Japanese Journal of Clinical Oncology 33:153-160 (2003)
© 2003 Foundation for Promotion of Cancer Research
Molecular Mechanisms of Myelodysplastic Syndrome
Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
ABSTRACT
Myelodysplastic syndrome (MDS) is a family of clonal disorders of hematopoietic stem cells that are characterized by ineffective hematopoiesis and susceptibility to acute myelogenous leukemias and are shown to be strikingly refractory to current therapeutic modalities. A substantial proportion of these complex diseases arises in the setting of exposures to environmental or occupational toxins, including cytotoxic therapy for a prior malignancy or other disorder. The conversion of a normal stem cell into a preleukemic and ultimately leukemic state is a multistep process requiring the accumulation of a number of genetic lesions. At the genomic level, MDS is typified by losses and translocations involving certain key gene segments, with disruption of the normal structure and function of genes that control the balance of proliferation and differentiation of hematopoietic precursors. More than half of the chromosomal abnormalities in MDS comprise deletions of chromosomes 5, 7, 11, 12, 13 and 20. This evidence suggests that as yet unidentified tumor-suppressor genes should have important roles in the molecular mechanisms of MDS. Further molecular approaches to such genetic lesions will identify the relevant tumor-suppressor genes. Over the past years, major signal transduction molecules have been identified and their genetic alterations have been extensively analyzed in both MDS and leukemias. These include receptors for growth factors, RAS signaling molecules, cell cycle regulators and transcription factors. Notable among them are transcription factors that regulate both proliferation and differentiation of hematopoietic stem cells. The disruption of the normal flow of the signal transduction pathways involving these molecules translates into ineffective multilineage hematopoiesis and bone marrow failure. Therefore, MDS provides a fertile testing ground on which we could study the molecular dissection implicated in the multistep leukemogenesis.
I. BIOLOGY
Myelodysplastic syndrome (MDS) is a family of clonal disorders characterized by dyshematopoiesis and susceptibility to acute myelogenous leukemia (AML). The natural history of these diseases ranges from a chronic course that may span years to a rapid course towards leukemic progression. Clonal proliferation is the consequence of acquired somatic mutation that confers a proliferative advantage to cells. Identification of clonal cells provides valuable information on the molecular pathogenesis of the disease, the processes that govern the transition to AML and diagnostic and prognostic information that is useful in the clinical approach to MDS. There are several methods for determining the clonality of cells; cytogenetic analysis and fluorescence in situ hybridization (FISH) analysis based on chromosomal abnormalities, Southern blot analysis and single-strand conformation polymorphism (SSCP) analysis based on genetic alterations and X-inactivation-based clonality analysis.
Clinically, patients with MDS present with variable cytopenias due to an ineffective hematopoiesis of unknown etiology. An important recent observation in this regard is the excessive intramedullary apoptotic death seen in the bone marrow biopsies of these patients, possibly accounting for the invariable cytopenias. One mechanism invoked to explain the apparent discrepancy between cellular marrow and peripheral blood cytopenias in patients with MDS is programmed cell death (apoptosis), which occurs with increased frequency in MDS marrow (1,2). Several cytokines or ligands known to have proapoptotic properties, such as interleukin-1ß (IL-1ß), tumor necrosis factor 
(TNF) and Fas-ligand are upregulated in many patients with MDS (1,3). It has been shown that blockade of TNF or Fas-ligand enhances hematopoietic colony transformation from MDS marrow in vitro and improves blood cell counts in vivo (1). However, it is clear from those studies that the regulation of hematopoiesis in MDS is complex and multiple factors are involved.
Hematopoietic stem cells of MDS are genetically unstable and thus susceptible to genetic lesions. Once a stem cell in MDS gains a dominant mutation over the normal cell growth, such a cell shows the clonal evolution to become much more susceptible to further multiple genetic mutations and finally to develop into leukemia. The initial genetic event of the presumed multistep pathogenesis of MDS is an unknown stem cell defect, which is followed by non-clonal karyotype instability. In approximately half of the patients, clonal karyotypic abnormalities, which are detectable at the stem cell level, occur (4). By unraveling loss of heterozygosity (LOH) and microsatellite instability (MSI), microsatellite polymorphic markers are useful to test the status of DNA mismatch repair (MMR) and cell cycle-conducting tumor-suppressor genes, both of which, if deficient, promote genetic instability (5).
LOH describes the homozygous state of a distinct chromosomal region and points to the presence of a closely located inactivated tumor-suppressor gene that might be involved in malignant transformation. MSI is defined by the occurrence of novel microsatellite alleles in neoplastic DNA when compared with DNA from non-malignant tissue of the same individual. It is a hallmark of patients with hereditary non-polyposis colorectal cancer (HNPCC) and related malignancies as a consequence of a defective DNA MMR machinery (6). MSI has also been observed in sporadic tumors and distinct hematological disorders, suggesting that DNA proofreading failure may be a common pathogenetic mechanism in neoplastic transformation. A disease-inherent genetic instability is thus supposed to be the basis for an accumulation of somatic mutations in MDS.
II. CYTOGENETICS AND GENETIC ALTERATIONS
Cytogenetic analysis is a cornerstone of the characterization of MDS and provides valuable clues to the molecular pathogenesis of MDS. Since more than 70% of patients with MDS have clonal cytogenetic abnormalities, cytogenetic studies play a pivotal role in defining the concept of primary MDS and therapy-related MDS, establishing the diagnosis, evaluating prognosis for survival and transformation to AML and approaching the molecular basis of the diseases. Some patients with MDS exhibit aneuploidy, in which a whole chromosome is either lost or gained, or structural abnormalities such as deletions, translocations, isochromosomes or marker chromosomes. The complexity of abnormal karyotypes is more common in therapy-related MDS than in primary MDS (7,8).
