New mechanisms of AML1 gene alteration in hematological malignancies


The human AML1 gene (also named CBFA2 or RUNX1), located in the 21q22 chromosomal band, encodes for one of the two subunits forming a heterodimeric transcription factor, the human core binding factor (CBF). AML1 protein contains a highly evolutionary conserved domain of 128 amino acids called runt domain, responsible for both heterodimerization with the β subunit of CBF and for DNA binding. AML1 is normally expressed in all hematopoietic lineages and acts to regulate the expression of various genes specific to hematopoiesis playing a pivotal role in myeloid differentiation. AML1 is one of the genes most frequently deregulated in leukemia through different mechanisms including translocation, mutation and amplification. Translocations lead to the formation of fusion genes encoding for chimerical proteins such as AML1-ETO which induces leukemogenesis. Recently, new mechanisms of AML1 deregulation by point mutations or amplification have been reported. To our knowledge, 51 patients (among 805 studied) with AML1 point mutations have been described. Forty of them have acute myeloid leukemia (AML) most often M0 AML. In this subtype of AML, the frequency of AML1 mutation is significantly higher; 21.5% of patients mutated (34/158). Mutations have also been found with lower frequency in other FAB subtype AML (6 cases), in myeloproliferative disorders (6 cases), in myelodysplastic syndrome (3 cases) and rarely in acute lymphoblastic leukemia (1 case). AML1 gene amplification has been found essentially in childhood ALL (12 cases) and more rarely in myeloid malignancies (4 cases). Here, we reviewed all these cases of AML1 point mutations and amplification and focused on the mechanisms of AML1 deregulation induced by these alterations.


The human AML1 gene (also named CBFA2 or RUNX1), located in the 21q22 chromosomal band, encodes for one of the two subunits forming the human core binding factor (CBF).1,2,3 AML1 protein contains a highly evolutionary conserved domain of 128 amino acids (aa) called runt domain, responsible for both heterodimerization with the β subunit of CBF and for DNA binding.4,5,6 AML1 is normally expressed in all hematopoietic lineages and acts to regulate the expression of various genes specific to hematopoiesis including the macrophage colony-stimulating factor receptor (M-CSFR), interleukin 3 (IL3), myeloperoxydase (MPO) and TCR β genes.7,8,9,10 Mice lacking AML1 or CBFβ have no fetal liver hematopoiesis, showing that the heterodimeric complex CBF is essential for definitive hematopoiesis of all lineages.11,12,13

AML1 is one of the genes most frequently deregulated in leukemia, mainly through translocations, point mutations and amplifications. Translocations of he AML1 gene occur in different leukemia subtypes, leading to the formation of fusion genes encoding for chimerical proteins including AML1-ETO (t(8;21)) in acute myeloid leukemia (AML), AML1-ETV6 (t(12;21)) in childhood acute lymphoblastic leukemia (ALL) and less often AML1-MDS1 (t(3;21)) in myelodysplastic syndrome (MDS) and blastic phase of chronic myeloid leukemia (CML), or other rare translocations.14,15 Many studies have demonstrated the role of these chimeric products in leukemogenesis. For example, by AML1-ETO knock in strategy it was shown that mice heterozygous for AML1-ETO had a similar phenotype to mice lacking AML1 or CBFβ indicating that AML1-ETO blocked normal AML1 functions.16 It has also been shown that the coiled-coil region of ETO could oligomerize with AML1-ETO protein, thus recruiting the nuclear corepressor N-CoR responsible for transcriptional repression of AML1 target genes and resulting in impaired differentiation of primary hematopoietic precursors.17,18

Recently, we and others have reported other mechanisms of inactivation of AML1 in hematological malignancies, through point mutations of the gene in AML and in MDS19,20,21,22,23,24,25 or through gene amplification,26,27,28,29,30,31,32 a rare event mainly observed in acute lymphoblastic leukemia (ALL). In this review, we focused on those two other mechanisms of AML1 deregulation in hematological malignancies.


