High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. in

A high incidence of somatically acquired point mutations in the AML1 / RUNX1 gene has been poorly acute majority of AML1 mutations identified in these diseases were localized in the amino (N)-terminal region, especially in the DNA-binding Runt we show that AML1 point mutations were found 26 (23.6%) of 110 patients with refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt) and AML following MDS (defined these three disease categories as MDS/AML). Among them, 9 (8.2%) mutations occurred in the carboxy (C)-terminal region, which were exclusively found in MDS/AML and were strongly correlated with sporadic MDS/AML. All MDS/AML patients with an AML1 mutation expressed wild type AML1 protein and had a significantly worse prognosis than those without AML1 mutations. Most mutants lost trans -activation regardless of their DNA binding potential. These data suggested that AML1 point mutation is one of the major driving force of MDS/AML, and these mutations may represent a distinct clinicopathologic-genetic entity. of hematopoietic malignancy.


Introduction
Somatically acquired point mutations of critical genes have been demonstrated to contribute to the development of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Genes encoding key regulatory factors for cell division, differentiation, or cell survival of hematopoietic progenitors, such as Ras, receptors for stem cell factor (c-Kit) and Flt-3 ligand, and transcription factors are frequent mutation targets. However, there is no strong correlation between point mutations of these genes and morphology, drug sensitivity or prognosis. This is in sharp contrast with certain chimeric oncoproteins that result from recurrent chromosomal translocations, and that form distinct clinicopathologic-genetic entities. 1 For instance, a majority of leukemias associated with AML1-ETO (MTG8) chimera are de novo AML with maturation, and it is relatively easy to obtain complete remission in these cases by standard chemotherapy, and these patients have favorable prognoses. 2 Thus, the AML1-ETO chimera is considered to be the decision factor of the biological features of leukemia, while the point mutations mentioned above might merely play a complementary role that contributes to the progression of disease.
The AML1 gene was also found to be altered by point mutations in AML and MDS.
Beginning with the first report by Osato et al., 3 the unique features of this mutation have been revealed by several studies. First, although the frequency of AML1 mutations in de novo AML is low (<5%), they have been detected at a substantially higher frequency in a specific subtype of AML, poorly differentiated AML M0 (12 % to 33 %). [3][4][5][6] We also reported a high frequency (42 %) of AML1 mutations among radiation-associated and therapy-related MDS and AML. 7 These results suggest that AML1 mutations might play, in certain situations, critical roles in developing hematopoietic malignancy. Second, germ line mutations of For personal use only. on August 20, 2017. by guest www.bloodjournal.org From 4 AML1 have been shown to occur in a rare autosomal dominant disorder, familial platelet disorder with predisposition to AML (FPD/AML). [8][9][10] All mutations with one exception identified in patients with AML and FPD/AML are in the amino (N)-terminal region of this transcription factor, especially in the Runt homology domain (RHD), which mediates its ability to bind to DNA and core binding factor (CBF ). Nearly half of them are missense mutations that replace amino acid residues directly contacting DNA, as shown by analysis of the crystal structure of the RHD-CBF -DNA ternary complex. [11][12][13][14] Most of the other mutations are frame shift or nonsense mutations that abolish the function of the RHD. 3,7 The majority of AML1 point mutations abrogate its DNA-binding potential, suggesting that loss of function is the main mechanism through which mutated AML1 contributes to the malignant transformation of hematopoietic progenitors.
Germ line mutations of the RUNX2 gene, which encodes another member of the Runx family of transcription factors that contains the RHD, have been detected in patients with Cleidocranial dysplasia (CCD), an autosomal dominant disorder characterized by skeletal anomalies (See review by Otto et al. 15 ). Although more than 70% of mutations detected in CCD are in the RHD, around one fourth of them are in the carboxy (C)-terminal region.