1. Chromosomal Deletions
One of the most prominent characteristics in chromosomal abnormalities observed in MDS is a predominance of chromosomal deletions, whereas de novo leukemias are typified by balanced reciprocal translocations. Frequent loss of genetic loci leads to the hypothesis that MDS may be caused by inactivation of tumor-suppressor genes. Based on this hypothetical model, one copy of a gene is deleted by chromosomal deletion and the other copy is inactivated by point mutation, minute deletion or loss of expression due to regional methylation. So far, however, this paradigm for MDS has not been proven, probably because of the difficulty in cloning tumor-suppressor genes within the large range of chromosomal deletions.
5q–
The 5q– chromosomal abnormality is the most frequent in MDS and is present in more than 20% of MDS patients. The 5q– syndrome is characterized by distinct clinical and morphological features including refractory macrocytic anemia with dyserythropoiesis, high prevalence in females, a normal or high platelet count, small megakaryocytes and a relatively good risk (9). The breakpoints within a large region of 5q are highly variable among patients, but the most critical region of deletion is supposed to lie between 5q31 and 5q33 (10). Several genes encoding hemopoietic growth factors and receptors, comprising IL-3, IL-4, IL-5, M-CSF (CSF-1), GM-CSF and the receptor for M-CSF (CSF-1R), are localized to the long arm of chromosome 5 and there has been much speculation that deletion of one or more of these genes may be critical to the pathogenesis of the associated MDS (11). The homozygous loss of some of these genes has still been considered as a possible mechanism in the pathogenesis of myeloid disorders with 5q deletion. IRF-1, a gene whose product manifests anti-oncogenic activity, is mapped to 5q31.1. IRF-1 lies between IL-5 and CDC25C and is centromeric to IL-3 and GM-CSF. Among these genes, IRF-1 is reported to be deleted at one or both alleles by an accelerated exon skipping mechanism in some cases of MDS with aberrations of 5q31 (12). Pur is a highly conserved, eukaryotic sequence-specific DNA- and RNA-binding protein involved in diverse cellular and viral functions including transcription, replication and cell growth. PURA gene, encoding Pur , is localized to chromosome bands 5q31.1, and is shown to be hemizygously deleted in MDS and AML. Frequent deletion of PURA indicates that PURA is one of the most commonly deleted genes in myeloid disorders characterized by del(5)(q31) (13).
Another approach to identifying a target gene, which is inactivated by 5q deletion, is to search for a gene that might be also a target for chromosomal translocations involving the region between 5q31 and 5q33. Several such genes have so far been cloned. The NPM–MLF1 chimeric protein is produced by the t(3;5)(q25.1;q34) chromosomal translocation, which is associated with MDS prior to progression into AML (14). Leukemia cells ectopically expressing NPM–MLF1, but not those with wild-type NPM or MLF1, were shown to undergo apoptosis. GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125FAK), which encodes a member of the Rho family of the GTPase-activating protein, was revealed to fuse with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia (15). The particular position of the human GRAF gene at 5q31 and the proposed tumor-suppressor properties of its avian homolog suggest that it also might be pathogenetically relevant for hematological malignancies with deletions of 5q. Moreover, some mutations within the GAP domain of the second GRAF allele resulting in inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q). Other than these genes, there also exist more genes including acyl-CoA synthetase 2 gene, ACS2, fused to TEL gene in t(5;12)(q31;p13) translocation in patients with MDS (16) and nuclear receptor-binding SET-domain-containing protein, NSD1, fused to NUP98 in t(5;11)(q35;p15.5) translocation associated with 5q deletion in childhood AML (17).
Monosomy 7
Monosomy 7 and 7q– are among the most frequent chromosomal abnormalities in MDS and are associated with poor prognosis in terms of either short survival or leukemic evolution. Although genes on chromosome 7 that are responsible for the disease phenotype are not identified, a critical region at 7q22.1 has been clarified (18). A potential myeloid tumor-suppressor gene, PIK3CG, has recently been identified in this locus, but is unlikely to act as a recessive tumor-suppressor gene in MDS with monosomy 7 (19). Monosomy 7 is also found in children with juvenile myelomonocytic leukemia (JMML), which has been referred to as monosomy 7 syndrome. JMML is a pediatric myelodysplastic syndrome that is associated with neurofibromatosis type 1 (NF1). The NF1 tumor-suppressor gene encodes neurofibromin, a GTPase-activating protein (GAP) for p21RAS. Children with NF1 are predisposed to JMML and both alleles of the NF1 gene are inactivated in leukemic cells in some patients with NF1 (20). NF1 gene mutations have been detected in ~30% of JMML cases. RAS gene mutations or inactivation of NF1 gene are thought to be critical events in the progression of MDS with monosomy 7 (21,22). In conjunction with monosomy 7, it is under investigation whether unbalanced translocation, [–7, +der(1;7)(q10;p10)], which is frequently detected in MDS, is biologically the same as monosomy 7.
20q–
A chromosomal 20q deletion is associated with ~5% of primary MDS and confers a relatively favorable prognosis. Erythrocytic and megakaryocytic lineages appear to be mainly involved in dysplastic changes. In some MDS cases with 20q–, clonal mature granulocytes from peripheral blood lack the anomaly. An increased bone marrow apoptosis of granulocytic precursors bearing 20q– may support these findings (23). Deletions are always interstitial and the crucial region deleted in MDS has been mapped between D20S174 and D20S17. Although the deletion is still fairly large by molecular standards, several candidate tumor-suppressor genes are mapped to this locus (24).
