Introduction

Many blood disorders are associated with ageing. An example of this is myeloid malignancy, where more than 60% of patients diagnosed with myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukaemia are over 60 years old.1 It has been proposed that DNA mutations accumulate during the life of an individual as a consequence of failure to correct errors introduced in the genome during the cell replicative process, leading to either activation of oncogenes or silencing of tumour suppressors that operate as driving factors in the initiation of the disease.2, 3 Indeed, links have been established between age-related MPNs and specific cytogenetic abnormalities,4 and older patients with MPN exhibit a higher frequency of chromosomal defects.5

Intriguingly, a percentage of patients with MDS, MPN and myeloid leukaemia present cytogenetic abnormalities that include a deletion in the long arm of chromosome 20 (del20q).6, 7 It has been proposed that one or more genes within this region might be acting as tumour suppressors, the deletion of which could relate to the onset and/or progression of the disease, although the identity of the gene(s) remains unknown. One gene encoded within del20q with a role in cellular proliferation, DNA replication, maintenance of genome stability and senescence is the transcription factor MYBL2 (B-Myb).8, 9, 10, 11, 12, 13, 14, 15 Complete deletion of MYBL2 is embryonic lethal.16 To assess its role in ageing within the haematopoietic system, we have examined the haematopoietic phenotype in Mybl2 haploinsufficient mice (Mybl2+/Δ) upon ageing.17 We demonstrate that, Mybl2+/Δ mice are prone to develop MDS, MPN and lethal myeloid neoplasm, in contrast to animals expressing normal levels of Mybl2. Moreover, the tumourigenic effects of Mybl2 haploinsufficiency on the haematopoietic system were accelerated when haematopoietic stem cells were subjected to the proliferative stress imposed by bone marrow transplantation. Consistent with this result, we find a strong correlation between MYBL2 expression and 23 genes that are related to DNA replication and cell cycle checkpoint regulation in patients with MDS, regardless of their Del 20q status. Finally, we demonstrate that low MYBL2 expression status correlates with a poor prognosis in patients with high grade MDS (refractory anaemia with excess blasts (RAEB)), regardless of the cytogenetic abnormality. Taken together, our data point to MYBL2 levels as critical in haematopoiesis and suggest a causal role for MYBL2 in the development of a range of haematological malignancies. Our faithful mouse model will not only further inform us of myeloid leukaemogenesis but may also provide a model for preclinical testing of therapeutics.

Materials and methods

Mice and genotyping

All animal experiments were performed under an animal project licence in accordance with UK legislation. Mice were maintained on a C57/BL6 background. Mice were genotyped by PCR analysis.

Myeloid disorders classification

Three clinicians (pathologist/haematologists) from the Birmingham Queen’s Elizabeth Hospital and Royal Shrewsbury Hospital independently reviewed the cases and agreed with the Bethesda criteria for classification of blood disorders observed in Mybl2+/Δ mice.

Blood counts

Adult mice were bled in acid-citrate-dextrose solution (citric acid 6.8 mM, trisodium citrate 11.2 mM, glucose 24 mM) and blood counts obtained with an ABX Pentra 60 (ABX Diagnostics, Irvine, CA, USA) automatic blood counter.

Bone marrow, spleen and liver sections

Histological analysis of paraffin sections of tibias, spleen and liver was performed using standard methods. H&E and reticulin-stained sections were analysed under bright field microscopy.

Antibodies, flow cytometry and cell sorting

Single cell suspensions of bone marrow were prepared by standard techniques. Red cells were depleted, when required, by selective lysis. Non-specific binding of antibodies to Fc receptors was prevented by the use of anti-CD16/CD32 Fc-block (BD Pharmingen, Franklin Lakes, NJ, USA). Antibodies used are described in Supplementary Table 1. Antibodies used for lineage cocktail were B220, Ter119, CD5, CD8a, Gr-1 and Mac-1. Stained cells were analysed on a Cyan flow cytometer using Summit software (Dako, Glostrup, Denmark). For cell sorting, red cells were ACK-lysed (Ammonium-Chloride-Potasium) and after staining with the required conjugated monoclonal antibodies, samples were filtered through a 70-μm strainer and sorted on a Cytomation XDP MoFlo machine (Beckman Coulter, Brea, CA, USA). Live cells were selected by forward/side scatter gating and doublet discrimination.