These data prompted us to test whether AML1 point mutations in the C-terminal region occur in hematopoietic malignancy, especially in MDS, because only 6 patients with MDS were so far tested for the C-terminal mutations of AML1. 3 Here we report a high frequency of AML1 mutations in the C-terminal, as well as in the N-terminal region of the protein in MDS patients, especially those with refractory anemia with excess blast (RAEB), RAEB in transformation (RAEBt), and AML following MDS. This suggests that AML1 point mutations are strongly associated with these specific types of hematopoietic malignancy. Hospital and its affiliated hospitals between 1995 and 2003. We divided these patients into 'sporadic' and 'secondary' disease categories according to their past history. The secondary group included (a) atomic-bomb survivors in Hiroshima, who were exposed within 3 Km of the hypocenter, 7 (b) patients who developed disease after radiotherapy and/or chemotherapy for malignancy or myeloproliferative disorder (MPD), (c) an individual who was occupationally exposed to Mustard gas. We also examined 51 cases of MPD in the chronic phase [15 myelofibrosis (MF), 21 essential thrombocythemia (ET), 13 polycythemia vera (PV), and 2 atypical MPD)], 23 cases of chronic myeloid leukemia (CML; 20 in the chronic phase and 3 in blast crisis), and 28 cases of acute lymphoid leukemia (ALL).
Diagnosis was based on morphologic and immunophenotypic studies according to the French-American-British (FAB) classification. 16,17 The majority of the patients in this study were treated in Hiroshima University Hospital or Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital with a protocol containing intensive chemotherapy and bone marrow transplantation. Cytogenetic analyses using standard procedures were performed according to the International System of Human Cytogenetic Nomenclature (1995). Patient samples were taken after obtaining informed consent and approval from the institutional review board at Hiroshima University. Mononuclear cells were isolated from bone marrow 6 or peripheral blood samples by Ficoll-Conray density gradient centrifugation. Genomic DNA was extracted with a Puregene Kit (Gentra, Minneapolis, MN) and total RNA was extracted using a TRIzol Kit (Gibco Life Technologies, Rockville, MD), according to the manufacturers' instructions.
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 a BigDye Terminator Cycle sequencing kit (Perkin-Elmer) and were analyzed on an ABI 7 Prism 310 Genetic Analyzer (Perkin-Elmer). To confirm the 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'/ Immunoprecipitations and immunoblot analysis. FLAG beads were blocked in phosphate-buffered saline (PBS) containing 1% bovine serum albumin prior to addition to the lysates. Immunoblot analysis was performed as reported previously. 7 The primary antibodies used in this study were anti-M2 antibody (Sigma), 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 (EMSA) and transcriptional assay.
Nuclear extracts from Cos-7 cells that were transiently transfected with the corresponding expression plasmid were prepared as described previously. 20 Protein concentrations were determined with Bradford reagents (Bio-Rad, Hercules, CA). EMSA was performed as described previously. 21 For supershift analysis, 1 µl of rabbit AML1 antiserum that was raised against a polypeptide (Arg-Ile-Pro-Val-Asp-Ala-Ser-Thr-Ser-Arg-Arg-Phe-Thr-Pro-Pro-Ser, corresponding to the N-terminal region of AML1 of human and mouse origin) was used. Transcription assays using HeLa and U937 cells were performed by the procedure described elesewhere. 7 9 Statistical methods.
Kaplan-Meyer analysis was used to estimate survival of patients with RAEB, RAEBt and AML following MDS with or without AML1 point mutations. 22 The log-rank test was used to compare the probabilities of survival for both groups.

High frequency of AML1 mutations in the N-terminal region in MDS and AML
We previously reported 11 cases of secondary MDS or AML (for definition of  Table 1 (cases 14 to 23).