Less frequent deletions
In addition, a number of less frequent but characteristic losses of a part or the whole of a chromosome have been identified in MDS, including del(13q), del(11q) and del(12p). Deletion of 13q consistently involves bands q14 and q21 and FISH analysis delineates a commonly deleted region flanked by YAC 833A2 and YAC 854D4 (25). Much interest has been focused on RB gene, which is located at 13q14 and deleted in some MDS cases with del(13q). Some MDS cases with a deletion of 11q are characterized by ringed sideroblasts and are diagnosed as acquired sideroblastic anemia according to the FAB classification (26). Genomic studies mapped the putative tumor-suppressor gene to q22.2–q23.3 of chromosome 11. MDS cases with a deletion of 12p are heterogeneous. Deletions are usually interstitial with loss of a region between bands p11 and p13, where KIP1(CDKN1B) and TEL (ETV6) are located (27).
2. Chromosomal Translocation
TEL(ETV6) fusion
A t(5;12)(q33;p13) translocation is a recurrent chromosomal abnormality in a subgroup of myeloid malignancies with features of both myeloproliferative disorder and MDS (Table 1) (28). The molecular consequence of t(5;12) is a fusion between the platelet-derived growth factor receptor-ß (PDGFR-ß) gene on chromosome 5 and an ETS-like gene, TEL (ETV6), on chromosome 12. Eosinophilia and/or monocytosis in bone marrow are predominant morphological features in MDS with t(5;12) (29). Oligomerization of TEL/PDGFR-ß through the TEL HLH domain leads to constitutive activation of the PDGFR-ß tyrosine kinase domain and thereby cellular transformation (30). Imatinib mesylate (formerly STI571), which specifically inhibits the kinase activity of ABL (including BCR/ABL), PDGFR-ß and c-KIT and thus shows efficacy in the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors, also induces durable responses in patients with chronic myeloproliferative diseases associated with TEL/PDGFR-ß (31). TEL gene is also fused to ARNT, MN1, EVI-1 and ACS2 in MDS cases carrying t(1;12)(q21;p13), t(12;22)(p13;q11), t(3;12)(q26;p13) and t(5;12)(q31;p13), respectively (32–35).
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MLL fusion
The human homolog of Drosophila trithorax gene, MLL, is a common target of chromosomal translocations involving 11q23, which are associated with acute leukemias showing a biphenotypic or a monocytic phenotype, infant leukemias or topoisomerase inhibitor-induced secondary leukemias (36). Although the most typical 11q23 translocations such as t(4;11) (q21;q23), t(9;11)(q21;q23) and t(11;19)(q23;p13.3) are not found in primary MDS, some 11q23 translocations including t(11;19)(q23;p13.1) and t(11;16)(q23;p13), which generate MLL/MEN (ELL) and MLL/CBP chimeric genes, respectively, are detected in primary or therapy-related MDS (37,38). Tandem duplication of the MLL gene is identified in some MDS with either normal karyotype or trisomy 11 (39).
Nucleoporin abnormality
NUP98 is a nucleoporin and is involved in nuclear import and export of proteins and RNAs (40). To date, a number of chimeric NUP98 genes have been identified in patients with therapy-related AML or MDS with chromosomal translocations involving 11p15.5. The resultant chimeric transcripts encode fusion proteins that juxtapose the N-terminal GLFG repeats of NUP98 to the C-terminus of the partner gene. The chromosomal aberrations t(7;11)(p15;p15), t(2;11)(q31;p15), t(11;17)(p15;q21), t(11;12)(p15;q13), t(11;20)(p15;q11) and inv(11)(p15q22) result in the generation of NUP98/HOXA9, NUP98/HOXD13, NUP98/HOXB, NUP98/HOXC, NUP98/TOP1 and NUP98/DDX10 fusion genes, respectively (41–45). These results indicate that NUP98 is a recurrent target in therapy-related leukemias and MDS.
Translocation (6;9)(p23;q34) is a cytogenetic aberration that can be found in specific subtypes of both AML and MDS (46). This translocation is associated with an unfavorable prognosis. CAN, also called NUP214, is a nucleoporin that contains multiple FG-peptide sequence motifs. It interacts at the nuclear pore complex with at least two other proteins, the nucleoporin NUP88 and hCRM1 (exportin 1), which was recently shown to function as a nuclear export receptor (47).
EVI-1 family
Structural alterations involving 3q21 and 3q26 bands occur in ~2% of patients with AML or MDS. The major alterations are inv(3)(q21q26) and t(3:3)(q21;q26) and are classified as the 3q21q26 syndrome with features of abnormalities of megakaryocytopoiesis, an elevated platelet count and poor prognosis (48). Aberrant expression of the ecotropic virus integration-1 (EVI-1) gene, located at 3q26, is mediated by enhancer elements of the Ribophorin I gene, located at 3q21 (49). The translocation t(1;3)(p36;q21) observed in a subset of MDS or AML, which is characterized by trilineage dysplasia, dysmegakaryocytopoiesis and poor prognosis. In this abnormality, MEL1 gene, which is located at 1p36 and highly homologous to the EVI-1 gene, is transcriptionally activated by the translocation of the 3q21 region with the Ribophorin I gene (50). The t(3;21)(q26;q22) translocation associated with therapy-related AML and MDS and blastic crisis of CML generates the AML1/EVI-1 chimeric gene, resulting in aberrant expression of an almost whole coding region of EVI-1 protein (51). As a mechanism of leukemogenesis induced by ectopic expression of EVI-1, EVI-1 was shown to repress Smad-induced transcription through recruiting a corepressor, C-terminal binding protein (CtBP), which binds to histone deacetylase (HDAc) (52,53).
