay using U937 monocytic cells, in which the activity of the M-CSFR promoter was trans-activated by transfecting AML1 alone in a
dose-dependent manner (Figure 3B). When the truncated type AML1 mutants
(mutants 1 to 3) were cotransfected with wild-type AML1, the promoter
activities were not affected. In contrast, 4 missense type AML1 mutants
(mutants 6, 8, 9, and 10), which lack the DNA-binding ability but
retain the potential to bind CBF (Figure 2A-B), suppressed the
trans-activation activity of wild-type AML1 in a
dose-dependent fashion. As expected, the Gly42Arg mutant induced the
promoter activity more efficiently than wild-type AML1. Thus, AML1
mutants identified in MDS/AML patients had significantly different
effects on the same M-CSFR promoter.
In this paper, we report that AML1 point mutations were detected in virtually half of late-onset MDS patients among the A-bomb survivors of Hiroshima. AML1 was also frequently mutated in MDS/AML patients after treatments that contain alkylating agents and/or local radiation for nonhematopoietic malignancies or MPD. Although we performed PCR-SSCP analysis under optimized conditions, we can't exclude the possibility that some mutants were not detectable by our method. Therefore, the actual mutation frequency could be higher. The incidence of acute and chronic leukemia among A-bomb survivors was
sharply dose-dependent, and the leukemia developed after a short
latency period. Leukemias appeared after a minimum latency period of
2-3 years, reached a maximum after 6-7 years, and then decreased slowly
with time (Figure 4).29 In
contrast, the dose-dependency of the risk of other tumors was less
prominent, and the incidence of these diseases increased after long
minimum latency periods of 10 or more years and continued to increase with time.29 Most of the MDS patients in our study
received a low radiation dose judging by their proximity to the
hypocenter, as shown in Table 2. Indeed, with the exception of 3 cases,
the estimated radiation dose of these patients was below 50 cGy, according to the DS86.22,23 This apparent difference
in the distribution of radiation dose between leukemia and late-onset
MDS among A-bomb survivors may be interpreted, at least in part, by
molecular mechanisms that contribute to the transformation of
hematopoietic progenitors. Leukemogenic fusion genes as a result of
nonrandom chromosomal translocations are detected in approximately half
of acute leukemia patients30 but are relatively rare in
MDS cases,31 many of which are generally considered
to develop as a result of the accumulation of gene deletions and
point mutations.32 Thus, chromosomal translocations caused by double-strand DNA breaks resulting from high dose radiation are likely to contribute to the development of acute and chronic leukemia after a short latency time, whereas point mutations of genes
induced by low-dose radiation may contribute to the development of MDS
among A-bomb survivors decades later.
This idea is supported by a report that investigated
"innocent" point mutations in hematopoietic progenitors of healthy
A-bomb survivors. Langlois et al33 reported the increased
frequency of somatic cell mutations at the glycophorin A (GPA) locus in red blood cells (RBCs) among A-bomb survivors. Although the variant frequencies (VFs) at the GPA locus increased in a dose-dependent fashion among survivors exposed to low-dose exposure (< 1.7 Gy), large
fluctuations in VFs were seen for moderate-dose survivors (1.7 to 5 Gy), and VFs were uniformly low for high-dose survivors (> 5 Gy). They
interpreted these apparently paradoxical results in terms of the
numbers of surviving hematopoietic stem cells, which decrease
exponentially with the radiation dose. Because a low-dose exposure of
50 cGy would produce minimal cell kill and induce a VF of around
10 × 10 A question may be raised that if the MDS patients among A-bomb survivors in this study acquired their AML1 mutations at the time of exposure, then 50 years is too long to develop MDS. The pedigrees of FPD/AML may provide the answer.11,12 AML is mainly an adult-onset disease in FPD/AML patients, suggesting that the accumulation of additional gene alterations required to transform hematopoietic progenitors takes decades, even though all stem cells of FPD/AML patients harbor AML1 gene mutation. Thus, it is not surprising that one stem cell that acquired AML1 mutation took a half century to develop MDS. Alkylating agents and topoisomerase II inhibitors are 2 major drugs that would induce distinctive therapy-related MDS/AML (t-MDS/AML). Patients who develop AML after exposure to alkylating agents mostly have long latency periods, and AML is often preceded by a myelodysplastic phase. Deletion or loss of chromosome 5 or 7,35 as well as mutations of genes such as p53, are frequently observed.36 In contrast, AML cases after exposure to topoisomerase II inhibitors typically have a short latency time without prior MDS and harbor balanced translocations involving chromosome bands 3q26, 11q23, and 21q22.35 All t-MDS/AML patients in our study who had an AML1 mutation were treated with alkylating agents, with or without local radiation, and had relatively long latency periods (range, 60-182 months; median, 123 months) without chromosomal translocations, suggesting that there is a close relation between AML1 mutation and t-MDS/AML associated with alkylating agents, although further large studies are necessary to confirm this. Also, it remains to be established whether AML1 mutations in t-AML secondary to MPD were induced in normal hematopoietic progenitors by alkylating agents or were acquired in abnormal progenitors as a second hit during the progression from preleukemic phase to overt leukemia. The crystal structure of the runt domain-CBF
