Ectopic Expression of ERG and FLI1 Enforce a Megakaryocyte Phenotype Independent of GATA-1

Abstract Specific genes that contribute to Transient Myeloproliferative Disorder (TMD) and DS Acute Megakaryocytic Leukemia (AMKL) remain largely undefined. To date, only the X-linked transcription factor GATA1, which is mutated in these disorders, has been implicated in contributing to DS hematopoietic malignancies. We recently reported that overexpression of either of the chromosome 21 ETS family members ERG or ETS2 led to a dramatic increase in megakaryopoeisis from wild-type or Gata1-knockdown fetal liver hematopoietic progenitors. Furthermore, we demonstrated that expression of ETS2 or ERG caused a significant increase in megakaryocyte colony formation and led to immortalization of Gata1-knockdown fetal liver hematopoietic progenitors in vitro. In new studies to define the mechanisms by which these ETS proteins promote megakaryopoiesis, we discovered that the ETS family member FLI1, which shares significant homology with ERG, significantly increased CD41 and CD42 expression and the degree of polyploidization of megakaryocytes in vitro in a manner that was nearly identical to that of ectopic ERG. Furthermore, FLI1 also immortalized Gata1-knockdown fetal liver progenitors. To characterize the effects of ectopic ETS family members on transcription, we performed genome wide expression profiling using RNA isolated from fourth generation immortalized Gata1-knockdown cells expressing ETS2, ERG, FLI1 or GFP alone. Principal component analysis showed that the ERG and FLI1 gene expression patterns clustered tightly with one another and were well separated from both the MIGR1 and ETS2 signatures. Unsupervised hierarchical clustering further revealed that 726 genes were differentially expressed in FLI1, ERG, ETS2 immortalized cells compared to MIGR1 control cells. Of these, genes associated with the erythroid lineage, including Klf1, Lmo2, glycophorin A, Egr1, Ank1 and Eraf were strongly repressed. These findings are consistent with recent publications showing that FLI1 promotes the development of megakaryocytes at the expense of red blood cells and suggest that ERG and ETS2 mimic FLI1 in our assays. Notably, we did not observe significant increases in the expression of Hox genes or Meis1, genes whose expression is often associated with transformed hematopoietic cells. However, we did detect increased expression of Bmi1, which has an essential role in regulating the proliferative capacity of both normal and malignant hematopoietic progenitors, as well as increased expression of Jak2 and Stat5, two genes whose activation is associated with myeloproliferative disease and megakaryocytic transformation, in ETS-family transduced cells. Next, to determine whether ETS-family expression affected the immunophenotype of Gata1-knockdown fetal liver progenitors, immortalized cells were assessed for cell surface expression of c-kit and CD41. ERG and FLI1 expressing colonies included a substantial proportion of CD41+/c-kit- cells, indicative of megakaryocytic maturation. Furthermore, colonies formed from progenitors transduced with ERG were comprised of greater than 90% CD41+ cells, confirming that nearly all of the cells within the immortalized colonies are of the megakaryocyte lineage. Finally, because our gene array data showed increased Jak2 and Stat5 mRNA levels in ETS-family transduced cells, and activating mutations in JAK3 are associated with AMKL, we assessed whether JAK/STAT signaling was altered in the immortalized cells. Using flow cytometry, we found that cells within the ERG and FLI1 cultures consistently showed significant increases in phospho-STAT3 staining in comparison to the MIGR1 control. The increases in STAT3 phosphorylation suggest that ERG and FLI1 promote immortalization of Gata1-knockdown fetal liver progenitors in part by activation of the JAK/STAT pathway. Together our data support the hypothesis that an increased dosage of ETS factors, including ETS2 and ERG, contribute to a pro-megakaryocytic phenotype and to the leukemogenic process in DS TMD and AMKL.