Hematopoietic stem cells (HSC) maintain a balance between self-renewal and lineage commitment, and the homeostatic mechanisms of this regulation are not well understood. Glycogen synthase kinase-3 (GSK-3) negatively regulates multiple signaling pathways in hematopoiesis, including Wnt signaling and cytokines such as Thrombopoietin (Tpo) and Erythropoietin, which signal through JAK2 to activate STAT5, Akt, and MAP kinase pathways. We have found GSK-3 plays a pivotal role in controlling the balance between HSC self-renewal and differentiation. Inhibition of Gsk3 in bone marrow transiently expands phenotypic HSCs and hematopoietic progenitor cells (HPCs) in a ss-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term assays, Gsk3 inhibition progressively depletes HSCs through activation of mTOR. Long-term HSC depletion is prevented by mTOR inhibition and exacerbated by ss-catenin knockout. Thus Gsk3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal, and we find that combined inhibition of mTOR and Gsk3 results in sustained HSC expansion in vivo. Furthermore, by combining GSK-3 and mTOR inhibitors, we are able to culture functional HSCs from mice or humans in cytokine- free medium and then reconstitute long-term, multilineage hematopoiesis in transplant recipients, providing a system for the ex vivo study of hematopoiesis and a potential method to expand human HSCs for clinical applications. The overall goal of this project is to examine the respective contributions of Wnt, mTOR, and Tpo/Mpl/JAK2 signaling in the response to GSK-3 inhibition and to define the role of GSK-3 in regulating these pathways within the hematopoietic system.
Specific aim 1 will rigorously test both the independent and overlapping functions of Gsk3a and Gsk3b in bone marrow cells using conditional gene knockouts.
Aim 2 will explore the role of Wnt and mTOR pathways downstream of GSK-3 in HSCs, and will identify the cell population that responds to GSK-3 and mTOR inhibitors under ex vivo conditions.
Aim 3 will investigate the upstream pathways that regulate GSK-3 and mTOR, including cytokine signaling through JAK2 and Akt-dependent pathways, and will specifically distinguish the respective contributions of Wnt and Akt signaling through GSK-3. These studies will define the function(s) of GSK-3 in normal hematopoiesis and may lead to the development of novel therapeutic approaches to expand HSCs in hematopoietic disorders.

Public Health Relevance

This proposal investigates the regulation of blood forming cells and specifically how the body maintains the ability to produce a high volume of mature blood cells and at the same time maintains the population of cells in the bone marrow needed for new blood formation. The proposal explores new approaches to enhance blood formation for bone marrow transplantation and in patients with low blood counts due to disease or chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL110806-01
Application #
8210662
Study Section
Special Emphasis Panel (ZRG1-CB-N (02))
Program Officer
Thomas, John
Project Start
2011-08-01
Project End
2015-04-30
Budget Start
2011-08-01
Budget End
2012-04-30
Support Year
1
Fiscal Year
2011
Total Cost
$542,962
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Shinde, Mansi Y; Sidoli, Simone; Kulej, Katarzyna et al. (2017) Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing. J Biol Chem 292:18240-18255
Nguyen-McCarty, Michelle; Klein, Peter S (2017) Autophagy is a signature of a signaling network that maintains hematopoietic stem cells. PLoS One 12:e0177054
Bhavanasi, Dheeraj; Wen, Kwun Wah; Liu, Xiaolei et al. (2017) Signaling mechanisms that regulate ex vivo survival of human acute myeloid leukemia initiating cells. Blood Cancer J 7:636
Zhang, Maomao; Skirkanich, Jennifer; Lampson, Michael A et al. (2017) Cell Cycle Remodeling and Zygotic Gene Activation at the Midblastula Transition. Adv Exp Med Biol 953:441-487
Bhavanasi, Dheeraj; Klein, Peter S (2016) Wnt Signaling in Normal and Malignant Stem Cells. Curr Stem Cell Rep 2:379-387
Bhavanasi, Dheeraj; Speer, Kelsey F; Klein, Peter S (2016) CKAP4 is identified as a receptor for Dickkopf in cancer cells. J Clin Invest 126:2419-21
Rozenova, Krasimira; Jiang, Jing; Donaghy, Ryan et al. (2015) MERIT40 deficiency expands hematopoietic stem cell pools by regulating thrombopoietin receptor signaling. Blood 125:1730-8
Land, Ruben H; Rayne, Anna K; Vanderbeck, Ashley N et al. (2015) The orphan nuclear receptor NR4A1 specifies a distinct subpopulation of quiescent myeloid-biased long-term HSCs. Stem Cells 33:278-88
Song, Yiwen; Jiang, Jing; Vermeren, Sonja et al. (2014) ARAP3 functions in hematopoietic stem cells. PLoS One 9:e116107
Valvezan, Alexander J; Huang, Jian; Lengner, Christopher J et al. (2014) Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish. Dis Model Mech 7:63-71

Showing the most recent 10 out of 20 publications