Megakaryocytopoiesis is the process by which egakaryocytes differentiate from biphenotypic megakaryocyte-erythroid precursors, to megakaryoblasts, which undergo endomitosis to become polyploid, and subsequently undergo maturation to mature megakaryocytes that can release platelets into the circulation. Each of these stages is critical for the cells to produce function platelets, which are critical for hemostasis. Despite great advances in our understanding of hematopoiesis, relatively little is known regarding how megakaryocytopoiesis is regulated, and how this may go awry in diseases of megakaryocytes affecting platelet formation and in acute megakaryoblastic leukemia (AMKL). This proposal is focused on determining the mechanism(s) by which MKL1 promotes megakaryocytic differentiation. MKL1 was first identified by its involvement in the t(1;22) translocation, which occurs almost exclusively in AMKL of newborns. To understand the mechanism by which the RBM15-MKL1 fusion protein encoded by the t(1;22) translocation promotes leukemia, we must understand the normal functions of RBM15 and MKL1 in hematopoiesis, which have only recently begun to be elucidated. My laboratory has discovered several important clues regarding the normal function(s) of MKL1: 1) MKL1 is differentially expressing during megakaryocytic differentiation with the highest levels in the most mature polyploid megakaryocytes (Mks), 2) overexpression of wildtype (WT) MKL1 promotes Mk differentiation of human cell lines as well as primary murine and human cells with increased numbers of Mk and increased ploidy of Mk, 3) MKL1 knockout mice have impaired megakaryocytic differentiation and decreased platelet numbers, 4) notch stimulation promotes Mk differentiation and 5) MKL and notch act synergistically on both SRF and notch responsive promoters.
The aims of this proposal build upon these observations to elucidate the mechanisms by which MKL1 promotes megakaryocytopoiesis.
Aim 1 is to determine the functional interactions of MKL1 with SRF and MKL2 in megakaryocytopoiesis using in vivo and in vitro approaches.
Aim 2 is to test hypothesis that MKL1 and notch act synergistically to promote megakaryocytic differentiation.
Aim 3 is to assess the mechanism by which MKL1 promotes polyploidization of megakaryocytes. The clinical relevance of the proposed work is clear - these studies will help to elucidate the mechanisms underlying normal megakaryocytopoiesis, which is key for normal formation of functional platelets. In addition, these studies will form the basis for future studies on the RBM15-MKL fusion protein found uniquely in AMKL, which accounts for 10% of AML cases in children.

Public Health Relevance

The platelets in our blood are critical for prevention f bleeding. Platelets are formed in the bone marrow from cells called megakaryocytes. The work proposed is focused on a obtaining a better understanding of the process of megakaryocytopoiesis with the long-term goals of deriving effective therapies for genetic and acquired diseases affecting these cells. Acute megakaryoblastic leukemia (AMKL) affects primarily newborn infants and children during the first year of life. When AMKL is associated with a t(1;22) chromosomal translation between the RBM15 gene on chromosome 1 and the MKL1 gene on chromosome 22, there are few effective treatment options, and the disease is nearly always fatal. In this new application, we propose to build on our published work and preliminary data elucidating the role of MKL1 in normal and malignant megakaryocyte expansion, polyploidization and maturation. Specifically, we are assessing the role of MKL1 in megakaryocyte differentiation, and the mechanisms underlying its action. The results of these studies will provide new insights into the role of MKL1 in normal as well as malignant cells.)

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK086267-03
Application #
8306979
Study Section
Special Emphasis Panel (ZRG1-VH-D (02))
Program Officer
Bishop, Terry Rogers
Project Start
2010-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
3
Fiscal Year
2012
Total Cost
$339,978
Indirect Cost
$134,553
Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Guo, Shangqin; Zi, Xiaoyuan; Schulz, Vincent P et al. (2014) Nonstochastic reprogramming from a privileged somatic cell state. Cell 156:649-62
Smith, Elenoe C; Teixeira, Alexandra M; Chen, Rachel C et al. (2013) Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation. Blood 121:1094-101
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
Megyola, Cynthia M; Gao, Yuan; Teixeira, Alexandra M et al. (2013) Dynamic migration and cell-cell interactions of early reprogramming revealed by high-resolution time-lapse imaging. Stem Cells 31:895-905
Gao, Yuan; Smith, Elenoe; Ker, Elmer et al. (2012) Role of RhoA-specific guanine exchange factors in regulation of endomitosis in megakaryocytes. Dev Cell 22:573-84