Bone marrow transplants (replication stress assays)

Transplantations to determine whether replication stress decreases the time of disease appearance were performed by injecting 5 × 105 reference wild-type ACK-treated bone marrow cells (B6 × B6:SJL F1, CD45.1/CD45.2) together with 5 × 105 test donor cells from either wild-type or Mybl2+/Δ mice (B6, CD45.2/CD45.2) into the tail vein of host animals (B6:SJL, CD45.1/CD45.1) that had been lethally irradiated (975 rads). Peripheral blood was analysed every 4 weeks following transplantation using antibodies to distinguish CD45.1 and CD45.2 and a range of lineage-specific markers. After transplantation, the animals were scored for engraftment by immunofluorescent flow cytometry. Host and donor animals were 8–10 weeks old. Five different donors were used.

Quantitative reverse transcriptase-PCR

Real time PCR was performed as previously described.11 Primers are listed in Supplementary Table 2.

Statistical analysis

When comparing data sets between wild-type and Mybl2+/Δ animals, two-tailed unpaired Student's t-test was used and the unequal variance correction was applied. The significance of difference between expected and observed results was assessed by the χ2 test. When comparing more than two groups at the same time, Kruskal–Wallis one-way analysis of variance on ranks was used to calculate the significance of our results. For the Kaplan–Meier survival curve, statistical significance was determined with the log rank test. For all tests, a P value lower than 0.05 was considered significant.

Microarray

Preprocessed gene expression data for CD34+ cells from 183 patients with MDS and 17 healthy controls were obtained from the Gene Expression Omnibus (GEO) Data sets (GSE19429).18

Databases

Pathway enrichment was determined through query to the Analytical Web portal for high-throughput biology (www.bioprofiling.de), using R spider Tool combining signaling and metabolic pathways from Reactome and KEGG databases.19

Results

The MYBL2 gene is expressed in human and mouse bone marrow progenitor cells

The deletion of the long arm of chromosome 20 (del20q) is variable, but all patients contain a common deleted region (CDR). To date, five CDRs have been described in relation to del20q, each of them containing the MYBL2 gene (Supplementary Figure 1a).5, 7, 20, 21, 22 To investigate whether any of these genes is expressed in haematopoietic cells, we measured the expression of all nine genes contained within the smallest CDR published so far7 (PTPRT, SFRS6, L3MBTL, SGK2, IFT52, MYBL2, GTSF1L, TOX2 and JPH2), in human CD34+ and CD34 cells and granulocytes, as well as in mouse lineage-negative progenitor cells and more committed lineage-positive cells. Only five of these nine genes, including MYBL2, are expressed in human CD34+ cells (Supplementary Figure 1b). Similarly, mouse bone marrow lineage-negative progenitor cells show expression of the same five genes (Supplementary Figure 1c).

Ageing Mybl2+/Δ mice develop a mixed haematological malignant phenotype

As MYBL2 is present in haematopoietic cells and the gene is contained within the CDR of del20q, we decided to use our genetic modification of the MYBL2 locus to determine the importance of Mybl2 haploinsufficiency in the development of myeloid malignancy during ageing. Mybl2+/Δ mice were first generated by crossing Mybl2+/F mice17 with Zp3Cre transgenic animals to bring about deletion of the Mybl2 floxed allele at the zygote stage,23 whereby germline transmission was achieved, generating mice heterozygous for the deleted allele. As shown in Supplementary Figure 2a, Mybl2 haploinsufficiency correlates with an almost 50% reduction of Mybl2 mRNA levels. However, full blood counts (Supplementary Figure 3a) and bone marrow progenitor analysis (Supplementary Figure 3b) did not show any statistically significant cellular changes in animals at 3, 6 or 12 months of age, indicating that a reduction of MYBL2 levels does not impair haematopoietic development or immediately lead to myeloid disorders.

To assess the impact of Mybl2 haploinsufficiency during ageing, a cohort of 24 mice (13 Mybl2+/Δ and 11 Mybl2+/+ littermate controls) was maintained for up to 22 months. At that time, mice were killed for analysis, although three of the animals were killed 1 month earlier upon presenting signs of distress (Supplementary Figure 4).

