Date of Award

5-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Derek A. Persons, MD, PhD

Committee

John V. Cox, PhD Erin G. Schuetz, PhD Brian P. Sorrentino, MD Gerard P. Zambetti, PhD

Abstract

The transcription factor GATA2 is highly expressed in hematopoietic stem cells (HSCs) and is downregulated during differentiation. Overexpression of GATA2 is frequently observed in acute myeloid leukemia. In previous studies, enforced expression of GATA2 using a MSCV GATA2 retroviral vector blocked differentiation of HSC and progenitors without inducing leukemia. We hypothesized that a lower dose of GATA2 can relieve the HSC block and eventually transform myeloid progenitors into leukemia stem cells. To test this hypothesis we generated a MSCV GATA2-ERT vector in which nuclear concentration of GATA2 can be regulated by Tamoxifen (TAM). The GATA2-ERT protein was confirmed to leak into nucleus of transduced BM cells even in the absence of TAM. This low nuclear GATA2-ERT led to enhanced self-renewal of myeloid progenitors in vitro and was able to immortalize primary bone marrow (BM) cells into IL-3 dependent myeloid cell lines. Continuous GATA2-ERT expression was also required for the proliferation of these immortalized lines. Nmyc and HoxA9 mRNA were significantly higher in GATA2-ERT immortalized lines compared to control immortalized lines. Repression of either Nmyc or Hoxa9 in GATA2-ERT immortalized cells impaired their proliferation suggesting that they are potential GATA2 targets mediating this effect.

Myeloid expansion and a block in T cell and B cell lineage differentiation was observed in transplant recipients of GATA2-ERT expressing BM cells without TAM. The myeloid expansion occurs after the Granulocyte Monocyte Progenitor (GMP) and the lymphoid block occurs after the Common Lymphoid Progenitor (CLP) in transgenic mice. Finally using the C352P mutant we showed that myeloid expansion and B lymphoid block require DNA binding activity of GATA2-ERT and were not due to the dominant negative effect of the cytosolic form of this protein.

We were the first to demonstrate that GATA2 overexpression confers increased selfrenewal of myeloid progenitors which correlates with increased GATA2 mRNA levels frequently observed in human AML patients. We believe that the lower dose of GATA2 is critical to this phenotype. Interestingly no myeloid leukemias were observed in both the transplant and transgenic animals.

DOI

10.21007/etd.cghs.2014.0222

Share

COinS