Although almost all the missense and truncated-type mutants of AML1 in the previous reports and in this paper lose DNA binding potential, and thus lack the trans-activation activity when tested by reporter assay using the M-CSFR promoter (Figure 3A), the missense mutations down-regulated the trans-activation potential of wild-type AML1 in a dominant-negative fashion, while the truncated-type mutations have no such effect (Figure 3B). This difference is important, because normal AML1 protein is expected to be expressed in MDS/AML cells because the normal allele for AML1 gene is usually retained. On the other hand, 2 missense mutations, His58Asn and Gly42Arg, bind DNA even more avidly than wild-type AML1 and enhance the trans-activation potential of normal AML1 (Osato et al8 and Figure 3B). These somewhat puzzling findings could be explained by the choice of promoter in the reporter assay, because M-CSFR is unlikely to be the true target gene of AML1 mutants in the transformation process of hematopoietic progenitors. Alternatively, subtle dysregulation of the trans-activation activity of AML1, even up-regulation or down-regulation, might provide a growth advantage to hematopoietic cells. In this regard, 2 silent mutations (Thr101syn, Pro157syn) identified in this study would have pathologic significance. Although they may most simply be taken to represent a polymorphism, we did not find any other silent mutations among more than 300 samples. Thus, we assumed that these mutations might somehow affect the expression levels of AML1 protein through a posttranscriptional mechanism. Because no mutations were detected in the C-terminal portion of AML1 (amino acid 178 to 453) in previous studies,8,11 we initially limited our SSCP analysis to the RHD. However, if subtle alteration of AML1 function could result in leukemogenic potential, mutations may exist also in the C-terminal portion that mediates trans-activating potential. Indeed, a substantial number of nonsense or insertion/deletion mutations were found in the C-terminal portion (amino acid 215 to 507) of AML3/RUNX2/CBFA1, another member of the Runx transcription factor family that shares the RHD, in patients with a hereditary bone disease, cleidocranial dysplasia, which is caused by mutation of AML3 mainly in the RHD (Otto et al37). Thus, we extended our efforts to detect mutations in the C-terminal portion of AML1 in AML/MDS patients. We found a few patients with insertion/deletion mutants that resulted in frameshift mutations, although no mutations were found in A-bomb survivors (data not shown). Careful functional analysis of mutations in the AML1 gene should be performed to clarify these unsolved questions using in vitro and in vivo experimental systems. To develop the latter, mice carrying mutated AML1 genes are currently being established in our laboratory.
The authors are grateful to Dr D. Zhang (Beth Israel Hospital and Harvard Medical School) for providing pM-CSF-R-luc vector, and Dr H. Hirai (University of Tokyo) for providing anti-AML1 antibody. Emi Kurita, Reina Matsumoto, and Ryoko Yamaguchi provided excellent technical support for these experiments. We also thank Dr Kiyoshi Miyakawa for helpful discussions and critical reading of the manuscript.
Submitted August 20, 2002; accepted August 21, 2002.
Prepublished online as Blood First Edition Paper, September 5, 2002; DOI 10.1182/blood-2002-04-1010.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hironori Harada, Department of Molecular Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan; e-mail: herf1{at}hiroshima-u.ac.jp.
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© 2003 by The American Society of Hematology.
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T. J. Ley, P. J. Minx, M. J. Walter, R. E. Ries, H. Sun, M. McLellan, J. F. DiPersio, D. C. Link, M. H. Tomasson, T. A. Graubert, et al. A pilot study of high-throughput, sequence-based mutational profiling of primary human acute myeloid leukemia cell genomes PNAS, November 25, 2003; 100(24): 14275 - 14280. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
6, the number of stem-cell mutations per A-bomb
survivor would be 10 to 100, while an exposure of 8 Gy would reduce the
stem-cell pool to about 102-103 cells, so that
even with an expected VF of 200 × 10