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. 2009 Apr 2;113(14):3337-47.
doi: 10.1182/blood-2008-08-174813. Epub 2009 Jan 23.

ETS2 and ERG promote megakaryopoiesis and synergize with alterations in GATA-1 to immortalize hematopoietic progenitor cells

Monika J Stankiewicz  1 , John D Crispino
Affiliations

ETS2 and ERG promote megakaryopoiesis and synergize with alterations in GATA-1 to immortalize hematopoietic progenitor cells

Monika J Stankiewicz et al. Blood. .

Abstract

ETS2 and ERG are transcription factors, encoded on human chromosome 21 (Hsa21), that have been implicated in human cancer. People with Down syndrome (DS), who are trisomic for Hsa21, are predisposed to acute megakaryoblastic leukemia (AMKL). DS-AMKL blasts harbor a mutation in GATA1, which leads to loss of full-length protein but expression of the GATA-1s isoform. To assess the consequences of ETS protein misexpression on megakaryopoiesis, we expressed ETS2, ERG, and the related protein FLI-1 in wild-type and Gata1 mutant murine fetal liver progenitors. These studies revealed that ETS2, ERG, and FLI-1 facilitated the expansion of megakaryocytes from wild-type, Gata1-knockdown, and Gata1s knockin progenitors, but none of the genes could overcome the differentiation block characteristic of the Gata1-knockdown megakaryocytes. Although overexpression of ETS proteins increased the proportion of CD41(+) cells generated from Gata1s-knockin progenitors, their expression led to a significant reduction in the more mature CD42 fraction. Serial replating assays revealed that overexpression of ERG or FLI-1 immortalized Gata1-knockdown and Gata1s knockin, but not wild-type, fetal liver progenitors. Immortalization was accompanied by activation of the JAK/STAT pathway, commonly seen in megakaryocytic malignancies. These findings provide evidence for synergy between alterations in GATA-1 and overexpression of ETS proteins in aberrant megakaryopoiesis.