3. Genetic Mutations
RAS gene
RAS is an important signaling component for cell proliferation and is activated by receptor tyrosine kinases (RTKs) stimulated with extracellular ligands (54). RAS functions as a relay switch that is positioned downstream of cell surface receptor tyrosine kinases and upstream of a cytoplasmic cascade of kinases including the mitogen-activated protein kinases (MAPKs). Activated MAPKs in turn regulate the activities of nuclear transcription factors. RAS has the GTP-binding activity and the GTP-hydrolyzing property. RAS/GDP is an inactive form and is converted to an active form of RAS/GTP by guanine nucleotide-exchange protein (GEP) upon activation of RTKs with extracellular ligand stimuli. RAS/GTP interacts with target proteins such as RAF, thereby activates the downstream signaling molecules, MAPKs, and is converted to an inactive form RAS/GDP by hydrolysis of bound GTP in the presence of GTPase-activating protein. Mutated RAS proteins do not show GTPase activity, thus accumulate RAS/GTP and thereby constitutively activate the downstream signaling.
RAS genes are known to be activated by point mutations at codon 12, 13 or 61 (55). Among RAS genes, mutations of the N-ras gene are most frequent and detected in 20–30% of human leukemias and 10–15% of MDS cases (Table 2) (56). An N-ras mutation in MDS is associated with short survival period and increased probability of developing AML (57). These observations suggest that activation of N-ras oncogene should be related to leukemic transformation at least in a fraction of MDS patients.
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FLT3 gene
FLT3 gene encodes receptor-type tyrosine kinase that is involved in proliferation and differentiation of hematopoietic precursor cells. An internal tandem duplication of the human FLT3 gene is found as a somatic mutation in 15–20% of AML and 5% of MDS (58). This abnormality seems to be a late genetic event during the disease course and patients with FLT3 mutations tend to have a poor prognosis, suggesting that FLT3 tandem duplication is associated with leukemic transformation from antecedent MDS (59).
p53 gene
The p53 gene is a hallmark of tumor-suppressor genes and its alterations are involved in various types of human malignancies. Inactivation of the p53 gene in both alleles by mutations or deletions has been shown to predispose the cells to neoplastic transformation. Inactivation of the p53 gene is detected in 5–10% of MDS and preferentially in clinically advanced stages and in karyotypically unstable cases with MDS, indicating that p53 mutations may play a role in leukemic progression of MDS (60).
AML1 gene
The AML1 (Runx1) gene encoding the heterodimeric transcription factor, which binds to DNA through the Runt domain, is frequently involved in chromosomal translocations associated with human leukemias. Heterozygous missense mutation of the AML1 gene is noted to be causative for familial platelet disorder (FPD) with predisposition to AML, in which haploinsufficiency of AML1 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia (61). Missense mutations, mainly in the Runt domain, of the AML1 gene, are also identified in ~5% of AML (62). It is notable that AML1 mutations are preferentially detected in AML of the M0 phenotype with high frequencies of 22% and that most of them are biallelic, since AML1 protein transcriptionally regulates the expression of the myeloperoxidase gene (63). Although less frequently, the AML1 gene is also a target of mutations in MDS and at least some of them show not only a loss-of-function phenotype of AML1 but also a dominant negative effect on normal AML1 function (64).
C/EBP gene
The transcription factor C/EBP (for CCAAT/enhancer binding protein-alpha) is crucial for the differentiation of granulocytes. Conditional expression of C/EBP triggers neutrophilic differentiation and no mature granulocytes are observed in C/EBP-mutant mice. Heterozygous mutations in C/EBP are found in 7–8% of AML and rarely in MDS (65,66).
4. Epigenetic Alteration
The cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and have been shown to be tumor-suppressor genes (67,68). p15INK4B gene is an inhibitor of CDK4 and CDK6 whose expression is induced by transforming growth factor (TGF)ß (69). Although genetic alterations in these genes may be limited to lymphoid malignancies (70), it has been reported that their inactivation by aberrant methylation of 5' CpG islands is involved in various hematologic malignancies (71). Recent reports suggest frequent methylation of the p15INK4B gene promoter in leukemia cells and it has been proposed that this methylation could be necessary for leukemic cells to escape TGFß regulation. p15INK4B methylation is observed in 30–50% of MDS cases and is correlated with the percentage of bone marrow blasts, the risk of disease evolution toward AML and the poor prognosis, suggesting that p15INK4B methylation plays an important role in the pathogenesis of high-risk MDS and is related to leukemic transformation of MDS (72,73). These suggest that proliferation of leukemic cells might require an escape of regulation of the G1 phase of the cell cycle and possibly of TGFß inhibitory effect.
Decitabine (5-aza-2'-deoxycytidine) acts as a powerful demethylating agent in vitro. Clinically, low-dose decitabine ameliorates cytopenias including induction of trilineage responses in ~50% of patients with high-risk MDS (74). It is reported that repeated courses of low-dose decitabine induce cytogenetic remissions in a substantial number of elderly MDS patients with pre-existing chromosomal abnormalties; these are associated with improved survival compared with patients in whom the cytogenetically abnormal clone persists (75). MDS patients with high-risk chromosomal abnormalities may particularly benefit from this treatment.