Haematological disorders were evident in 12 out of the 13 Mybl2+/Δ mice, compared to only 1 of the controls (χ2=16.62, P-value 0.000045) with 9 out of 13 Mybl2+/Δ mice having enlarged spleens compared to only 1 of the controls (χ2=8.86, P-value 0.0029). The average spleen weight in wild-type mice (0.1±0.02 g) being in sharp contrast to the mean of 0.24±0.12 g in 8 out of 12 of the Mybl2+/Δ animals, and an extreme case with a spleen of 1.3 g. Using the Bethesda classification criteria,24 we ascertained that six of the animals developed MDS (6/13, 46%), five had a MPN (5/13, 38%) and one animal developed a myeloid leukaemia (1/13, 7%) (Supplementary Table 3).

In grouping the animals by type of disease, we could determine that the decrease in WBC and RBC observed in MDS mice were statistically significant, mirroring the common anaemia and leucopenia seen in the human disease. In addition, in the animals with MPN, a statistically significant increase in WBC and a decrease of RBC were observed mirroring haematological indices in some human MPN (Figure 1a). By contrast, platelet numbers varied greatly within each disease-group, with some animals showing thrombocytopenia and others thrombocytosis (Figure 1a). However, this platelet heterogeneity is not uncommon in human MPN and MDS subtypes. By analysing the progression of WBC, RBC and platelet numbers at established time-points during the life of the animals, we could determine that signs of anaemia and changes in WBC counts were detectable between 12 and 18 months of age and exacerbated by 22 months (Figure 1b). We also observed that, regardless of the type of disease developed, no re-expression of Mybl2 or compensation from the other allele took place (Supplementary Figure 2b). It was possible that the loss of sequences within the Mybl2 locus in our mouse model removed a regulatory element important for the expression of neighbouring genes. If that would be the case, the affected gene might effectively participate with Mybl2 in the development of the disease. To rule out this possibility, we measured mRNA expression of these genes in diseased mice. Out of four potentially expressed neighbouring genes, expression of L3mbtl and Gtsf1l could not be detected in wild-type mice, Srsf6 and Itf52 were expressed, but the level of mRNA was not altered in the ageing mice with either the MPN or MDS phenotypes when compared to equivalent age wild-type littermate animals (Supplementary Figure 5), confirming that the only gene deregulated in diseased mice was Mybl2.

Figure 1
figure 1

Blood values in ageing mice by disease. (a) Cell counts of WBC (left panel), RBC (middle panel) and platelets (right panel) from the wt (black bars), Mybl2+/Δ mice that developed MDS (white bars) and the Mybl2+/Δ mice that developed MPN (grey bars). Kruskal–Wallis test was used to calculate P-value. (b) Charts showing blood values progression of WBC (left panel), RBC (middle panel) and platelets (right panel) at different time-points during the life of three Mybl2+/Δ mice that developed MDS (blue), three Mybl2+/Δ mice that developed MPN (red) and three wild-type mice (green). Each colour represents one mouse.

Haploinsufficiency of Mybl2 (Mybl2+/Δ) predisposes for multiple myeloid disorders

As described above, during ageing, Mybl2+/Δ mice developed MDS, MPN and myeloid neoplasms. The haematological characteristics of mice within each disease-group are summarised below.

MDS in aged Mybl2 +/Δ mice

Six out of thirteen Mybl2+/Δ mice developed MDS, mirroring the haematological characteristics and heterogeneity of human MDS disease. The peripheral blood of these animals exhibited neutropenia, and displayed dyserythropoiesis that included size variation (anysocytosis), shape variation (poikilocytosis) and the presence of Howell–Jolly bodies (Figure 2a). Platelet counts were also affected, with animals showing either thrombocytopenia (390 × 103/mm3 platelets) or thrombocytosis (2900 × 103/mm3 platelets).

Figure 2
figure 2

Ageing Mybl2+/Δ mice are prone to develop MDS. (a) Peripheral blood smear from a Mybl2+/Δ mouse stained with DiffQuick where anysocytosis and poikilocytosis are present. (b) Characterisation using Gr-1 and Mac-1 surface markers of peripheral blood and spleen from wild-type and two different Mybl2+/Δ animals. (c) Charts showing the percentage of myeloid cells on peripheral blood and spleen from wild-type and Mybl2+/Δ animals. (d) Characterisation using CD71 and Ter119 surface markers of spleen erythroid cells from wild-type and two different Mybl2+/Δ animals.