In our previous paper, we analyzed 300 patients. Because all the mutations detected are observed in M0 AML and myeloid malignancies with acquired trisomy 21, we focused our new screening especially on these hematological disorders including 27 new M0 AML, and eight new myeloid disorders with acquired trisomy 21. In addition, we also analyzed 16 paired bone marrow samples (diagnosis and progression in AML) of myelodysplastic syndrome patients because we observed absence of mutation in this disorder in our previous paper and Imai et al21 reported two mutations in 37 patients. We also studied 15 bone marrow samples from M7 AML. Finally, we studied 30 cases with myeloproliferative disorders (MPD) including 18 essential thrombocythemia and 12 atypical CML because in our first report, the majority of the mutated patients, excluding M0 AML presented myeloproliferative disorders, but at this time, we focused our screening only on patients with acquired trisomy 21. Diagnosis of AML and MDS was based on morphologic and immunophenotypic analysis according to French–American–British (FAB) criteria.33 All patients had given informed consent.


Detection of AML1 gene runt domain mutations was performed firstly on DNA extracted from bone marrow by single-strand conformation polymorphism (SSCP) analysis of exons 3 to 5 of the AML1 gene, as previously described.20 PCR was performed in a total reaction volume of 20 μl that contained 50 ng DNA, 2.5 mmol/l MgCl2, 0.3 μmol/l of each primer, 10 mmol/l Tris HCl, 50 mmol/l KCl, 200 μmol/l of each dNTP (Pharmacia, Stockholm, Sweden), 1 U of Taq DNA polymerase (Promega, Charbonnières, France), and 1 μCi 32P-labeled deoxycytidine trisphosphate. The size of the PCR products was 312 base pairs (bp), 249 bp and 246 bp for exons 3 to 5, respectively. Direct sequencing was performed in all cases with abnormal SSCP profiles using the ABI protocol for Taq-Dye Terminator Sequencing on an automated ABI377 sequencer. Sequences were analyzed with the Sequence Analysis software V3.3 and the Sequence Navigator software V1.0.1 (Applied Biosystems, Foster City, CA, USA). Sequencing was performed on both strands.

In addition, to exclude any mutations outside of the runt domain as described by Yeoh et al,24 we performed direct sequencing on the complete coding sequence of AML1 cDNA, in the cases where higher frequency of AML1 mutation have been reported, ie M0 AML, hematological malignancies with acquired trisomy 21, or in patients with AML1 amplification. The coding sequence of the AML1 cDNA was sequenced as previously reported.34 Briefly, total RNA was extracted from frozen aliquots of 107 bone marrow cells with Trizol reagent (Invitrogen, Life Technologies, Karlsruhe, Germany) according to the manufacturer's instruction. RNA pellets were resuspended in 10 μl of RNAse-free water and quantity was estimated by ultraviolet spectrofluorometry. cDNA was synthesized from 1 μg of total RNA in a 20 μl reaction mixture as previously described.34 Three PCR amplifications were performed in parallel; each containing 2 μl of cDNA (corresponding to 100 ng of total RNA), 1× TaqGold reaction buffer (Applied Biosystems), 1.5 mM MgCl2, 250 μM each dATP, dCTP, dGTP, dTTP (Pharmacia), 0.5 U of AmpliTaq Gold polymerase (Applied Biosystems) and 50 pmol of each primer. Thermocycling conditions used were 12 min at 94°C followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 minute and a final extension step of 5 min at 72°C. After purification on QIAquick PCR purification columns (Qiagen, Hilden, Germany), 577 bp, 465 bp and 426 bp PCR fragments were sequenced using the ABI protocol for Taq-Dye Terminator Sequencing on an automated ABI377 sequencer.

Point mutations of the AML1 gene

In the last 2 years, 41 cases of hematological malignancies with acquired point mutations of AML1 gene have been reported to our knowledge, and we here report 10 additional new cases. Thus, point mutations of the AML1 gene have now been reported in 51 of the 805 patients with hematological malignancies studied (6.4%). Those 805 patients included 414 AML (of which 158 were M0AML), 169 MDS, 93 ALL and 129 MPD including 92 blast crisis CML. The 51 cases of mutations were observed in 40 AML (including 34 M0AML) three MDS, two ALL and six MPD (including 2 blast crisis CML) (Table 1).