III. MOLECULAR PATHOGENESIS
In spite of a multiplicity of endeavors to elucidate the molecular mechanisms of MDS, little is known about the pathogenesis of the first trigger or the early stage of MDS. Based on the accumulated knowledge on the genes involved in chromosomal translocations and somatic mutations, several signal transduction pathways emerge to be focused. Over the last decade, major signal transduction pathways triggered by exogenous stimuli including growth factors were identified and characterized. In relation to oncogenesis, one of the notable signal transduction pathways is now known as the RAS signaling pathway since RAS oncogene product is a key molecule in the development of a wide variety of tumors (76). Genetic alterations of some molecules on the RAS signaling pathway are revealed to be responsible for pathogenesis of MDS as well as leukemias. They are divided into two categories, receptor tyrosine kinases including FLT3, FMS and KIT and RAS and GAP-related protein, NF1, which are downstream of RTKs.
Normal cell growth through the cell cycle is regulated by the sequential formation, activation and subsequent inactivation of a series of cyclin–cyclin-dependent kinase (CDK) complexes. The mechanisms underlying the expression of cyclins and the activation of the different cyclin–CDK complexes required for progression through the successive cell cycle transitions are now well understood. In addition to positive regulation by the activation of cyclin–CDK complexes, negative regulation is also required at several checkpoints of the cell cycle. It is now well known that tumors can be caused by loss of the normal brakes, tumor-suppressor genes. p53 stimulates production of p21 which blocks cyclin–CDK complexes and therefore causes G1 arrest (77). Similarly, p15INK4B, p16INK4A and RB form complexes with cyclin or CDK and contribute to cell growth suppression. Among the cell cycle regulators, p15INK4B is most frequently and p53 less frequently involved in the pathogenesis of MDS, although not in the early stage but in the late stage of the disease.
In terms of the pathogenesis of the initial development of MDS, the most highlighted molecules are nuclear proteins, especially transcription factors that play important roles both in cellular development and regulation of cell lineage-specific gene expression. Among the genes encoding transcriptional regulators, of which alterations are detected in MDS or MDS/AML, are AML1, C/EBP TEL(ETV6), MLL and EVI-1. AML1, C/EBP and TEL(ETV6) are demonstrated to be essential in the hematopoietic cell development or differentiation by the gene targeting method in mice. AML1-binding sites were identified in the upstream of genes encoding factors and receptors that determine the lineage specificity of hematopoietic cells. AML1-targeted mice showed embryonic lethality at mid-gestation due to hemorrhage in the central nervous system and complete block of definitive hematopoiesis (78,79). C/EBP is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte colony-stimulating factor (G-CSF) receptor, neutrophil elastase and myeloperoxidase. Indeed, C/EBP knockout mice display a profound block in granulocyte differentiation (80). TEL-disrupted mice are embryonic lethal because of a yolk sac angiogenic defect. In the mouse chimeras with TEL-disrupted ES cells, TEL function is essential for the establishment of hematopoiesis of all lineages in the bone marrow, suggesting a critical role for TEL in the normal transition of hematopoietic activity from fetal liver to bone marrow (81).
It should be noted that quantitative or qualitative aberrations of these transcription factors are detected in primary MDS in addition to MDS/AML. Since such transcription factors elaborately regulate expression of cell lineage-specific genes that are required for hematopoietic cell differentiation, it is conceivable that their abnormalities could induce imbalance or blockage of hematopoietic cell differentiation, which might be observed as dyshematopoiesis or ineffective hematopoiesis in MDS. To demonstrate further the early genetic events in the development of MDS, it is necessary to generate MDS model mice genetically modified with such genetic lesions.
FOOTNOTES
+ Abbreviations: AML, acute myelogenous leukemia; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; FISH, fluorescence in situ hybridization; FPD, familial platelet disorder; GAP, GTPase-activating protein; G-CSF, granulocyte colony-stimulating factor; GEP, guanine nucleotide-exchange protein; GM-CSF, granulocyte–macrophage colony-stimulating factor; HDAc, histone deacetylase; HNPCC, hereditary non-polyposis colorectal cancer; IL, interleukin; IRF-1, interferon regulatory factor-1; JMML, juvenile myelomonocytic leukemia; LOH, loss of heterozygosity; MAPK, mitogen-activated protein kinase; M-CSF, macrophage colony-stimulating factor; MDS, myelodysplastic syndrome; MMR, mismatch repair; MSI, microsatellite instability; PDGFR-ß, platelet-derived growth factor receptor-ß; RTK, receptor tyrosine kinase; SSCP, single-strand conformation polymorphism; TGF ß, transforming growth factor ß; TNF, tumor necrosis factor 
For reprints and all correspondence: Hisamaru Hirai, Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Hongo, Tokyo 113-8655, Japan. E-mail: hhirai-tky{at}umin.ac.jp
REFERENCES
1 Gersuk GM, Beckham C, Loken MR, Kiener P, Anderson JE, Farrand A, et al. A role for tumour necrosis factor-alpha, Fas and Fas-Ligand in marrow failure associated with myelodysplastic syndrome. Br J Haematol 1998;103:176–88.[CrossRef][Web of Science][Medline]
2 Raza A, Gezer S, Mundle S, Gao XZ, Alvi S, Borok R, et al. Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood 1995;86:268–76.