Flow cytometric analyses of peripheral blood and spleen revealed an increase in myeloid populations in Mybl2+/Δ mice compared with controls. The affected myeloid population from diseased mice differed in its surface marker signature, varying between Mac1+Gr1, Mac1Gr1+ or Mac1+Gr1+ (Figures 2b and c). Further, one animal displayed a striking case of dyserythropoiesis in the spleen, with an abnormal erythroid maturation profile made up of cells expressing lower levels of both Ter119 and CD71 (red circle) (Figure 2d). To determine the aggressiveness of MDS in these animals, we performed bone marrow transplants into sublethally irradiated hosts. The transplanted cells engrafted poorly, even from animals with a more severe phenotype, and did not show any proliferative advantage 8 weeks after transplantation (Supplementary Figure 7a), mimicking the inability of human MDS cells to induce clinical disease in xenotransplants.

MPN in aged Mybl2 +/Δ animals

Five out of thirteen Mybl2+/Δ animals developed a MPN. H&E staining of bone marrow sections revealed that while aged wild-type mice displayed accumulation of fat cells, Mybl2+/Δ animals exhibited myeloproliferation with high cellularity and practically no fat cells (Figure 3a). Reticulin staining of bone marrow sections was negative, indicating lack of fibrosis (Supplementary Figure 6b). The heterogeneity observed in MPN patients was also observed in MPN Mybl2+/Δ mice. Thus, three mice had leukocytosis with WBC counts as high as 25 × 103/mm3, with or without anaemia, with or without thrombocytopenia. Flow cytometric analyses revealed an increase in the myeloid population in peripheral blood, bone marrow and spleen (Figure 3b).

Figure 3
figure 3

Ageing Mybl2+/Δ mice develop MPN. (a) H&E staining of bone marrow paraffin sections from wild-type littermate controls (left panels) and Mybl2+/Δ showing myeloproliferation (right panels). (b) Characterisation using Gr1 and Mac-1 surface markers of peripheral blood (PB), bone marrow (BM) and spleen (Sp) from wild-type and Mybl2+/Δ MPN animals. (c) H&E staining of spleen paraffin sections from wild-type littermate controls (left panels) and Mybl2+/Δ showing myeloproliferation (right panels). (d) Characterisation using CD41 and c-Kit surface markers of BM from wild-type and a Mybl2+/Δ animal showing high number of platelets in peripheral blood.

The remaining two MPN mice constituted a separate group displaying severe thrombocytosis resembling human essential thrombocythemia, with platelet values as high as 3000 and 6990 × 103/mm3 (normal levels in ageing mice being 1000–1500 × 103/mm3) and a MPV of 13.2 μm3 (normal values around 6 μm3). A high content of megakaryocytes was detected in the spleen by H&E staining of tissue sections (Figure 3c) and in the bone marrow by flow cytometry (Figure 3d).

Bone marrow transplantation into sublethally irradiated hosts showed a good engraftment (50%) of the MPN cells at 8 weeks after transplantation, in clear contrast with the poor engraftment of the MDS cells (Supplementary Figure 7b). Nevertheless, no signs of MPN disease were observed. No increase in the percentage of the MPN transplanted population was detected even 8 months after transplantation and the transplanted animals did not die as a consequence of the transplanted cells.

Lethal myeloid neoplasm in one Mybl2 +/Δ mouse

One of the aged Mybl2+/Δ mice developed a myeloid neoplasm with marked leukocytosis, anaemia and an increase in the number of platelets. A mild increase in the number of platelets and the tendency to anaemia could be observed at 12 and 18 months of age, whereas the massive increase of WBC started after 18 months (Supplementary Figure 8a), only 3 months before the animal had to be killed due to signs of distress.

Blood smears showed the presence of immature myeloid cells (Supplementary Figure 8b). The profound anaemia in this Mybl2+/Δ mouse was reflected by the white appearance of the bones (Supplementary Figure 8c). This mouse also developed splenomegaly (Supplementary Figure 8d), and H&E staining showed a disruption of spleen architecture, with loss of lymphocytic follicles and an increase in the number of megakaryocytes (Supplementary Figure 8e). Likewise, staining of liver sections showed infiltration of myeloid cells (Supplementary Figure 8f).