Table 1 AML1 gene mutations in hematological malignancies

Mutations and type of hematological malignancy

Acute leukemia:

Of the 93 ALL studied, two had AML1 point mutation. One of them was a silent mutation (ATC→ATA)19 and the other25 was a frame shift mutation inducing premature chain termination.

Of the 414 AML studied, 40 (10%) had AML1 gene point mutation. All mutated cases were newly diagnosed AML except for one of them, with a M3AML in relapse. Mutations were found in 34 of 158 M0 AML (21.5%), none of 32 M1AML, two of 63 M2AML (3.2%), one of 14 M3 AML (7%), one of 52 M4 AML (2%), one of 32 M5 AML (3.1%), none of two M6 AML and one of 36 M7 AML (2.8%). Thus, except in M0AML, the incidence of the AML1 gene mutations appears to be very low. However, the number of patient studied in some AML subtypes was too low for definite conclusions.

Myeloproliferative disorders:

Of the 129 MPD cases tested, mutations were found in six cases including two blast crisis chronic myeloid leukemia (CML) (Ref. 19 and this study), one essential thrombocytosis (ET),20 one case of myelofibrosis progressing to AML (this study) and two cases of atypical CML (aCML).20 The patient with ET rapidly progressed to M0 AML after analysis.

Myelodysplastic syndromes:

Of the 169 MDS studied, three had AML1 mutations. One of them, with RA, had progressed to AML at the time of AML1 mutation analysis.21 One of them presented with chronic myelomonocytic leukemia (CMML)21 and there were limited informations in the last patient.25 AML1 mutations seem therefore a very rare event in typical MDS, possibly associated with transformation. However, Osato et al19 found no mutation in six leukemic transformation of MDS and by analysis of 16 samples of MDS at diagnosis and at transformation time, we observed no mutations.

Thus, AML1 mutations seemed to predominate in M0 AML (34 cases) and are more rarely observed in other type of AML, MPD or MDS (17 cases).

Lymphoid maligancies:

To our knowledge, except for ALL, AML1 mutations have not been studied in lymphoid malignancies.

Correlation between AML1 mutation and other findings (Table 2)

Table 2 Clinical, cytogenetic and molecular data of the 51 patients with AML1 point mutations

In the 34 mutated cases with M0 AML, karyotype was available in 17 patients. A normal karyotype was observed in eight cases, complex karyotype in one case, hyperdiploid karyotype in one case, del (20q) in three cases, trisomy 13 in three cases, monosomy 7 in one case. No patient had structural or numeric alteration of chromosome 21. In the 17 remaining mutated patients (with AML, MPD or MDS), karyotype was available in seven cases and all cases except two with silent mutation showed trisomy or tetrasomy for chromosome 21, suggesting a link between additional chromosome 21 and AML1 gene mutation in patients with myeloid disorders.

In a retrospective multicenter study of 59 M0 AML patients, we found no significant differences between patients with and without AML1 mutation for sex, age, platelet count, Hb level, circulating blast count, myelodysplastic features in bone marrow, cytogenetic findings, incidence of FLT3 duplication, complete remission (CR) achievement with chemotherapy and survival. On the other hand, mutated cases had significantly higher leukocytes count, higher percent bone marrow blasts, lower CD33 expression, higher HLA DR expression and more frequent heavy chain immunoglobulin (Ig H) or T cell receptor (TCR) gene rearrangement that non-mutated cases (unpublished data).