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Figure 1
Figure 1
ERG and ETS promote megakaryopoiesis in wild-type fetal liver progenitors. (A) Fetal liver cells transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG were differentiated for 72 hours and the GFP+ fraction was assessed for CD41 expression by flow cytometry. (B) The degree of polyploidization was determined by DAPI staining of GFP, CD41 double-positive differentiated fetal liver cells. (C) CD42 expression within the GFP-positive population was assessed by flow cytometry. Fold changes for CD41, percentage of cells with DNA content of 8N or more, and CD42 expression in the transduced cells relative to MIGR1 control-infected cells are shown to the right of the representative flow plots. Graphs display average fold changes plus or minus standard error. * P less than .02. For all experiments, n = 4. (D) qRT-PCR analysis of expression of genes that characterize megakaryocyte maturation in wild-type lineage-depleted fetal liver hematopoietic progenitors transduced with MIGR1, MIGR1-ETS2, and MIGR1-ERG. Data depict mean plus or minus standard error for a minimum of 3 independent experiments performed in triplicate. * P less than .05.
Figure 2
Figure 2
ERG and ETS promote megakaryopoiesis in Gata1-knockdown progenitors but cannot overcome block in maturation associated with a GATA-1 deficiency. (A) Gata1-knockdown fetal liver cells transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG were differentiated for 72 hours and the GFP+ fraction was assessed for CD41 expression by flow cytometry. (B) The degree of polyploidization was determined by DAPI staining of GFP, CD41 double-positive differentiated fetal liver cells. (C) CD42 expression within the GFP-positive population was assessed by flow cytometry. Fold changes for CD41, percentage of cells with DNA content of 8N or more, and CD42 expression in the transduced cells relative to MIGR1 control-infected cells are shown to the right of the representative flow plots. Graphs display average fold changes plus or minus SE. * P less than .01. For all experiments, n = 4. (D) qRT-PCR analysis of expression of genes that characterize megakaryocyte maturation in Gata1-knockdown lineage-depleted fetal liver hematopoietic progenitors transduced with MIGR1, MIGR1-ETS2, and MIGR1-ERG. Data depict mean plus or minus SE for a minimum of 3 independent experiments performed in triplicate. * P less than .05.
Figure 3
Figure 3
ETS2, ERG, and FLI-1 promote megakaryopoiesis in Gata1s knockin progenitors. (A) Gata1s knockin fetal liver cells transduced with MIGR1, MIGR1-ETS2, MIGR1-ERG, or MIGR1-FLI-1 were differentiated for 72 hours and the GFP+ fraction was assessed for CD41 expression by flow cytometry. (B) The degree of polyploidization was determined by DAPI staining of GFP, CD41 double-positive differentiated fetal liver cells. (C) CD42 expression within the GFP-positive population was assessed by flow cytometry. (D) The degree of polyploidization was determined by DAPI staining of GFP, CD42 double-positive differentiated fetal liver cells. Fold changes for CD41 (E), CD42 (F) expression, and percentage of cells with DNA content of 8N or more (G) in the transduced cells relative to MIGR1 control-infected cells are shown below the representative flow plots. Graphs display average fold changes plus or minus SE. * P less than or equal to .03. For all experiments, n = 3.
Figure 4
Figure 4
ETS2 and ERG promote CFU-Mk formation from wild-type but not Gata1-knockdown progenitors. (A) The number of megakaryocyte colonies generated by GFP+ wild-type fetal liver progenitors transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG. * P less than .05. (B) The number of megakaryocyte colonies generated by GFP+ Gata1-knockdown fetal liver progenitors transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG. Note that none of the differences are statistically significant.
Figure 5
Figure 5
Overexpression of ETS2 and ERG promote immortalization of Gata1-knockdown and Gata1s knockin fetal liver hematopoietic progenitors. (A) The numbers of total colonies derived from plating sorted GFP+ wild-type fetal liver progenitors transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG for 3 generations. Ten thousand cells were plated each time. No differences were significant except for the ERG third replating. P value less than .03; n = 3 in all experiments. (B) The numbers of total colonies derived from plating sorted GFP+ Gata1-knockdown fetal liver progenitors transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG for 3 generations. Five thousand cells were plated each time. * P less than .05. (C) The numbers of total colonies derived from plating sorted GFP+ Gata1s knockin fetal liver progenitors transduced with MIGR1, MIGR1-ETS2, MIGR1-ERG, or MIGR1-FLI1 for 3 generations. Five thousand cells were plated each time. * P less than or equal to .05.
Figure 6
Figure 6
Expression profiling reveals that ERG and FLI-1 have signatures that are distinct from both ETS2 and MIGR1. (A) RNA for genome-wide expression profiling was extracted from fourth-generation Gata1-knockdown colonies transduced with the MIGR1, MIGR1-ERG, MIGR1-ETS2, and MIGR1-FLI-1. (B) Heat map generated by unsupervised hierarchic clustering reveals that 726 genes discriminate ETS2-, ERG-, and FLI1-overexpressing colonies from MIGR1-transduced cells. (C) Principal component analysis reveals that the ERG and FLI-1 signatures are close to each other and are well separated from MIGR1 and ETS2. This implies that ERG and FLI-1 are more similar to each other than to MIGR1. (D) Heat map showing the top 50 hematopoiesis genes that are differentially expressed in the samples. Red indicates increased expression by 2-fold or more, whereas green depicts decreased expression by 2-fold or more. Genes within blue boxes are those whose expression differs among the ETS family–transduced cells. (E) qRT-PCR confirmation of several differentially expressed genes in the colonies derived from Gata1-knockdown fetal liver hematopoietic cells. Graph depicts mean plus or minus SE for a minimum of 3 independent experiments performed in triplicate. All differences are significant at the 5% level.
Figure 7
Figure 7
ETS-, ERG-, and FLI-1–immortalized cells express CD41 and c-kit and show enhanced STAT-3 phosphorylation. (A) Comparison of CD41 and c-kit expression in ETS2-, ERG-, and FLI-1–overexpressing Gata1-knockdown fetal liver hematopoietic cells isolated from methylcellulose after 4 weeks of culture. Plots are representative of 3 independent experiments. Significance levels for differences for ERG and FLI-1 in comparison with MIGR1 are for CD41 expression (singly positive) only: ERG (P < .01), FLI-1 (P < .04); for CD41/c-kit double-positive gate: ERG (P < .02); c-kit expression: ERG (P < .008), FLI-1 (P < .03). (B,C) Comparison of phospho-STAT3 and phospho-STAT5(a/b) staining of fourth-generation ETS2-, ERG-, and FLI-1-overexpressing Gata1-knockdown fetal liver hematopoietic cells. Significance levels for differences in phospho-STAT3 signaling in comparison with MIGR1: ERG (P < .01), FLI-1 (P < .009), and ETS2 (not significant). P values were calculated by comparing the percentage of cells stained for phospho-STAT3 in the different groups for 3 independent experiments.
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