The overall profile of bone marrow represented by the side versus forward scatter plots revealed that the Mybl2+/Δ cells were homogeneous, a feature typical of myeloid leukaemia (Supplementary Figure 8g). Furthermore, we detected a dramatic increase in the percentage of monocytes and granulocytes, with Gr1+Mac1+ cells constituting more than 50% of the total population in peripheral blood, bone marrow, spleen and liver (Supplementary Figure 8h), which is in agreement with the observations from blood smears and sections. Together with the high platelet numbers and increased spleen megakaryocytes, these features were collectively suggestive of a MPN that had progressed to a myeloid leukaemia, a progression that also occurs in human MPN patients.

Transplantation of bone marrow Mybl2+/Δ cells accelerates development of MDS

As MYBL2 has a role in DNA replication and maintenance of genome integrity, we speculated that the haematological disorders observed in Mybl2+/Δ animals could be the consequence of an accumulation of replication errors. We reasoned that if the phenotype originated in haematopoietic stem cell, as is suggested by the abnormalities of multiple myeloid lineages, then by subjecting the haematopoietic system to replicative stress, we might accelerate disease development. We therefore performed transplantation of total bone marrow cells into lethally irradiated recipients. To detect changes in relative proliferation of the test-donor cells (Mybl2+/+ or Mybl2+/Δ), we injected these as a 1:1 mixture with wild-type reference cells (Mybl2+/+). Test-donor and wild-type reference cells could be distinguished by their differential expression of CD45 allelic variants, that is, CD45.2+ (test donor) versus CD45.1+/CD45.2+ (reference donor). Host mice were CD45.1+, which allowed discrimination from transplanted cells. In total, 25 transplanted animals were analysed, of which 10 and 15 had received wild-type or Mybl2+/Δ CD45.2+ donor cells, respectively.

Five out of fifteen (33%) animals that received Mybl2+/Δ donor cells exhibited a relative increase in the ratio of test:reference reconstitution (χ2=4.16, P-value=0.041) (Figures 4a and b). The mice with increased reconstitution developed MDS 6–9 months after transplantation, whereas none of the 10 animals that were transplanted with control cells showed signs of such a change. Recipients exhibiting the proliferative advantage of the Mybl2+/Δ test cells displayed a phenotype like the ageing mice described above, with splenomegaly (Figure 4c), low WBC counts, anaemia (Figure 4d) and thrombocytopenia. Blood smears showed dyserythropoiesis with signs of polychromasia, anysocytosis and poikilocytosis (Figure 4e).

Figure 4
figure 4

Animals transplanted with Mybl2+/Δ donor cells are more prone to develop MDS disorders. (a) Chart graph shows donor test:reference ratio at different time-points. Two different sets of wild-type donors (blue) and three sets of Mybl2+/Δ donors (red/orange), each carried out in duplicate, are shown. (b) Reference/donor profiles of peripheral blood (PB), bone marrow (BM) and spleen (Sp) at 32 weeks after transplantation from one wild-type donor and two Mybl2+/Δ donors that developed MDS. (c) Picture showing splenomegaly in animals transplanted with Mybl2+/Δ bone marrow cells (right) compared with animals transplanted with wild-type bone marrow cells (left). (d) Pictures showing pale blood from one mouse transplanted with Mybl2+/Δ bone marrow cells (right) compared with normal colour blood from an animal transplanted with wild-type bone marrow cells (left). (e) Diffquick staining of blood film from a mouse transplanted with Mybl2+/Δ bone marrow cells showing polychromasia and anysocitosis and poikilocytosis. (f) Analysis of donor (CD45.1/CD45.2+) contribution to the erythroid lineage in BM and Sp in animals transplanted with wild-type donor and two animals transplanted with MYBL2+/Δ donor cells. (g) Analysis of donor (CD45.1/CD45.2+) contribution to myeloid lineages in PB, BM and Sp in animals transplanted with wild-type donor and Mybl2+/Δ donor cells. (h-i) Chart graphs show the percentage of donor (CD45.1/CD45.2+) contribution to (h) KSL cells and (i) multipotential progenitors (MPP) in five animals transplanted with wild-type donor cells and the five animals transplanted with Mybl2+/Δ donor cells that developed MDS. (j) Chart graph shows MPP/KSL ratios. Each symbol refers to individual animals. Red bars represent the average value (n=5).

