GATA1s Induces Hyperproliferation of Eosinophil Precursors.

Abstract 2318

Transient leukemia (TL) is evident in 5–10% of all neonates with Down syndrome (DS) and closely linked to characteristic N-terminal truncating GATA1-mutations (GATA1s). Here we investigated the relationship between GATA1s-mutation and clonal eosinophilia observed in a cohort of TL patients (23%, n=60 DS neonates diagnosed with TL).

TL-blasts exhibited an activated eosinophilic genetic program (as demonstrated by GSEA and validated by qRT-PCR) and had an increased propensity to differentiate along the eosinophilic lineage in vitro. Sorted patient TL-eosinophils carried the same mutation in GATA1 as the TL-blasts showing their clonal origin.

To delineate the role of GATA1s in the TL-associated eosinophilia, we examined the myeloid differentiation of human fetal hematopoietic progenitors ectopically expressing GATA1 or GATA1s. We used a modified lentiviral LeGO-iG vector simultaneously expressing murine Gata1s or full-length Gata1 and an shRNA against endogenous GATA1 (65% knockdown efficiency, p<0.05), ensuring the investigation of the net effect of GATA1s in cells with low endogenous GATA1 expression.

Ectopic expression of Gata1s in human CD34+ fetal hematopoietic stem and progenitor cells (HSPCs) induced 10-fold higher (p<0.01) proliferation rate of granulated progenitor cells in the myeloid in vitro differentiation medium accompanied by a monocytic differentiation block and reduction of neutrophils. In methylcellulose colony-forming assays, we observed formation of atypical colonies (29% of all CFUs), consisting of hyperproliferative myeloid progenitors with eosinophilic and basophilic granulation. These cells predominated in replating experiments, leading to a 15-fold higher cumulative number of CFUs after the fourth round of replating for Gata1s-transduced cells compared to the empty vector-transduced cells (p<0.001). To elucidate the lineage nature of these myeloid progenitors, we used high throughput chip cytometry that allowed us to study expression of 20 surface and intracellular markers of the cells from a picked Gata1s-colony. We observed high expression of the early myeloid marker CD13 and of IL-3 receptor (CD123). In conjunction with histochemical stainings chip cytometry allowed us to exclude monocytic, erythroid, lymphoid and basophilic/mast cell origin of these cells as all markers for these lineages were low or absent. Global gene expression profiling using microarray technology of Gata1s-, Gata1- and empty vector-transduced HSPCs, grown for four days in the myeloid differentiation medium and of empty vector transduced cells, before we transferred them into the myeloid differentiation medium demonstrated enrichment (GSEA) of eosinophilic genes in Gata1- and Gata1s-transduced cells and downregulation of monocytic, neutrophil, basophil, mast cell and HSPC gene sets. Thus, global gene expression analyses, chip cytometry and histochemical methods identified Gata1s-transduced myeloid progenitors as atypical eosinophilic promyelocytes with abnormal pro-eosinophilic granulation.

However, though GATA1s retained the function of GATA1 to promote differentiation of HSPCs along the eosinophil lineage while repressing the neutrophil/monocytic program, only Gata1s-transduced cells demonstrated a hyperproliferative phenotype. GSEA of known E2F-bound target genes and qRT-PCR validation demonstrated that, unlike Gata1, Gata1s was not able to repress a number of GATA1 target genes, such as MYC, and the pro-proliferative E2F transcription network perturbing the balance between differentiation and proliferation. This might result in the hyperproliferation of eosinophil precursors at the expense of the neutrophil/monocytic lineage. As E2F target genes were also upregulated in sorted TL-eosinophils (n=3), we propose the same molecular mechanism of GATA1s-induced clonal eosinophilia in TL.