Type of mutations reported

Germline and acquired AML1 mutations in hematological malignancies?:

Germline mutation of the AML1 gene has been reported by Song et al25 in very rare cases of familial platelet disorder with predisposition to acute myelogeneous leukemia (FPD/AML). In all cases, mutations were mono-allelic and resulted in haploinsufficiency. Affected FPD/AML individuals had thrombocytopenia with platelet function defect. In our experience, all AML1 mutations were acquired as shown by the comparison of SSCP profiles and direct sequencing of AML1 locus in three mutated patients at the time of diagnosis and in complete remission. Indeed, no mutations were detected in complete remission

Molecular characteristics of AML1 mutations:

In the 51 mutated cases reported, 71 AML1 mutations were found (as different biallelic mutations were observed in some cases). Three of them were silent mutations (two cases of I187I19 and one of S21S23). Twenty-seven were nonsense mutations inducing frameshift or stop codon and consequently truncated AML1 protein. In four cases, mutations occurred at a splicing site between exon 4 and intron 4 in three cases inducing abnormal splicing and premature termination of mRNA. In all but three cases, chain termination occurred in the runt domain between aa 60 and 177 and seemed clustered in four groups: four cases with mutation between aa 60 and 72, at the N-terminal part of the runt domain, seven cases with mutation between aa 105 and 115 in the middle part of the runt domain, eight cases between aa 135 and 140, four cases between aa 174 and 177 and one case in aa 198. In all those cases, truncated AML1 protein lacked the transactivation domain (aa 242 to 453). The remaining two mutations involved aa 24, and 319 in the transactivation domain. Interestingly, the same mutations were found in FPD/AML and in acquired hematological disorders.

Twenty-five missense mutations were observed in 22 patients. Three mutation hotspots were apparent: between aa 79 and 83 (3 cases), between aa 135 and 139 (6 cases) and between aa 171 and 177 (9 cases), respectively, the other mutations involved aa 29, 49, 58, 63, 69, 150 and 187.

Correlation with type of hematological malignancy:

In M0AML, alteration of both alleles was observed in 21 of the 34 mutated cases. Mutation of the first allele was associated with point mutation (10 cases) or with loss (11 cases) of the second allele. Three of the mutated cases could be studied both at diagnosis and in complete remission. Analysis of six microsatellite polymorphic markers located on chromosome 21 and FISH using cCMP21 probe on those three patients showed loss of chromosome 21 (confirming the conventional cytogenetic data), but also loss of the wild-type (WT) allele of the AML1 gene with partial duplication of the mutated allele through gene conversion or mitotic recombination.20 The mechanism leading to this alteration of the second allele remains to be elucidated. In the 13 remaining mutated cases with M0AML, 12 had mutation of only one allele and status of the second AML1 allele in the last patient was uncertain.

All the other 17 AML1 mutations reported were monoallelic, including the six cases of MPD. Interestingly, in the patient with ET progressing to M0AML, only one allele was mutated during the ET phase whereas both AML1 alleles were altered (ie mutation and deletion) at the time of progression. In MDS, two of the three mutated cases had mono-allelic AML1 mutation and the last patient, with RA that had progressed to AML, had a biallelic alteration of AML1 gene. The FAB subtype of AML in this patient was not known

Functional consequences of AML1 gene mutation:

They have been studied by Imai et al,21 Osato et al,19 and Bravo et al35 who found that AML1 mutant proteins had lost their activities. This functional consequence of AML1 mutation in the runt domain has been clearly demonstrated using a reporter plasmid containing M-CSF promoter. In all cases of AML1 mutations, except H58N, loss of transactivating properties of AML1 was observed.

The runt domain is also implicated in heterodimerization with CBFβ and in DNA binding on consensus DNA sequences of AML1 target genes. Osato, Bravo and Imai demonstrated that all mutants analyzed except for H58N abolished DNA binding. Structural alterations of the AML1 protein induced lack of transcriptional activities of AML1 mutants by inhibition of the ability of the runt domain to interact and bind to DNA consensus sequences. Interestingly, many of the mutations reported affect either aa residues in contact with DNA (R80, K83, R135, R139, D171, R174, R177) and induce loss of hydrogen bonding interaction to DNA, or aa residues which participate in stabilization of the structure of the folds of the runt domain.35 Moreover, coimmunoprecipitation experiments showed that, in R139G missense AML1 mutations, the capacity to heterodimerize with CBFβ was conserved and more efficient that with wild-type AML1. So, by competing for heterodimerization with CBFβ, R139G acts as a dominant negative inhibitor of wild-type AML1. On the other hand, heterodimerization with CBFβ was abolished in nonsense mutations which induced grossly truncated AML1 proteins.