3 Shetty V, Mundle S, Alvi S, Showel M, Broady-Robinson L, Dar S, et al. Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes. Leukemia Res 1996;20:891–900.[CrossRef][Web of Science][Medline]
4 Haase D, Feuring-Buske M, Schafer C, Schoch C, Troff C, Gahn B, et al. Cytogenetic analysis of CD34+ subpopulations in AML and MDS characterized by the expression of CD38 and CD117. Leukemia 1997;11:674–9.[CrossRef][Web of Science][Medline]
5 Loeb LA. Cancer cells exhibit a mutator phenotype. Adv Cancer Res 1998;72:25–56.[Web of Science][Medline]
6 Arzimanoglou II, Gilbert F, Barber HR. Microsatellite instability in human solid tumors. Cancer 1998;82:1808–20.[CrossRef][Web of Science][Medline]
7 Third MIC Cooperative Group Study. Recommendations for a morphologic, immunologic and cytogenetic (MIC) working classification of the primary and therapy-related myelodysplastic disorders. Cancer Genet Cytogenet 1988;32:1–10.[CrossRef][Web of Science][Medline]
8 Mecucci C. Molecular features of primary MDS with cytogenetic changes. Leukemia Res 1998;22:293–302.[CrossRef][Web of Science][Medline]
9 Van den Berghe H, Vermaelen K, Mecucci C, Barbieri D, Tricot G. The 5q– anomaly. Cancer Genet Cytogenet 1985;17:189–255.[CrossRef][Web of Science][Medline]
10 Boultwood J, Lewis S, Wainscoat JS. The 5q– syndrome. Blood 1994;84:3253–60.
11 Boultwood J, Rack K, Kelly S, Madden J, Sakaguchi AY, Wang LM, et al. Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion. Proc Natl Acad Sci USA 1991;88:6176–80.
12 Willman CL, Sever CE, Pallavicini MG, Harada H, Tanaka N, Slovak ML, et al. Deletion of IRF-1, mapping to chromosome 5q31.1, in human leukemia and preleukemic myelodysplasia. Science 1993;259:968–71.[Abstract]
13 Lezon-Geyda K, Najfeld V, Johnson EM. Deletions of PURA, at 5q31 and PURB, at 7p13, in myelodysplastic syndrome and progression to acute myelogenous leukemia. Leukemia 2001;15:954–62.[CrossRef][Web of Science][Medline]
14 Yoneda-Kato N, Look AT, Kirstein MN, Valentine MB, Raimondi SC, Cohen KJ, et al. The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM–MLF1. Oncogene 1996;12:265–75.[Web of Science][Medline]
15 Borkhardt A, Bojesen S, Haas OA, Fuchs U, Bartelheimer D, Loncarevic IF, et al. The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q. Proc Natl Acad Sci USA 2000;97:9168–73.
16 Yagasaki F, Jinnai I, Yoshida S, Yokoyama Y, Matsuda A, Kusumoto S, et al. Fusion of TEL/ETV6 to a novel ACS2 in myelodysplastic syndrome and acute myelogenous leukemia with t(5;12)(q31;p13). Genes Chromosom Cancer 1999;26:192–202.[CrossRef][Web of Science][Medline]
17 Jaju RJ, Fidler C, Haas OA, Strickson AJ, Watkins F, Clark K, et al. A novel gene, NSD1, is fused to NUP98 in the t(5;11)(q35;p15.5) in de novo childhood acute myeloid leukemia. Blood 2001;98:1264–7.
18 Johnson EJ, Scherer SW, Osborne L, Tsui LC, Oscier D, Mould S, et al. Molecular definition of a narrow interval at 7q22.1 associated with myelodysplasia. Blood 1996;87:3579–86.
19 Kratz CP, Emerling BM, Bonifas J, Wang W, Green ED, Beau MM, et al. Genomic structure of the PIK3CG gene on chromosome band 7q22 and evaluation as a candidate myeloid tumor suppressor. Blood 2002;99:372–4.
20 Side L, Taylor B, Cayouette M, Conner E, Thompson P, Luce M, et al. Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders. N Engl J Med 1997;336:1713–20.
21 Stephenson J, Lizhen H, Mufti GJ. Possible co-existence of RAS activation and monosomy 7 in the leukaemic transformation of myelodysplastic syndromes. Leukemia Res 1995;19:741–8.[CrossRef][Web of Science][Medline]
22 Luna-Fineman S, Shannon KM, Lange BJ. Childhood monosomy 7: epidemiology, biology and mechanistic implications. Blood 1995;85:1985–99.
23 Asimakopoulos FA, Holloway TL, Nacheva EP, Scott MA, Fenaux P, Green AR. Detection of chromosome 20q deletions in bone marrow metaphases but not peripheral blood granulocytes in patients with myeloproliferative disorders or myelodysplastic syndromes. Blood 1996;87:1561–70.
24 Asimakopoulos FA, Green AR. Deletions of chromosome 20q and the pathogenesis of myeloproliferative disorders. Br J Haematol 1996;95:219–26.[CrossRef][Web of Science][Medline]
25 La Starza R, Wlodarska I, Aventin A, Falzetti D, Crescenzi B, Martelli MF, et al. Molecular delineation of 13q deletion boundaries in 20 patients with myeloid malignancies. Blood 1998;91:231–7.
26 Mecucci C, Van Orshoven A, Vermaelen K, Michaux JL, Tricot G, Louwagie A, et al. 11q– chromosome is associated with abnormal iron stores in myelodysplastic syndromes. Cancer Genet Cytogenet 1987;27:39–44.[CrossRef][Web of Science][Medline]
27 Hoglund M, Johansson B, Pedersen-Bjergaard J, Marynen P, Mitelman F. Molecular characterization of 12p abnormalities in hematologic malignancies: deletion of KIP1, rearrangement of TEL and amplification of CCND2. Blood 1996;87:324–30.