The overall incidence of N-terminal mutations in sporadic and secondary RAEB, RAEBt and AML following MDS was 8% (7/88) and 45.4% (10/22), respectively (Table2). We found only one patient (1.1%) with an N-terminal AML1 mutation among 95 sporadic AML patients without antecedent MDS, and no N-terminal AML1 mutations were found in the ALL, CML or MPD patients. These data confirmed findings previously reported by us and others that AML1 mutations in the N-terminal region are not associated with ALL, MPD or CML, but they are implicated in sporadic AML (except M0) at low frequency, and they are frequently found in secondary AML and MDS ( Figure 1 and Table 2). 3, 4, 7, 8, 23

Frequent occurrence of C-terminal mutations of the AML1 gene in MDS
To investigate the AML1 mutations in the C-terminal portion of AML1, we analyzed All of the C-terminal mutations (cases 24 to 32) resulted in frame shifts similar to the Runx2 mutations identified in CCD. 15 The consequences of the C-terminal mutations were unusual ( Figure 2A). Usually, frame shift mutations are created by insertions or deletions of nucleotides in exons or by loss of splice donor or acceptor sites, and they result in truncation 12 of the authentic protein followed by a relatively short additional stretch of amino acid residues originating from the wrong reading frame or from intronic sequences. Among the nine C-terminal AML1 mutations found, only 3 cases (cases 24, 27 and 29) obeyed this general rule. In 4 cases (cases 25, 28, 31 and 32), small nucleotide insertions or deletions (4 to 14bp) occurred in exons, but the stretches of additional amino acids resulting from the wrong reading frame were 195 to 335 residues in length (Table 1), because the wrong frame contained an in-frame termination codon 353 bp downstream (3') of the authentic termination codon ( Figure 2B). Thus the mutated AML1 proteins in these four cases were even longer than wild type AML1 and appeared to be fusion proteins rather than truncations of AML1 Consequently, the mutated AML1 protein had an insertion of 36 amino acid residues that originated from intronic sequences in the middle of the trans-activation domain of AML1. 13 We observed virtually equal intensities of normal and shifted bands in the PCR-SSCP analysis of all the samples with AML1 mutations, including case 30, and obtained comparable frequencies of normal and mutated sequences by the sequence analysis. In addition, germ line genomic DNA sequences were examined in specimens obtained from nonleukemic organs in six cases (cases 4, 5, 6, 9, 10 and 12), and these were found to be normal (data not shown), suggesting that mutations of the AML1 gene were monoallelic at the somatic level.
To test whether AML1 point mutations affect the prognosis of MDS, we tracked the overall survival of patients with RAEB, RAEBt, and AML following MDS, comparing survival of those associated with AML1 mutations (n=26) with that of patients without the mutations (n=81) (Figure 4). There was no significant difference in the distribution of age To test whether AML1 mutants are able to interact with CBF , we performed immunoblot analysis after immunoprecipitation. Cos-7 cells were cotransfected the FLAG-tagged wild-type or mutated AML1 ( Figure 5B, upper panel), together with CBF .
CBF was coimmunoprecipitated with FLAG-tagged wild-type AML1 by beads coated with anti -FLAG antibody (lower panel, lane 9). CBF was also coimmunoprecipitated with AML1 proteins having mutations in the C-terminal domain (lanes 1 to 8), indicating that these mutants interacted with CBF . Taking account of expression levels of mutant proteins, the binding potential of mutants 24 and 27 seemed to be increased.

Transcriptional potential of C-terminal AML1 mutants
To investigate the transcriptional activities of the C-terminal AML1 mutants, reporter experiments were performed using the promoter of macrophage colony-stimulating factor receptor (M-CSFR), which is known to be transcriptionally regulated by AML1. 19 When wild-type AML1 and CBF were cotransfected in HeLa cells, the promoter activity was induced 7-fold compared to transfection with CBF alone ( Figure 6A). In contrast, none of the C-terminal mutants induced significant trans-activation, regardless of their DNA-binding potentials ( Figure 5A). 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 6B). As a positive control, we used CBF -MYH11, which is formed as a result of inv (16) and is a well-established negative regulator of AML1. 24 All mutants suppressed the trans-activation activity of wild-type AML1 in a dose-dependent fashion. Inhibition of the trans-activation seemed to be related to DNA-and CBF -binding potential ( Figures 5A and 5B). Thus, the C-terminal AML1 mutants identified in this study lacked trans-activation potential and could act as dominant negative inhibitors of wild type AML1.