Osato et al showed abnormal subcellular localization of mutant proteins in the cytoplasm whereas the WT proteins are nuclear. This was mainly observed for frame shift or nonsense mutations where the truncated protein resulting from the mutation had lost arginines localized in the C-terminal part of the runt domain critical for nuclear localization and only in one missense mutation R177Q.

AML1 mutations and leukemogenesis

AML1 acts as a key regulator of hematopoiesis through the regulation of various hematopoietic genes including (granulocyte–macrophage stimulating factor (GM-CSF), myeloperoxidase (MPO), interleukin 3 (IL3), T cell receptor β (TCR), M-CSF receptor, neutrophil elastase and multi drug resistance (MDR) genes). By interaction with other DNA binding proteins and the histone acetyl transferase complex, it contributes to activate transcription of target genes, but on the other hand, AML1 can also associate with mSin3 and groucho/TLE proteins to repress transcription.36 So AML1 plays a pivotal role in myeloid differentiation and deregulation of this gene may contribute to leukemogenesis in different ways.

Haploinsufficiency of CBFA2 plays a role in familial thrombocytopenia (FPD/AML) and in susceptibility of those patients to develop acute leukemia.25 As there was no evidence for an association between mutation of the other AML1 allele and progression to AML, Song et al suggested that the simple inactivation of one allele (haploinsufficiency) was sufficient to induce predisposition to acute leukemia (FPD/AML). Interestingly, a gene dosage effect of AML1 has also been reported in the development of hematopoietic system and haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.37 In acquired hematological malignancies, haploinsufficiency of AML1 may also have a pathogenic role and monoallelic alteration of AML1 gene was indeed found in 26 of the 51 reported cases of AML1 point mutations. Interestingly, patients with monoallelic alteration could be divided into two groups: patients with M0 AML and patients with other AML FAB subtypes, MDS or MPD. This last group, when karyotype data were available, showed high frequency of trisomy of chromosome 21 and in one case tetrasomy 21. How could haploinsufficiency play a role in these patients? In our experience in M0 FAB subtype, we found a propensity of alteration of the first allele to promote alteration of the second allele by mitotic recombination. This could also be true in cases of polysomy of chromosome 21 where only one allele of AML1 could be functional (data not shown) and haploinsufficiency could participate in leukemogenesis. On the other hand, as seen later, AML1 amplification seems to contribute to leukemogenesis in ALL cases, but in these cases amplification occurs by intrachromosomal amplification or presence of AML1 copies on extrachromosomes (markers, double minute, ring) and in our experience no mutation of AML1 gene was observed in these cases.

As seen above, functional consequences of bi-allelic AML1 point mutation through absence of DNA binding of the core complex are abolishment of transactivation of genes involved in myeloid differentiation and maturation including M-CSF receptor, MPO, GM-CSF, IL3 etc. Moreover, some of the mutations act in a dominant negative manner and prevent an effect of the remaining WT AML1 in the case of mono-allelic alteration. This hypothesis was confirmed by Osato and Imai, who demonstrated that some missense mutations cotransfected with WT AML1 abolished transactivation of the M-CSF receptor promoter. Moreover, Imai demonstrated that this dominant negative effect for R139G was due to the fact that the mutated protein did not bind DNA but had enhanced capacity to bind CBFβ and, by sequestering it, inhibited action of WT AML1, as observed for chimeric products of t(3;21) or t(8;21) translocations.17 In this hypothesis, the remaining WT AML1 could not act on its target gene, and alteration of both alleles was not required to completely inhibit transactivation properties of AML1. MPO belongs to these target genes and it is not surprising to find a predominant FAB subtype M0 AML in patients with complete inactivation of the AML1 gene through bi-allelic mutation or mono-allelic mutation associated with deletion of the other allele or with mono-allelic mutation acting as dominant negative as the R139G mutation.