28 Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell 1994;77:307–16.[CrossRef][Web of Science][Medline]
29 Wlodarska I, Mecucci C, Marynen P, Guo C, Franckx D, La Starza R, et al. TEL gene is involved in myelodysplastic syndromes with either the typical t(5;12)(q33;p13) translocation or its variant t(10;12)(q24;p13). Blood 1995;85:2848–52.
30 Carroll M, Tomasson MH, Barker GF, Golub TR, Gilliland DG. The TEL/platelet-derived growth factor beta receptor (PDGF beta R) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGF beta R kinase-dependent signaling pathways. Proc Natl Acad Sci USA 1996;93:14845–50.
31 Apperley JF, Gardembas M, Melo JV, Russell-Jones R, Bain BJ, Baxter EJ, et al. Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta. New Engl J Med 2002;347:481–7.
32 Salomon-Nguyen F, Della-Valle V, Mauchauffe M, Busson-Le Coniat M, Ghysdael J, Berger R, et al. The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL–ARNT fusion. Proc Natl Acad Sci USA 2000;97:6757–62.
33 Buijs A, Sherr S, van Baal S, van Bezouw S, van der Plas D, Geurts van Kessel A, et al. Translocation (12;22) (p13;q11) in myeloproliferative disorders results in fusion of the ETS-like TEL gene on 12p13 to the MN1 gene on 22q11. Oncogene 1995;10:1511–9.[Web of Science][Medline]
34 Raynaud SD, Baens M, Grosgeorge J, Rodgers K, Reid CD, Dainton M, et al. Fluorescence in situ hybridization analysis of t(3;12)(q26;p13): a recurring chromosomal abnormality involving the TEL gene (ETV6) in myelodysplastic syndromes. Blood 1996;88:682–9.
35 Yagasaki F, Jinnai I, Yoshida S, Yokoyama Y, Matsuda A, Kusumoto S, et al. Fusion of TEL/ETV6 to a novel ACS2 in myelodysplastic syndrome and acute myelogenous leukemia with t(5;12)(q31;p13). Genes Chromosom Cancer 1999;26:192–202.[CrossRef][Web of Science][Medline]
36 Ayton PM, Cleary ML. Molecular mechanism of leukemogenesis mediated by MLL fusion proteins. Oncogene 2001;20:5695–707.[CrossRef][Web of Science][Medline]
37 Mitani K, Kanda Y, Ogawa S, Tanaka T, Inazawa J, Yazaki Y, et al. Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation. Blood 1995;85:2017–24.
38 Taki T, Sako M, Tsuchida M, Hayashi Y. The t(11;16)(q23;p13) translocation in myelodysplastic syndrome fuses the MLL gene to the CBP gene. Blood 1997;89:3945–50.
39 Caligiuri MA, Strout MP, Schichman SA, Mrozek K, Arthur DC, Herzig GP, et al. Partial tandem duplication of ALL1 as a recurrent molecular defect in acute myeloid leukemia with trisomy 11. Cancer Res 1996;56:1418–25.
40 Radu A, Blobel G, Moore MS. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci USA 1995;92:1769–73.
41 Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, Toyama K, et al. Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nature Genet 1996;12:154–8.[CrossRef][Web of Science][Medline]
42 Raza-Egilmez SZ, Jani-Sait SN, Grossi M, Higgins MJ, Shows TB, Aplan PD. NUP98–HOXD13 gene fusion in therapy-related acute myelogenous leukemia. Cancer Res 1998;58:4269–73.
43 Nishiyama M, Arai Y, Tsunematsu Y, Kobayashi H, Asami K, Yabe M, et al. 11p15 translocations involving the NUP98 gene in childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome. Genes Chromosom Cancer 1999;26:215–20.[CrossRef][Web of Science][Medline]
44 Ahuja HG, Felix CA, Aplan PD. The t(11;20)(p15;q11) chromosomal translocation associated with therapy-related myelodysplastic syndrome results in an NUP98–TOP1 fusion. Blood 1999;94:3258–61.
45 Arai Y, Hosoda F, Kobayashi H, Arai K, Hayashi Y, Kamada N, et al. The inv(11)(p15q22) chromosome translocation of de novo and therapy-related myeloid malignancies results in fusion of the nucleoporin gene, NUP98, with the putative RNA helicase gene, DDX10. Blood 1997;89:3936–44.
46 Soekarman D, von Lindern M, Daenen S, de Jong B, Fonatsch C, Heinze B, et al. The translocation (6;9) (p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features. Blood 1992;79:2990–7.
47 Boer J, Bonten-Surtel J, Grosveld G. Overexpression of the nucleoporin CAN/NUP214 induces growth arrest, nucleocytoplasmic transport defects and apoptosis. Mol Cell Biol 1998;18:1236–47.
48 Jotterand Bellomo M, Parlier V, Muhlematter D, Grob JP, Beris P. Three new cases of chromosome 3 rearrangement in bands q21 and q26 with abnormal thrombopoiesis bring further evidence to the existence of a 3q21q26 syndrome. Cancer Genet Cytogenet 1992;59:138–60.[CrossRef][Web of Science][Medline]
49 Suzukawa K, Parganas E, Gajjar A, Abe T, Takahashi S, Tani K, et al. Identification of a breakpoint cluster region 3' of the ribophorin I gene at 3q21 associated with the transcriptional activation of the EVI1 gene in acute myelogenous leukemias with inv(3)(q21q26). Blood 1994;84:2681–8.