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Discussion
In this study, we established strong correlations between point mutations of the AML1 gene and subgroups of MDS and AML, i.e., RAEB, RAEBt and AML following MDS. Here we define these three disease categories as MDS/AML. Of the 110 patients with MDS/AML who were tested, 26 (23.6%) had an AML1 mutation (Table 2). Conversely, of 32 patients with AML1 point mutation, 26 (81 %) belonged to this category (Table 1). Moreover, the prognosis for the patients with AML1 mutations was significantly worse than for those without AML1 mutations ( Figure 4).
The MDS/AML category we defined above is characterized by (1)  patients were reported to have a total homozygous deletion of AML1 . 6 By contrast, all 26 MDS/AML patients with AML1 mutations in this study and an additional three patients reported by others expressed wild type AML1, 3,23 suggesting that this transcription factor differentiates not only normal hematopoietic progenitors in the process of development, but also pathological blasts in leukemogenesis. This hypothesis is supported by the fact that wild type AML1 is usually expressed in leukemic blasts harboring AML1-ETO chimeras, which cause AML with maturation (M2). Another possibility is that different AML1 mutations result in different phenotypes. Indeed, C-terminal mutations we found so far only in MDS/AML, suggesting that this type of mutation may induce MDS/AML but not de novo AML. However, there seemed to be little difference in N-terminal mutations between MDS/AML, AML (M0) and FPD/AML (Table 3). A limited number of amino acid residues were replaced by missense/insertion mutations, and these types of mutations were found in each of the three disease categories. Nonsense/frame shift mutations in each disease category also resulted in similar, or even identical, truncated forms. This suggested that, in the case of N-terminal AML1 mutations, the phenotype of the resulting leukemia would be determined by the presence of wild type AML1 or other genetic abnormalities that contributed to leukemogenesis in cooperation with the AML1 mutation.
Most amino acid residues replaced by missense or insertion type mutations in the N-terminal region of AML1 were located in three protein loops, called loop( A'-B), 19 loop( E-F), and G'tail, which mediate the DNA-binding potential, as demonstrated by analysis of the crystal structure of the RHD-CBF -DNA ternary complex (Table 3). [11][12][13][14] Nonsense and frame shift mutations in the N-terminal region result in partial deletion of the Runt homology domain. Thus these mutants are predicted to lose DNA-binding ability and trans-activating potential (we designate this type of mutation as Type a-1), and indeed, we and others have confirmed this for 18 mutants (Table 3). 3, 7, 10 , 23 However, there are four missense mutations (Leu29Ser, Gly42Arg, His58Asn, and Ile150Thr) that replace amino acids out of the three loops. The Gly42Arg and His58Asn mutant proteins were shown to bind to DNA more avidly than wild type AML1, and to have enhanced trans-activation potential (Type b). 3,7 Analysis of C-terminal mutants provided three additional types of mutation: attenuated DNA-binding potential without trans-activation potential (Type a-2, mutants 25, 31, and 32), enhanced DNA-binding ability but no trans-activation potential (Type a-3, mutants 24, 26, 27 and 30b), and DNA-binding ability equivalent to wild type AML1 but no trans-activation potential (Type a-4, mutant 30a). Mutants belonging to Type a-3 have a similar structure to that of an alternative splice variant, AML1a, which also binds to DNA more avidly than wild type AML1b but lacks trans-activation potential. 31 Currently, we can offer no adequate interpretation for the question as to why mutations resulting in such divergent biochemical features contribute equally to the same type of myeloid malignancy.
However, we can say that, with a few exceptions such as Gly42Arg or His58Asn, AML1 mutants lose their trans-activation potential, suggesting a loss of function mechanism. We believe that further careful analysis of these AML1 mutants will reveal the key molecular system that induces myeloid malignancy.   a-1, a-2, a-3, a-4,and b indicate the biochemical functions affected by each mutation (see text). Boxes show the identical mutation Gray backgrounds indicate missense/insertion mutations that replace amino acid residues in loops directly contacting DNA or nonsense/frame shift mutants resulting in similar truncated forms of the protein.           Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society