Trisomy of Erg is required for myeloproliferation in a mouse model of Down syndrome

Down syndrome (DS) is uniquely associated with a transient myeloproliferative disorder (DS-TMD) and acute megakaryocytic leukemia (DS-AMKL).¹  Increased dosage of genes within chromosome 21 (Hsa21), including AML1/RUNX1, ETS2, and ERG, has been postulated to cooperate with an acquired GATA1 mutation to generate DS-TMD and DS-AMKL,²  although direct evidence identifying critical genes is lacking.

A member of the ETS family of transcription factors,³ ERG is necessary for normal platelet development and stem cell function⁴  and has been implicated in the pathogenesis of human leukemia.^5-7  Located on the long arm of Hsa21,⁸  evidence for a role of ERG in the development of human DS-TMD and DS-AMKL arose from detection of ERG expression in human acute megakaryoblastic leukemia cell lines and primary human samples of DS-AMKL and DS-TMD.²  In vitro, overexpression of ERG can induce megakaryocytic differentiation of human cell lines and murine fetal liver progenitors,^4,9,10  and can interact with GATA1 mutations to immortalize fetal liver progenitors.^9,10 

The Ts(17¹⁶)65Dn mouse is a well-characterized model of DS containing a trisomic chromosomal Down syndrome critical region (DSCR) syntenic to Hsa21, including 94 orthologs of the 170 genes within Hsa21.¹¹  Ts(17¹⁶)65Dn mice develop a highly penetrant myeloproliferative disorder (MPD),¹²  supporting the contention that trisomy of critical gene(s) within Hsa21 can lead to hyperproliferation and megakaryocytic differentiation, and may therefore predispose to the development and phenotype of the megakaryoblasts observed with human DS-TMD and DS-AMKL.

To determine whether trisomy of functional Erg drives the development of the myeloproliferative phenotype observed in Ts(17¹⁶)65Dn mice, trisomic Ts(17¹⁶)65Dn mice were crossed to mice carrying the loss-of-function Erg^mld2 mutation that contains a S329P nontransactivating missense mutation in the DNA-binding region of Erg,⁴  to generate mice disomic for functional Erg but trisomic for all other genes within the DSCR of Ts(17¹⁶)65Dn.

Derivation and genotyping of the Erg^mld2 mutant allele has been described.⁴  Ts(17¹⁶)65Dn mice (The Jackson Laboratory) were maintained on an F1 background of C57BL/6JEiJ and C3HHeSnJ. All mice were derived from the first-generation progeny of matings between Erg^+/mld2 and Ts(17¹⁶)65Dn mice. Genotyping for segmental trisomy of chromosome 16 was performed using the MLPA Reaction Kit (MRC Holland).¹³  Experiments were performed with procedures approved by the Melbourne Health Research Directorate or The Walter and Eliza Hall Institute of Medical Research Animal Ethics Committees.

Blood was collected into tubes containing ethylenediaminetetraacetic acid (Becton Dickinson) and analyzed with an Advia 120 analyzer (Bayer). Single-cell suspensions from 1 femur and spleen were collected in balanced salts solution (0.15M NaCl, 4mM KCl, 2mM CaCl₂, 1mM MgSO₄, 1mM KH₂PO₄, 0.8mM K₂HPO₄, and 15mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid supplemented with 2% [vol/vol] bovine calf serum). Bone marrow cells (2.5 × 10⁴) were cultured in 1-mL volumes of 0.3% agar in Iscove modified Dulbecco medium containing 20% newborn calf serum and stem cell factor (50 ng/mL), erythropoietin (2 U/mL), and interleukin-3 (10 ng/mL), incubated for 7 days in a fully humidified atmosphere of 5% CO₂ in air, fixed, dried onto glass slides, and stained for acetylcholinesterase, Luxol fast blue, and hematoxylin, and the number and type of colonies were determined. Organs were fixed in 10% buffered formalin and embedded in paraffin, and 1- to 3-μm sections were stained with hematoxylin and eosin or silver nitrate for reticulin visualization. Megakaryocytes were enumerated by microscopic examination of bone marrow and spleen sections. Megakaryocyte ploidy analyses were performed as described.¹⁴ 

Lineage⁻c-Kit⁺Sca1⁺ (LSK) staining used biotinylated anti-TER119, anti-GR1, anti-Mac1, anti-B220, anti-CD4, and anti-CD8 antibodies (BD Pharmingen), in addition to anti–c-Kit–allophycocyanin and anti-Sca1–phycoerythrin—cyanine 7 (eBioscience) with secondary staining using streptavidin peridinin-chlorophyll-protein complex—cyanine 5.5 (BD Pharmingen). Mature lineage staining used fluorochrome-conjugated anti-TER119, anti-CD41, anti-GR1, anti-MAC1, anti-B220, anti-CD4, and anti-CD8 antibodies (BD Pharmingen).

Student unpaired 2-tailed t tests with Holm modification of Bonferroni correction for multiple testing¹⁵  were used.