In the transplanted mice that developed MDS, we determined the contribution of test–donor cells to the erythroid and myeloid lineages by gating on the CD45.1/CD45.2+ population. Erythroid lineage maturation appeared to be the most affected, with accumulation of erythroblasts (CD71+ Ter119) in the bone marrow, erythropoiesis detected in the spleen and marked dyserythropoiesis (Figure 4f). Unlike reference cells, which yielded lineage cells at the expected turnover, Mybl2+/Δ cells contributed to a marked increase in the proportion of myeloid cells in peripheral blood, bone marrow and spleen (Figure 4g). In addition, animals developing MDS upon transplantation with Mybl2+/Δ cells showed an increased percentage of the Kit+Sca1+Lin (KSL) (Figure 4h) or Kit+Sca1Lin (Figure 4i) progenitor populations compared with animals transplanted with control cells, skewing the ratio of KSL:multipotential progenitors from the typical 1:10 to above (1:3) or below (1:23) the normal ratio (Figure 4j), indicating that the proportion of progenitor cells and KSL cells is deregulated in animals that developed MDS upon transplantation with Mybl2+/Δ cells.

Low MYBL2 expression is detected in human MDS patients suffering RAEB

To test the relevance of our findings in the mouse model for human myeloid disorders, we investigated whether low levels of MYBL2 expression were associated with MPN and MDS in humans, regardless of del20q cytogenetic abnormality. For this purpose, we made use of MDS patient gene-profiling arrays previously published by Pellagatti et al.18 Based on the French–American–British (FAB) cooperative study group classification and its subsequent modifications by the World Health Organisation (WHO), a cohort of 183 MDS patients was subdivided into three categories based on phenotypic characteristics, that is, MDS with refractory anaemia, refractory anaemia with ring sideroblasts and RAEB, the latter being further subclassified into RAEB1 and RAEB2 subgroups according to the percentage of bone marrow blasts (Supplementary Table 4).

Comparison of MYBL2 levels in CD34+ cells, from normal donors versus patients within each subgroup, revealed a significant decrease in MYBL2 expression in MDS patients with RAEB, with levels down to 54% in MDS RAEB2 patients (Figure 5a). In contrast, very little variation was observed in the expression of the other four genes that are associated with del20q in MDS patients (Figure 5b). These findings therefore show a decreased expression of the MYBL2 gene in human MDS subgroups that have been associated with poorer prognosis.25

Figure 5
figure 5

Low MYBL2 expression is detected in human MDS patients with poor prognosis. (a) Chart graph shows MYBL2 expression levels in CD34+ cells from 183 MDS patients, subdivided into different subclassifications according to WHO, compared to CD34+ cells from healthy donors. P values are indicated for statistically significant differences (two-tail Student’s t-test unpaired unequal variances). (b) Chart graph shows percentage of mean expression of genes contained within del20q minimum common deleted region (MYBL2, SFSR6, ITF52, GTSF1L and L3MBTL) in CD34+ cells from 183 MDS patients, subdivided into different subclassifications according to WHO, compared with CD34+ cells from healthy donors (*P<0.05; **P<0.01; ***P<0.001).

Although MPN data is scarce, by using arrays made public by Guglielmelli et al.,26 we were able to compare MYBL2 expression in CD34+ cells from three normal donors versus three patients with idiopathic myelofibrosis. Similar to the MDS patients described above, MYBL2 expression appears to be significantly lower in MPN patients (Supplementary Figure 9).

MYBL2 expression correlates with DNA replication and checkpoint proteins in human MDS

In an effort to better understand which biological processes may associate with the low expression level of MYBL2 in MDS, we sought to identify genes whose expression correlated specifically with that of MYBL2 in human MDS. We used the Pearson correlation coefficient as a measure of relationships between levels of expression of different genes, and evaluated the link between expression of each gene against the level of the MYBL2 transcript. A threshold Pearson correlation coefficient of 0.78 (and P value <0.001) was set as a filter to isolate strong and highly significant correlations.