AML1 mutation could be involved in leukemogenesis through other mechanisms. Indeed, many of the AML1 alterations induce the truncated form of the protein which had lost the C-term domain crucial for interaction with the TLE/groucho corepressor gene.38 In all of those cases, the mutated protein could not act to repress transactivation of many genes such as MDR, TCRα or TCRβ.

AML1 amplification

Gene amplification is a common mechanism of oncogene deregulation found mainly in solid tumors but also sometimes in hematological malignancies (C-Myc amplification in AML and myeloma and MLL amplification in AML and MDS especially therapy-related cases).39,40

Gene amplification can appear cytogenetically as homogeneously staining regions (HSR) or as extra chromosomal elements (markers or double minute) but it is often difficult to identify with conventional cytogenetics. Molecular cytogenetic techniques including spectral karyotyping (SKY), fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) analysis have helped in this identification.

AML1 gene amplification can occur through polysomy of chromosome 21 or through true high-level amplification. The latter due to intrachromosomal amplification by tandem repeat and/or presence of AML1 copies on extrachromosomes (ring or markers) has been reported mainly in childhood acute lymphoblastic leukemia (ALL) and less often in AML and MDS. To our knowledge, 16 cases of high-level amplification of the AML1 gene have been described (Table 3) including four cases at our institution; 12 of the 16 cases concerned childhood ALL.26,27,30,31,34 In these 12 cases, four to 15 copies of the AML1 gene were generally found by FISH. Interestingly, AML1 gene expression had been studied in two cases with AML1 amplification and shown an increase in AML1 transcripts parallel to the increase in AML1 gene copies in both cases.

Table 3 Clinical and cytogenetic data of the 16 patients with AML1 amplification

Amplification of AML1 gene was also reported in two cases of myeloid malignancies including one AML and one MDS transforming to AML.28,29 In both cases AML1 gene copies were found on a ring chromosome. In two of our patients diagnosed as MDS with complex karyotype, we have also found by FISH analysis, high-level AML1 amplification (unpublished data). In those two cases, AML1 gene extra-copies were found on a ring chromosome in one case, and on a derivative chromosome 21 from a t(17;21) in the other case. In seven cases of AML1 amplification, including five ALL, one MDS and one AML, no mutation in the runt domain of AML1 gene was observed, possibly suggesting that amplification per se could contribute to leukemogenesis.31,41 This finding, associated with the specific cytogenetic features (AML1 extra-copies often on extra-chromosomes) suggests to us that cases of high-level amplification should be distinguished from cases of polysomy of chromosome 21 where AML1 gene mutation seems frequently to occur. Consequently, AML1 gene deregulation in these two groups of patients is probably different in terms of contribution to leukemogenesis. In the case of polysomy of chromosome 21, implication of other oncogenes located on chromosome 21 cannot be excluded. In the cases of true high-level amplification, AML1 seems to be the only amplified gene and its implication in leukemogenesis appears probable.

Functional consequences of AML1 amplification and its role in leukemogenesis are still unknown. Generally, gene amplification induces overexpression of the gene. As seen above, correlation between the number of AML1 copies and level of expression of the gene has been shown in the case of ALL. It was also shown that AML1 can be a transforming gene when overexpressed in fibroblasts using AML1b retroviral infection of NIH3T3 cells.41 Thus, AML1 overexpression due to amplification could contribute to transformation of hematopoietic cells.


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Supported by the Centre Hospitalier de Lille (PHRC 1997), and the Ligue National contre le Cancer (Comité du Nord et de l’Aisne) and the Fondation de France (Comité leucémie).

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Correspondence to C Preudhomme.

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Roumier, C., Fenaux, P., Lafage, M. et al. New mechanisms of AML1 gene alteration in hematological malignancies. Leukemia 17, 9–16 (2003).

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  • AML1
  • mutation
  • amplification
  • undifferentiated acute myeloid leukemia

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