Abstract
Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder characterized by overproduction of myelomonocytic cells. Activating mutations of PTPN11, which encodes the protein tyrosine phosphatase, Shp2, are found in 35% of JMML patients. Murine bone marrow low density mononuclear cells (LDMNCs) expressing activating Shp2 mutants preferentially undergo myelomonocytic differentiation despite being subjected to conditions that typically support only mast cell development. Evaluation of hematopoietic cell-specific transcription factor expression revealed that GATA2 expression, needed for mast cell differentiation, is dramatically reduced, while, surprisingly, PU.1 expression is unchanged in cells expressing activating Shp2 mutants. In addition to lineage-specific transcription factors such as PU.1, however, c-jun also promotes monocytic differentiation by functioning as a co-activator of PU.1. Thus, we hypothesized that activating Shp2 mutations (Shp2D61Y or Shp2 E76K) induce increased c-jun expression permitting, in collaboration with PU.1, excessive monocytic differentiation and reduced GATA2 expression in hematopoietic progenitors. As a corollary, we hypothesized that ectopic expression of GATA2, but not of GATA2 lacking the C-terminal zinc finger (GATA2del330–407), which is needed for disruption of the PU.1-c-jun interaction, would normalize aberrant myelomonocytic differentiation induced by activating Shp2 mutants. Consistent with our hypothesis, quantitative RT-PCR studies revealed 5-fold higher c-jun levels in cells expressing Shp2D61Y or Shp2E76K compared to cells expressing WT Shp2. We next utilized retroviral co-transduction of murine bone marrow LDMNCs to generate six groups:
pMIEG3-Shp2WT plus pCD4 (empty vector); pMIEG3-Shp2WT plus pCD4-GATA2; pMIEG3-Shp2WT plus pCD4-GATA2del330-407; pMIEG3-Shp2E76K plus pCD4; pMIEG3-Shp2E76K plus pCD4-GATA2; and pMIEG3-Shp-2E76K plus pCD4-GATA2del330-407.
Transduced cells were stained with anti-human CD4 conjugated to allophycocyanin (APC), sorted for EGFP+APC+ cells, and plated into progenitor assays. Colonies were scored for colony forming unit (CFU)-granulocyte-macrophage (GM), monocyte (M), granulocyte (G), and granulocyte-erythroid-monocyte-megakaryocyte (GEMM). As predicted, cells co-transduced with activating Shp2E76K plus pCD4 produced significantly more CFU-M than cells co-transduced with WT Shp2 plus pCD4. Upon co-transduction with GATA2, the number of CFU-M generated from Shp2E76K-expressing cells was significantly reduced and was similar to that observed in cells expressing WT Shp2. In contrast, co-transduction of GATA2del330-407 failed to normalize the number of CFU-M produced by Shp2E76K-expressing cells. Quantitative RT-PCR verified ectopic GATA2 and GATA2del330-407 expression in the co-transduced cells. These findings demonstrate that restoration of GATA2 expression normalizes the propensity toward monocytic differentiation induced by Shp2E76K. The lack of correction conferred by GATA2del330-407 in combination with the observed increased c-jun expression support a model in which GATA2 and c-jun compete for binding to PU.1 to direct cell differentiation decisions in hematopoietic progenitors bearing activating Shp2 mutants. Collectively, these findings imply that normalization of transcription factor expression may provide a novel approach to differentiation-mediated therapy in JMML.
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