MDS/MPDs are a group of disorders whose features overlap those of CMPDs and MDSs and are thus difficult to assign to either group. The etiology and molecular basis of MDS/MPDs remain unknown. Clinical and laboratory findings can fall within a continuum ranging from those associated with MDSs to those found with CMPDs. Laboratory findings for Dido
-targeted mice are similarly difficult to assign to any of the murine nonlymphoid hematopoietic neoplasm categories proposed to date, which include nonlymphoid leukemias, nonlymphoid hematopoietic sarcomas, myeloid dysplasias, and myeloid proliferation (18
). The disease developed by Dido
-targeted mice has features of both myeloid dysplasias and myeloid proliferation and is therefore similar to MDS/MPDs. We found no CLL patients with abnormal hDido
expression and no Dido
-targeted mice with lymphocytosis. Instead, these mice showed increased myeloid cell numbers in marrow and spleen, which occasionally resulted in augmented numbers of circulating cells and enlarged spleens, symptoms compatible with nonlymphoid leukemias, myeloid proliferation, and MDS/MPDs. We ruled out nonlymphoid leukemias, since the percentage of blast cells in blood and bone marrow is always higher in leukemias (> 20% of all nucleated cells) than that observed in the Dido
-targeted mice. Diseased mice were often anemic, and a large number of their spleen and bone marrow cells had atypical nuclei (both symptoms compatible with a diagnosis of myeloid dysplasia or MDS/MPDs). In addition, granulocyte-macrophage and erythroid progenitors showed decreased colony formation accompanied by increased cluster formation potential, a pattern used to diagnose MDSs (11
) and MDS/MPDs (9
). Altogether, these findings in affected Dido
-targeted mice suggest that the disease is similar to human MDS/MPDs.
We are unaware of other reports to date of animal models for MDS/MPDs. A few murine models have been developed for CMPDs, by expression of a mutant K-rasG12D
protein from the endogenous locus (24
), by germline or somatic inactivation of Nf1 in hematopoietic cells (27
), or by expression of the oncogenic fusion gene AML1-ETO (31
). None of these mice can be considered MDS/MPD models; although they develop progressive myeloid proliferation, they show no evidence of dysplasia. Signal-induced proliferation–associated gene 1
–deficient) mice develop CML in chronic phase, CML in blast crisis, or MDSs, although none of these mice shows a mixed proliferative and dysplastic phenotype (25
). A murine model for MDSs was also developed recently by constitutively expressing EVI1 in bone marrow cells (33
). Notably, these mice do not show myeloproliferative symptoms.
Tumor suppressor genes can be inactivated during tumorigenesis through different mechanisms, including loss of expression, acquisition of inactivating mutations, or acquisition of mutations that make the protein gain a new function or work in a dominant-negative fashion. Our data indicate that 100% of MDS/MPD patients, 64% of MDS and CMPD patients, and 33% of AML patients showed clear loss of hDido
expression. Although the number of patients examined is relatively small, our data suggest that hDido
expression alterations might be associated with myeloid neoplasms more frequently than is any other known genetic lesion. Less than 40% of CMML and less than 20% of JMML patients have point mutations in Ras
genes at diagnosis or in the course of disease (34
), less than 30% of JMML patients show NF1
gene abnormalities (36
), and only some JMML patients have PTPN11
). The percentage of individuals with myeloid disorders displaying abnormal hDido
activity would be even higher if hDido
gene lesions that are undetectable in our assays (i.e., point mutations) were present in patients with normal hDido
expression levels. We have begun sequencing hDido cDNA from patients diagnosed with myeloid neoplasms who showed relatively normal hDido
expression levels; at least 1 contained a 143-bp insertion that caused a frameshift and led to premature translation termination (our unpublished observations).
The percentage of Dido
-targeted mice with MDS/MPD symptoms was nonetheless lower than that of patients with hDido
alterations, and a gene dosage effect was apparent. A dosage effect in tumorigenicity (haploinsufficiency) has been shown for a number of tumor suppressor genes (39
). Lower penetrance in mice than in humans could be attributed to variation between these 2 species, including distinct genetic requirements for aberrant proliferation, differential expression of tumor suppressor genes, or uncharacterized differences. Examples of these variations have been detailed in the case, for example, of retinoblastoma tumors and the retinoblastoma gene (rb
). The RB
gene was initially identified as a locus associated with retinoblastoma development (41
). Although individuals who inherit 1 mutant RB
allele develop this tumor with nearly 100% probability, rb+/–
mice develop pituitary and thyroid tumors but not retinoblastoma (42
Although the mechanism of MDS/MPD pathogenesis remains unknown, several recurring clonal cytogenetic abnormalities are found in MDS/MPD patients. Deletion of the long arm of chromosome 20 is the most common structural abnormality in myeloid malignancies, although it is rarely seen in lymphoid neoplasms (20
). Karyotypic analysis showed that 1 patient in this study diagnosed with MDS/MPDs had a deletion within 20q12 (our unpublished observations). Karyotypic analysis only detects large chromosomal aberrations; other patients might thus have small deletions affecting 20q. This genetic lesion is proposed to elicit loss of 1 or more unidentified tumor suppressor genes (21
). We report that the hDido
locus was found within this region and that its expression was reduced in all MDS/MPD patients studied and most of those with MDSs alone or CMPDs, as compared with controls. Furthermore, we show data suggesting that Dido
gene alterations contribute to myeloid but not to lymphoid tumor formation in mice. We speculate that hDido
is a gene on this chromosome whose loss could increase susceptibility to myeloid malignancies. As tumors arise after a latency period of several months, additional genetic changes are probably necessary for tumorigenic conversion of Dido
-targeted cells. Long latency periods are also reported for development of myeloproliferative diseases in SPA-1
– or ICSBP
-deficient mice and in mice bearing the oncogenic fusion gene AML1-ETO
). The possible cooperation of Dido
deficiency with other genetic alterations is currently under study.
