EVI1 induces myelodysplastic syndrome in mice

Abstract

Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

Figure 1

EVI1 causes a fatal disease in reconstituted mice. (A) Retroviral DNA constructs used in Phoenix cell line. The 5′ long terminal repeat (LTR) provides the promoter for a transcript that includes EVI1 and a gene encoding resistance to G418 (NeoR, indicated by the arrow). Internal ribosome entry site (IRES) is required for the translation of the NeoR transcript. HIII is the unique HindIII restriction site present in the EVI1 cDNA. The location of the probe used for Southern blot analysis is indicated. HA, hemagglutinin epitope. (B) Western blot analysis of EVI1-producing packaging Phoenix cells (lane 1), vector-producing packaging Phoenix cells (lane 2), control mouse BM cells (lane 3), and EVI1-positive BM cells (lanes 4 and 5) confirms the appropriate expression of EVI1 only in EVI1-positive samples (lanes 1, 4, and 5). (C) Southern blot analysis of BM cells from reconstituted EVI1-positive mice (lanes 4 and 5) and control mice (lane 3). (D) Counts of wbc’s (diamonds, × 10³/μl), rbc’s (squares, × 10⁶/μl), platelets (horizontal bars, × 10⁵/μl), and levels of hemoglobin (crosses, g/dl) in PB of EVI1-positive mice (left) and in control mice (right). (E) The solid line shows the Kaplan-Meier survival curve of EVI1-positive reconstituted mice. All EVI1-positive mice died or were killed because of disease conditions. The dashed line represents the survival of the control mice.

Figure 2

EVI1 induces BM hypercellularity, dyserythropoiesis, erythroid and megakaryocytic hyperplasia, and apoptosis. Sections of normal spleen tissue (A and C) show normal white and red pulp. In comparison, EVI1-positive spleen tissue shows an expansion of red pulp and erythroid hyperplasia (B and dark cells in D). (E) Prussian blue iron staining of normal spleen tissue does not identify extensive iron deposition. (F) Iron depositions are evident in a section of EVI1-positive spleen tissue stained with Prussian blue. (H and J) Sections of EVI1-positive spleen tissue stained with cleaved caspase-3 antibody demonstrate the presence of apoptosis. (G and I) Normal spleen stained with cleaved caspase-3 antibody. (K and L) Control BM biopsy specimen shows normal cellularity and trilineage hematopoiesis. (M) In contrast, the EVI1-positive BM appears hypercellular, with erythroid and megakaryocytic hyperplasia. The BM dyserythropoiesis in EVI1-positive BM aspirates is shown (N), where the arrows point to nuclear irregularity and nuclear budding of erythroid precursors. (O) A PB smear of a control mouse is shown. In contrast, the PB smear of an EVI1-positive mouse shows anisopoikilocytosis (P), increased number of polychromatophilic rbc’s (Q), and Howell-Jolly bodies (R). Magnification, ×10 (A, B, E, F, G, H, K, M); ×40 (C, D, I, J, L, N, O, P, Q); ×100 (R).

Figure 3

EVI1 alters the response to cytokines and significantly increases the number of immature erythroid cells. (A_C) A total of 15,000 lineage-negative cells were isolated from control mice BM (black bars) or BM cells of moribund EVI1-positive mice (white bars). The cells were plated in duplicate in methylcellulose and were cultured with Epo (E) or GM-CSF (GM). After 3 days (Epo) or 7 days (GM-CSF) in culture, the colonies (left panels) and the cells (right panels) in each plate were isolated and counted. (A) The decrease in the number of colonies and cells of EVI1-positive mice shows that the BM cells of these animals have impaired in vitro differentiation. (B) The same assay was carried out with lineage-negative normal BM cells freshly infected with empty retrovirus (black bars) or EVI1-containing retrovirus (white bars). In contrast to the cells obtained from the moribund mice, EVI1 represses only the response to Epo in freshly infected BM cells. (C) The same assay was carried out with lineage-negative BM cells isolated from EVI1-positive mice 3 months after transplantation (white bars) or from age-matched controls (black bars). EVI1 represses only the response to Epo. (D) Cytospin preparations of control murine BM cells (left) or BM cells of moribund EVI1-positive mice (right) stained with Wright-Giemsa stain show that EVI1 delays in vitro differentiation as indicated by the smaller size and larger, less compact nuclei of the cells. (E) The spleens and BM of EVI1-positive mice (E, top panels) have a higher number of Ter119-positive cells than the organs of a control animal (C, bottom panels). Cells were stained with Ter119-PE and CD34-FITC. The percentage of positive cells for each quadrant is noted in the upper left corner of the quadrants. (F) RT-PCR analysis shows the expression of EVI1 in total BM cells of 3 moribund mice (lanes 1, 2, and 3), but not in the BM of a control mouse (lane 4). Analysis of sorted Ter119-positive cells of a moribund mouse (lane 5) confirms the expression of EVI1. EVI1 was not detected in Ter119-positive cells of the control (lane 6). For lane 7, no cDNA was added to the reaction. Gapdh was used as an internal standard.

Figure 4

EVI1 represses the expression of EpoR and c-Mpl. Real-time quantification of EpoR (squares) and c-Mpl (circles) normalized to Gapdh shows a significant decrease of these genes’ median expression in the BM of EVI1-positive mice 4 months after BMT or at the time of their disease-induced death. The results are plotted as the ratio between EpoR or c-Mpl and Gapdh multiplied by 100.