Erythropoiesis is tightly controlled at every phase of differentiation. Although much is known in terms of signaling and cell survival during the initial phase, very little is known with respect to signaling during late- stages of erythrocyte membrane remodeling, enucleation and reticulocyte maturation. Moreover, in conditions such as myelodysplastic syndromes (MDS), where anemia is the most common presentation and predominant cause of morbidity, the focus has mainly been to study the early phases of hematopoiesis. In an attempt to uncover genes aberrantly expressed in MDS, we performed an integrative genomic analysis of primary hematopoietic cells from MDS patients. These studies revealed that DOCK4, a guanine exchange factor was significantly under-expressed and hypermethylated in MDS stem and progenitor cells. DOCK4 is an important cofactor for various GTPases and is located on the chromosome 7q segment and is a common deletion in MDS. In addition to aberrant DNA methylation, our preliminary analysis reveals that DOCK4 is mutated in different sets of anemic patients as well, thus implicating it as an important gene in the pathogenesis of anemia. However, there is no knowledge of its functions during normal terminal erythroid development. We have used a dynamic model of human erythropoiesis to demonstrate that DOCK4 is highly expressed during late stages of normal erythropoiesis and knockdown of DOCK4 disrupts the F-actin assembly and alters the osmotic fragility of erythroblasts. Based on these data we hypothesize that DOCK4 is an important signaling intermediate in late-stage erythroblasts that is instrumental in maintaining erythroblast membrane homeostasis. Furthermore we hypothesize that aberrant silencing of DOCK4 contributes to ineffective erythropoiesis seen in MDS. We will test these hypotheses by carrying out the following aims.
In Aim 1 we will determine the functional role of DOCK4 in late stages of erythroid differentiation using an in vitro human model that recapitulates all the stages of erythropoiesis. Following knockdown of DOCK4 by lentiviral shRNA, we will examine its impact on cell viability/proliferation, membrane homeostasis, enucleation and reticulocyte maturation.
In Aim 2 we will determine the involvement of DOCK4 in key signaling pathways associated with erythropoiesis including its role in activation of downstream Rac and Rap GTPases. Novel downstream effectors will also be determined by phosphoproteomic analysis.
In Aim 3 we will determine involvement of DOCK4 in the pathogenesis of anemia associated with MDS using primary erythroid cells isolated from MDS patients. Several approaches such as mutational analysis, promoter DNA methylation and FISH analysis in primary late-stage erythroblasts will be performed. Finally, we will determine whether restoration of DOCK4 expression in MDS erythroblasts will reverse the defects associated with F-actin skeleton and other membrane dynamics. Altogether, these studies will conclusively establish the role of DOCK4 in normal erythropoiesis as well as its significance in the pathogenesis of anemia in MDS.
We have found that the gene for the DOCK4 protein is not properly expressed in a category of anemia patients with the disease myelodysplastic syndromes (MDS). We also have discovered that this protein is needed for formation of the actin framework of red cell membrane during the later stages of red blood cell formation in the marrow. Our proposed studies will investigate the role of DOCK4 in proper formation of red blood cells, the signaling pathways that are activated by this protein and the impact of suppression of DOCK4 expression in causing anemia in MDS. MDS is a common cause of anemia in the elderly and these studies will lead to newer insights into their pathogenesis.
|Bhagat, Tushar D; Chen, Si; Bartenstein, Matthias et al. (2017) Epigenetically Aberrant Stroma in MDS Propagates Disease via Wnt/?-Catenin Activation. Cancer Res 77:4846-4857|
|Sundaravel, Sriram; Duggan, Ryan; Bhagat, Tushar et al. (2015) Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes. Proc Natl Acad Sci U S A 112:E6359-68|
|Schinke, Carolina; Giricz, Orsolya; Li, Weijuan et al. (2015) IL8-CXCR2 pathway inhibition as a therapeutic strategy against MDS and AML stem cells. Blood 125:3144-52|
|Madzo, Jozef; Liu, Hui; Rodriguez, Alexis et al. (2014) Hydroxymethylation at gene regulatory regions directs stem/early progenitor cell commitment during erythropoiesis. Cell Rep 6:231-244|
|Yu, Yiting; Mo, Yongkai; Ebenezer, David et al. (2013) High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis. J Biol Chem 288:8805-14|
|Bhagat, Tushar D; Zhou, Li; Sokol, Lubomir et al. (2013) miR-21 mediates hematopoietic suppression in MDS by activating TGF-? signaling. Blood 121:2875-81|
|Bhattacharyya, Sanchari; Yu, Yiting; Suzuki, Masako et al. (2013) Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer. Nucleic Acids Res 41:e157|