ETS2 and ERG1 Promote Megakaryopoiesis and Immortalize GATA1 Knockdown (ΔneoΔHS) Fetal Liver Progenitors: Implications for Leukemogenesis in Down Syndrome.

ETS2 and ERG are two ETS family transcription factors encoded on chromosome 21 that have been implicated in leukemia and other types of cancer. In the setting of trisomy 21 (e.g. in individuals with Down syndrome) there is a significant increase in the propensity for leukemia, including AMKL (acute megakaryoblastic leukemia). It has been theorized that genes located on chromosome 21 may contribute to the predisposition of children with Down syndrome for pediatric AMKL, possibly by collaborating with mutations in the transcriptional regulator GATA1 . To test the oncogenic potential of these genes in the hematopoietic system, ETS2 and the ERG isoform, ERG1, were over-expressed in both wild-type and GATA1 knockdown (ΔneoΔHS) fetal liver hematopoietic progenitors and the effects on growth and differentiation were studied. We discovered that over-expression of either ETS2 or ERG1 in wild-type progenitors increased the formation of CD41 positive megakaryocytes by 2–3 fold in liquid culture. However, whereas ETS2 appeared to primarily promote the early stages of megakaryopoiesis, ERG1 over-expression led to a dramatic rise in the percentage of cells expressing CD42, a marker of more mature megakaryocytes. Ectopic ERG1 expression also affected the ploidy profile, with nearly all cells containing ≥4N DNA content. Quantitative real time RT-PCR analysis of lineage specific genes confirmed that expression of megakaryocyte genes, including GPIIb (CD41), platelet factor 4 (PF4), and s1-tubulin, was significantly increased in undifferentiated cells over-expressing either ETS2 or ERG1. These results show that over-expression of ETS2 or ERG1 rapidly initiates the megakaryocyte program. Over-expression of either ETS2 or ERG1 in GATA1 knockdown fetal liver cells similarly led to a 2.5–5 fold increase in the formation of CD41 positive megakaryocytes in liquid culture; however, neither ETS2 or ERG1 could circumvent the differentiation block that is characteristic of the GATA1 knockdown strain, including the inability to form proplatelet extensions. We next assessed the capacity of ETS2 or ERG1 over-expression to alter the colony forming ability of wild-type and GATA1 knockdown fetal liver progenitors. Both ETS2 and ERG1 expression led to a significant increase in CFU-MK derived from wild-type progenitors, as evidenced by an increase in the number of acetylcholinesterase positive colonies. In contrast, neither gene altered the formation of CFU-MK when over-expressed in the GATA1 knockdown progenitors, which already show a prominent enhancement in megakaryocyte colony formation over wild-type fetal liver cells. Finally, serial re-plating assays performed with GATA1 knockdown fetal liver cells showed that ectopic expression of either ETS2 or ERG1 could immortalize hematopoietic progenitors, whereas only ERG1 could enhance colony re-plating of wild-type cells. These findings provide evidence for a specific synergy between GATA-1 deficiency and ETS2 over-expression and suggest a distinct oncogenic potential for each factor. Overall, these results in primary murine fetal liver cells demonstrate that ETS2 and ERG1 promote the megakaryocyte lineage and facilitate immortalization of GATA1 knockdown cells, consistent with a potential oncogenic role in Down syndrome AMKL for these two ETS family genes located on chromosome 21.