To determine the specific contribution of trisomy of the Erg gene in the myeloproliferative phenotype associated with Down syndrome, mice disomic for functional Erg but otherwise trisomic on the Ts(17¹⁶)65Dn background were analyzed. As previously described, at 12 months of age trisomic Ts(17¹⁶)65Dn mice (hereafter referred to as Ts65Dn/Erg^+/+/+) demonstrated progressive thrombocytosis (Figure 1A), megakaryocytosis (Figure 1B), and megakaryocytic dysplasia within the bone marrow (Figure 1C), extramedullary hematopoiesis in the spleen with disrupted splenic architecture, expansion of the red pulp, and focal areas of myelopoiesis with prominent megakaryocytosis (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Trisomic Ts(17¹⁶)65Dn mice, with only 2 functional alleles of Erg (Ts65Dn/Erg^+/+/mld2), demonstrated amelioration of the histopathologic myeloproliferative features that were evident in trisomic littermates, with correction of thrombocytosis, bone marrow megakaryocytosis, and extramedullary hematopoiesis to levels observed in wild-type disomic littermate controls (Erg^+/+, Figure 1). Significant reticulin fibrosis of the bone marrow was noted in 3 of 9 Ts65Dn/Erg^+/+/+ mice analyzed at 12 months, but not in other genotypes.

Alterations in the numbers of hematopoietic stem and progenitor cells that characterize the myeloproliferative phenotype in trisomic mice were also corrected when trisomy at the Erg locus was reduced to functional disomy. The expanded bone marrow Lin⁻c-Kit⁺Sca1⁺ (LSK) compartment observed in trisomic Ts65Dn/Erg^+/+/+ mice returned to normal levels observed in disomic littermate controls in the Ts65Dn/Erg^+/+/mld2 mice (Figure 2A). Clonogenic culture assays assessing bone marrow and spleen hematopoietic progenitor cell frequency using stimulation with a cytokine cocktail of stem cell factor, interleukin-3, and erythropoietin demonstrated that increased preprogenitor blast colony¹⁶  and total colony frequency from Ts65Dn/Erg^+/+/+ bone marrow were both corrected to disomic littermate control frequencies in the Ts65Dn/Erg^+/+mld2 mice (Figure 2B). Consistent with a multilineage myeloproliferative phenotype in trisomic mice, flow cytometric analysis of mature hematopoietic lineages from bone marrow suspensions showed an increased proportion of granulocytes and megakaryocytes in Ts65Dn/Erg^+/+/+ mice whereas proportions in Ts65Dn/Erg^+/+/mld2 mice were similar to those in control Erg^+/+ mice (supplemental Figure 2). There was no difference in megakaryocyte ploidy between the different genotypes (supplemental Figure 3).

Thus, using the loss-of-function Erg^mld2 allele to return the functional Erg gene dosage to disomy, while maintaining trisomy of other genes, including AML1/Runx1 and Ets2 in the DSCR, we directly implicate Erg in myeloproliferative disease pathogenesis in trisomic Ts(17¹⁶)65Dn mice. Notably, despite the presence of Erg in the trisomic interval of the Ts1Cje DS model, these mice do not develop MPD, which may be ascribable to the effect of strain-specific genetic background and potential interaction of other genes in the Ts65Dn segmental trisomy that are not contained in the Ts1Cje segment.^17,18 

Whereas the MPD in Ts65Dn mice occurs in the absence of GATA1 mutations, which are characteristic of disease in humans, acute myeloproliferation and megakaryocytic differentiation are key pathologic features of hematologic disease associated with DS. In addition, the critical chromosomal region in human DS-AMKL/DS-TMD has been localized to an 8.3-Mb segment of Hsa21 containing the ERG gene.¹⁹  Our genetic evidence establishing the need for Erg trisomy for such pathology in Ts(17¹⁶)65Dn mice implies that ERG may also be a critical gene in human trisomy 21, playing a fundamental role in the pathogenesis of DS-TMD and DS-AMKL in human DS.

We thank Louise Inglis, Tracy Kemp, Meagan Blake, Kim Burchall, and Erik Dressler for excellent animal husbandry; Steven Mihajlovic and Ellen Tsui for histologic sections; and Jason Corbin for automated peripheral blood analysis.

This work was supported by a program grant (461219), project grant (516726), fellowships (W.S.A., B.T.K., and A.P.N.), and Independent Research Institutes Infrastructure Support Scheme grant (361646) from the Australian National Health and Medical Research Council; a Fellowship from the Australian Research Council (B.T.K.); a Fellowship from the Sylvia and Charles Viertel Charitable Foundation (B.T.K.); the Carden Fellowship Fund of the Cancer Council, Victoria (D.M.); the Australian Department of Education, Science and Training (scholarship to S.J.L.); a Haematology Society of Australia and New Zealand/Amgen New Investigator Scholarship (A.P.N.); a Fellowship from the Leukemia Foundation of Australia (C.L.C.); the Australian Cancer Research Fund; and a Victorian State Government Operational Infrastructure Support grant.

Contribution: A.P.N, C.D.H., D.M., C.L.C., S.J.L., L.D.R., B.T.K., and W.S.A. are responsible for the whole work including the conception, design, and conduction of the study, analysis and interpretation of the data, and drafting and revising the paper; all authors were involved in the discussion and revision of the paper and gave their permission for the final version submitted for publication.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Warren S. Alexander, Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, 3050 Australia; email: alexandw@wehi.edu.au.