This analysis identified 23 MYBL2-correlated genes in CD34+ cells from patients with MDS RAEB (Figure 6a and Supplementary Figure 10). This list of genes was used to query protein reaction and pathway databases via the R spider tool,19 resulting in the mapping of 16 genes to its reference global network demonstrating a strong enrichment in mitotic (P=0.01), DNA replication (P=0.01) and cell cycle checkpoints (P=0.015) pathways (Figure 6b). Hence, MYBL2 particularly associates with genes regulating the cell cycle of MDS RAEB CD34+ cells. To assess whether MYBL2 expression influenced mRNA levels of these genes, we tested their respective expression in MDS cells from our Mybl2+/Δ ageing mice model. As shown in Figure 6c, the expression of the cell cycle proteins cdc6, Birc 5, cdc20, Ube2C, cycA2 and Orc1L were also downregulated in Mybl2+/Δ cells paralleling the lower expression levels of these genes in human MDS patients.

Figure 6
figure 6

MYBL2 expression signature in MDS. (a) List of genes whose expression correlates highly with MYBL2 expression in MDS, as measured by the Pearson correlation coefficient. (b) Enriched pathways in MYBL2 high correlated genes. (c) Relative gene expression analysis relative to β2-microglobulin in Mybl2+/Δ Mac-1+ cells coming from ageing MDS mice. Six cell cycle genes that show high correlation to MYBL2 in human MDS patients were analysed using quantitative PCR.

Discussion

The work described here shows a direct correlation between MYBL2 expression levels and the occurrence of haematological pathologies such as MPN, MDS and myeloid leukaemia, therefore establishing deregulation of MYBL2 expression as a contributory factor in these myeloid disorders. In humans, these conditions have a higher occurrence in the elderly and a variable tendency of progression to acute myeloid leukaemia in patients diagnosed with MDS or MPN. Strikingly, we observe the same phenotype in mice expressing half the normal levels of Mybl2, providing in vivo evidence that Mybl2 haploinsufficiency triggers haematopoietic defects and induces myeloid malignancies.

The observed phenotype was reminiscent of human disease in more than one aspect. First, transplantation of bone marrow cells from the MDS or MPN mice into sublethally irradiated animals did not result in disease after 8 weeks despite positive engraftment, paralleling the inability of human MDS and MPN cells to induce clinical disease in xenotransplants.27, 28 Second, we were able to accelerate disease development by inducing replication stress in haematopoietic cells, suggesting that, corresponding to its role in protecting chromosome integrity,10 MYBL2 acts as a stabilising factor to prevent accumulation of random secondary mutations that contribute to the development of myeloid malignancies. The random nature of these mutations would also explain the partially penetrant MPN and MDS phenotypes that we have demonstrated. However, the accelerated appearance of hematological disorders only resulted in the appearance of MDS. We speculate that the lack of animals showing MPN disease could be because they required a longer period of time to develop. This could be possible as in our experiments once the first mouse from the cohort showed signs of disease the whole cohort was killed. The MDS animals presented an increase in the percentage of the Kit+Lin populations, which is also a feature in patients with MDS.27, 28

During ageing, there are two major changes in the haemopoietic system: the diminution of the adaptive immune system and an increase in the appearance of myeloproliferative disease.29 A simple explanation for the lack of lymphoid malignancies in Mybl2 haploinsufficient mice could be precisely the fact that with age, the haemopoietic system is skewed towards the production of myeloid cells at the expense of the lymphoid lineage. Alternatively, lower levels of Mybl2 may impact differently on specific cell types, having its greatest effect in committed myeloid progenitors.

Importantly, our model of Mybl2 haploinsufficiency recapitulates the appearance of blood disorders in humans during ageing in a way that has distinct advantages over many previously described models. For instance, Pten+/−Ship−/− mice30 and animals overexpressing Evi131 have lifespans of only 5 to 11 weeks, preventing their utilisation in long-term studies, as we have described here. Furthermore, blood disorders in these mice cannot be classified as MDS or leukaemia, so that the models fall short of expectations for comparison with human pathologies. In a recent SNP array-based karyotyping study of MPN, haploinsufficiency has been detected at high frequency for genes encoding transcription factors such as CUX1, IKZF1, ETV6, FOXP1 and RUNX1.5

Del20q is an interstitial deletion commonly found in these disorders,32, 33 and the minimal CDR has been shown to encompass a number of genes, among which the gene encoding polycomb protein L3MBTL1 is perhaps the best characterised. Interestingly, L3MBTL1 expression appears to be normal in human myeloid malignancies,20 which is consistent with our analysis in 183 MDS patients. Moreover, studies using L3mbtl1 knockout mice showed that these do not develop haematological disorders, even when allowed to age for more than 2 years,34 indicating that haploinsufficiency of L3mbtl1 does not contribute to the development of haematopoietic disorders in vivo, and that it is not a critical tumour suppressor in the context of del20q in human myeloid malignancies.