Since many patients diagnosed with myeloid neoplasms show reduced hDido2 and hDido3 but normal hDido1 levels, we propose that inactivation of hDido2 and hDido3 but not of hDido1 might be involved in the formation of these tumors. To confirm this hypothesis, we are currently targeting exons shared by murine Dido2 and Dido3. In contrast, many human and murine cell lines of other origins show reduced Dido1 but relatively normal Dido2 and Dido3 levels. These data suggest that loss of Dido2 and/or Dido3 might be specific to myeloid tumors. The decrease in hDido2 and hDido3 in these patients is probably not due to variability in bone marrow cell composition, since in vitro–cultured murine bone marrow erythrocyte, granulocyte-monocyte, and B lymphocyte precursors express similar mDido1, mDido2, and mDido3 levels (our unpublished observations). Preferential loss of certain Dido isoforms could be explained by neoplasia-associated splicing aberrations or differences in mRNA stability.
As Dido1 was previously implicated in early onset of apoptosis (28
), we looked for increased or decreased apoptosis in several tissues and systems in Dido
-targeted mice but found no differences among genotypes (our unpublished observations). It is nonetheless possible that single targeting of mDido1
had an effect on apoptosis; alternatively, cell lineage or developmental stage may play a role in Dido-induced apoptosis. Dido2 or Dido3 overexpression in different cell types did not induce cell death (our unpublished observations); these proteins may instead be involved in genomic stabilization. We recently found that mDido3 localizes to the meiotic synaptonemal central element and contacts chromatin structures by direct binding to histones. The Dido common N terminal domain mediates histone interaction, which suggests that Dido1 and Dido2 also bind directly to histones. The truncated mDido3 protein in Dido
-targeted mice lacks the N terminal region and cannot interact with histones (van Wely et al., unpublished observations). The presence of this truncated Dido3 form in the mutant mice raises the possibility that their hematopoietic phenotype could be attributed to loss-of-function, dominant-negative, or gain-of-function effects exerted by the mutant protein. Although we cannot definitively rule out any of these possibilities, we believe that loss of function probably accounts for MDS/MPD development in mice since loss of function was identified in truncated mDido3 (histone binding) and loss of hDido function (by loss of expression) was found in all MDS/MPD and most CMPD, MDS, and AML patients analyzed. As a dominant-negative mutation can be even more disruptive to normal cell function than a null mutation (45
), we would expect the penetrance of a dominant-negative mutant in heterozygosity to be at least as high as that of a nullizygous mouse.
Compared with hematopoietic progenitors from WT mice, those from most diseased Dido
-targeted mice show decreased colony but increased cluster formation potential. This resembles a pattern associated with MDS and MDS/MPD patients, which is characterized by increased micro- and macrocluster formation, defective colony maturation, and a decrease in or lack of colony formation (9
). The meaning of this growth pattern is unknown, but it might indicate cell growth conditions that facilitate a hyperproliferative state since it correlates positively with increased risk of leukemic transformation (46
). Although the exact mechanism underlying the anomalies in MDS/MPD and MDS patients has not been identified, these defects are postulated to reflect an imbalance between cell proliferation and differentiation, as well as increased apoptosis.
Dido-targeted mice develop a disease similar to MDS/MPDs after a 7- to 8-month latency period, suggesting that additional genetic lesions are required. Once Dido-targeted cells accumulate these lesions and are transformed, they develop disease, as shown in our cell transfer experiments. These results could be explained by a role in genomic stabilization for Dido, as its loss of function would facilitate induction of additional genetic lesions affecting distinct proliferation, differentiation, or survival pathways. In this model, Dido+/neo mice, which express lower levels of fully functional mDido3 than WT animals, may be subject to an intermediate degree of genomic destabilization, between that of WT and Didoneo/neo mice, and thus to lower tumor penetrance than Didoneo/neo mice. Loss of heterozygosity would not be necessary for tumor formation in heterozygous mice. Our findings suggest that Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPDs and testing new approaches to their diagnosis and treatment.