This proposal is focused on determining the basic molecular mechanisms underlying polyploidization and maturation of megakaryocytes, which are essential for effective platelet formation by adult megakaryocytes (Mk). In a tightly controlle differentiation process, diploid megakaryoblasts undergo a progressive increase in ploidy by repeated DNA replication without cell division, a process termed endomitosis, resulting in large multilobulated, polyploid nuclei. In myelodysplastic diseases (MDS) and other forms of BM failure, low ploidy megakaryoblasts often predominate. My laboratory has identified a link between the MKL1/SRF signal transduction pathway and polyploidization. SRF (Serum Response Factor) is a ubiquitous transcription factor that regulates cytoskeleton-associated genes. The transcriptional cofactor MKL1 binds to and activates the SRF protein. We have shown that MKL1 expression is upregulated during Mk maturation, and that MKL1 is essential for normal Mk polyploidization. In addition, we have shown that KO of SRF in the Mk lineage leads to a more severe phenotype than the MKL1 KO mice with a greater decrease in platelets, and significantly decreased ploidy of Mk with abnormal nuclear and cytoplasmic ultrastructure by electron microscopy. Prior studies using time-lapse microscopy to observe endomitotic Mk suggest that the initial endomitotic cleavage event in which cells progress from 2N to 4N occurs due to failure at late cytokinesis, whereas later endomitotic events (4N to 8N, 8N to16N, etc.) up to 128N do not show significant cleavage furrow formation. GEF-H1 and ECT2, two guanine exchange factors that are essential for activation and recruitment of RhoA to the cleavage furrow for completion of cytokinesis, must be downregulated sequentially for Mk to undergo polyploidization. Linking GEFH1 regulation, MKL1, and polyploidization, we have now determined that MKL1 induces GEF-H1 downregulation, and that shRNA-mediated GEF-H1 knockdown rescues the ploidy defect in Mkl-/- megakaryocytes. In order to better elucidate the biological mechanisms by which MKL promotes megakaryocyte polyploidization and maturation, we propose to 1) Determine the genetic mechanism by which MKL1 regulates GEFH1 expression to promote polyploidization of Mk;2) Determine the role of actin dynamics in MKL1 localization and transcriptional activation, and 3) Determine the mechanism by which MKL1 promotes megakaryocyte maturation. These studies will help to elucidate the mechanisms regulating normal Mk formation, which is critical for platelet formation and function. In addition, the data obtained will reveal the molecular regulation and functions of MKL1, a transcriptional cofactor in the RhoA/SRF pathway, which is dysregulated in Myelodysplasia (MDS) especially that associated with 5q- syndrome.

Public Health Relevance

Blood stem cells in the bone marrow are responsible for maintaining a population of cells, termed megakaryocytes, which release platelets into the blood. Platelets are needed to prevent bleeding. It is very important that the number of platelets in the blood be regulated. Too few platelets put patients at risk for bleeding, and too many platelets can cause blood clots. The work proposed in this grant focuses on novel findings that have elucidated how the megakaryocytes grow and mature in order to produce normal platelets. The aims of this work are to determine the basic molecular mechanisms this maturation of megakaryocytes, which is essential for effective platelet formation. The results of these studies will help us to understand normal platelet formation and function, which are affected during bone marrow transplantation putting patients at risk for bleeding, and which are abnormal in blood diseases in which megakaryocyte maturation and platelet formation are abnormal including Myelodysplasia. The studies may also lead to improved methods for manufacturing platelets for transfusion medicine.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK094934-02
Application #
8547064
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2012-09-21
Project End
2017-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$349,464
Indirect Cost
$139,576
Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Krause, Diane S; Crispino, John D (2013) Molecular pathways: induction of polyploidy as a novel differentiation therapy for leukemia. Clin Cancer Res 19:6084-8