Our results demonstrate that (i) the earlier appearance of blood disorders when Mybl2+/Δ cells are subjected to proliferation stress; (ii) lower MYBL2 expression levels in MDS patients with no del20q abnormality; and (iii) significant correlation between expression levels of MYBL2 and a subset of DNA replication/cell cycle regulation genes in human MDS patients indicate that MYBL2 indeed acts to protect against haematological malignancy. In MDS human patient samples, a deregulation of cell survival and cell cycle regulation pathways has been reported.18 In fact, some of the genes that signature correlates with MYBL2, such as birc5 and cdc20, have been described to be downregulated in MDS patients.35, 36 This positive correlation is in agreement with a known role for MYBL2 in cell proliferation and genome stability.8, 9, 10, 11, 12, 13, 14, 15 Although these two genes, birc 5 and cdc20, have not been shown to be direct targets of MYBL2, they are targets of FoxM1, a gene which is a master regulator of the G2/M phase and previously shown to be transcriptionally regulated by MYBL2 in embryonic stem cells.11

Just recently, Down et al.37 have described that in the absence of MYBL2, FoxM1 cannot bind to nor activate birc5. This study proposes that MYBL2 is required as a pioneer factor to enable FoxM1 binding to G2/M gene promoters.37 This could explain the large number of FoxM1 known target genes (Ube2c, cdc20, birc5, cenpF, cyclin B2) that correlated with the MYBL2 signature in MDS. In this way, the control of cell division might be impaired by low levels of MYBL2, altering cell fates established during cell division in early haematopoietic stem and progenitor cells that lead to clonal expansion with imbalance or impaired differentiation.

Our results linking MYBL2 deficiency with development of MPN and MDS are consistent with recent data from the group of Thomas Look (http://ash.confex.com/ash/2010/webprogram/Paper31436.html), who showed that MYBL2 levels were downregulated in about 70% of MDS patients. Moreover, two different point mutations in the MYBL2 gene were identified in a subset of patients with MDS who lacked del20q.

MYBL2 has recently been reported to be negatively regulated by two different miRNAs, miR-29 and miR-30.15, 38 In this context, Slape and colleagues showed up regulation of both miRNAs during leukaemic transformation. It is possible that downregulation of MYBL2 upon overexpression of miR-29 may account for the development of haematological neoplasms in human patients that lack del20q deletions or mutations of MYBL2. This observation has clinical implications, as it illustrates how individual factors may still intervene as a molecular switch in the onset of blood pathologies without prior damage to their primary gene structure, thus rendering the gene undetectable to genome-wide sequencing approaches that aim to define the molecular basis of the disease and to find specific gene mutations with diagnostic or prognostic value. Indeed, our observation that MYBL2 levels are downregulated in those MDS patients with poor prognosis (RAEB), regardless of cytogenetic abnormalities, indicates that mechanisms other than gene deletion must operate that bring MYBL2 levels down.

Recent reports have pointed out that mutations in the splicing factor gene SF3B1 are present in 20% of MDS patients, and with a high frequency (65%) in those patients that present ring sideroblasts, which is the subtype with better prognosis.39, 40, 41 This is in clear contrast with MYBL2 gene, which is significantly downregulated in those patients with a worse prognosis (RAEB). Thus, mutations in SF3B1 might be an indicative of a more favourable prognosis in MDS, whereas downregulation of MYBL2 might suggest a worse prognosis. If validated through clinical observation, both genes could potentially serve as prognostic biomarkers of disease progression and have an impact in prospective therapeutic strategies for MDS patients.

In summary, we have generated mice haploinsufficient for Mybl2 that display age-related haematological neoplasms reminiscent of human disease. MYBL2 seems to be acting as a triggering factor in the onset of blood malignancies through its ability to affect the expression of genes linked to DNA-replication and check-point control, ultimately impairing the physiological balance of blood cell turnover. Our model system could represent a valuable tool for identifying collaborative mutations that define the development of a particular MPN, MDS or transformation to leukaemia.