Abstract
GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML.
Highlights
GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality
acute myeloid leukemia (AML) (n = 2611) and control (n = 77) patient datasets were downloaded from Gene Expression Omnibus (GEO) to create a case/ control cohort hybridised to the same array (Affymetrix Human Genome U133 Plus 2.0 GeneChip) and analyzed through R using bio-conductor packages, where data was normalized using Robust Multi-array Average (RMA)
We found that GATA2 expression was higher in AML patients compared to healthy controls (BM MNCs) and was overexpressed in 25% of AML samples (Fig. 1A,B)
Summary
Lenvitiral vectors containing shRNA against human GATA2 (or scramble control) were diluted in H2O, and mixed with calcium chloride (Sigma). This mix was added drop-wise to 2x hepes buffered saline (HBS; Sigma) and after 15′ incubation, added to the media of HEK293T cells at 70% confluency in a 10-cm dish. AML cells were fixed with 1% PFA (Thermofisher) for 12′ on ice. Cells were permeabilised with 2% BSA (Thermofisher) and 0.1% Triton-X (Sigma) for 20′ on ice, washed, and stained for 30′ on ice in the dark with anti-human GATA2-PE (IC2046P) (R&D systems). HL60, K562 and NOMO1 cells were transduced with lentiviruses encoding a short hairpin against human GATA2 (or scramble control) and a GFP reporter.
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