50 Mochizuki N, Shimizu S, Nagasawa T, Tanaka H, Taniwaki M, Yokota J, et al. A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells. Blood 2000;96:3209–14.
51 Mitani K, Ogawa S, Tanaka T, Miyoshi H, Kurokawa M, Mano H, et al. Generation of the AML1–EVI-1 fusion gene in the t(3;21)(q26;q22) causes blastic crisis in chronic myelocytic leukemia. EMBO J 1994;13:504–10.[Web of Science][Medline]
52 Kurokawa M, Mitani K, Irie K, Matsuyama T, Takahashi T, Chiba S, et al. The oncoprotein Evi-1 represses TGF-beta signalling by inhibiting Smad3. Nature 1998;394:92–6.[CrossRef][Medline]
53 Izutsu K, Kurokawa M, Imai Y, Maki K, Mitani K, Hirai H. The corepressor CtBP interacts with Evi-1 to repress transforming growth factor beta signaling. Blood 2001;97:2815–22.
54 Marshall MS. Ras target proteins in eukaryotic cells. FASEB J 1995;9:1311–8.[Abstract]
55 Hirai H, Kobayashi Y, Mano H, Hagiwara K, Maru Y, Omine M, et al. A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome. Nature 1987;327:430–2.[CrossRef][Medline]
56 Paquette RL, Landaw EM, Pierre RV, Kahan J, Lubbert M, Lazcano O, et al. N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome. Blood 1993;82:590–9.
57 Hirai H, Okada M, Mizoguchi H, Mano H, Kobayashi Y, Nishida J, et al. Relationship between an activated N-ras oncogene and chromosomal abnormality during leukemic progression from myelodysplastic syndrome. Blood 1988;71:256–8.
58 Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T, et al. Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines. Leukemia 1997;11:1605–9.[CrossRef][Web of Science][Medline]
59 Horiike S, Yokota S, Nakao M, Iwai T, Sasai Y, Kaneko H, et al. Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia. Leukemia 1997;11:1442–6.[CrossRef][Web of Science][Medline]
60 Sugimoto K, Hirano N, Toyoshima H, Chiba S, Mano H, Takaku F, et al. Mutations of the p53 gene in myelodysplastic syndrome (MDS) and MDS-derived leukemia. Blood 1993;81:3022–6.
61 Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D, et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nature Genet 1999;23:166–75.[CrossRef][Web of Science][Medline]
62 Osato M, Asou N, Abdalla E, Hoshino K, Yamasaki H, Okubo T, et al. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood 1999;93:1817–24.
63 Preudhomme C, Warot-Loze D, Roumier C, Grardel-Duflos N, Garand R, Lai JL, et al. High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21. Blood 2000;96:2862–9.
64 Imai Y, Kurokawa M, Izutsu K, Hangaishi A, Takeuchi K, Maki K, et al. Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis. Blood 2000;96:3154–60.
65 Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S, et al. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nature Genet 2001;27:263–70.[CrossRef][Web of Science][Medline]
66 Gombart AF, Hofmann WK, Kawano S, Takeuchi S, Krug U, Kwok SH, et al. Mutations in the gene encoding the transcription factor CCAAT/enhancer binding protein alpha in myelodysplastic syndromes and acute myeloid leukemias. Blood 2002;99:1332–40.
67 Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436–9.
68 Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature 1994;368:753–6.[CrossRef][Medline]
69 Hannon GJ, Beach D. p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. Nature 1994;371:257–61.[CrossRef][Medline]
70 Ogawa S, Hangaishi A, Miyawaki S, Hirosawa S, Miura Y, Takeyama K, et al. Loss of the cyclin-dependent kinase 4-inhibitor (CDK4I; p16; MTS1) gene is frequent in and highly specific to lymphoid tumors in human hematopoietic malignancies. Blood 1995;86:1548–56.
71 Herman JG, Jen J, Merlo A, Baylin SB. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B. Cancer Res 1996;56:722–7.
72 Uchida T, Kinoshita T, Nagai H, Nakahara Y, Saito H, Hotta T, et al. Hypermethylation of the p15INK4B gene in myelodysplastic syndromes. Blood 1997;90:1403–9.
73 Quesnel B, Guillerm G, Vereecque R, Wattel E, Preudhomme C, Bauters F, et al. Methylation of the p15(INK4b) gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood 1998;91:2985–90.
74 Wijermans P, Lubbert M, Verhoef G, Bosly A, Ravoet C, Andre M, et al. Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol 2000;18:956–62.
75 Lubbert M, Wijermans P, Kunzmann R, Verhoef G, Bosly A, Ravoet C, et al. Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. Br J Haematol 2001;114:349–57.[CrossRef][Web of Science][Medline]
76 Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–9.
77 Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 1995;82:675–84.[CrossRef][Web of Science][Medline]
78 Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 1996;84:321–30.[CrossRef][Web of Science][Medline]
79 Wang Q, Stacy T, Miller JD, Lewis AF, Gu TL, Huang X, et al. The CBFbeta subunit is essential for CBFalpha2 (AML1) function in vivo. Cell 1996;87:697–708.[CrossRef][Web of Science][Medline]
80 Collins SJ, Ulmer J, Purton LE, Darlington G. Multipotent hematopoietic cell lines derived from C/EBPalpha(–/–) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte-colony-stimulating factor and retinoic acid-induced granulocytic differentiation. Blood 2001;98:2382–8.
81 Wang LC, Swat W, Fujiwara Y, Davidson L, Visvader J, Kuo F, et al. The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow. Genes Dev 1998;12:2392–402.
Received January 21, 2003; accepted March 18, 2003
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