Efficient recovery of the immune system following bone marrow or stem cell transplantation requires optimal function of all obligatory supportive components of the marrow microenvironment. Work completed during previous periods of funding was focused on bone marrow stromal cells (BMSC) function subsequent to dose escalated chemotherapy. These studies demonstrated that BMSC VCAM-1 and CXCL-12 (SDF-1) proteins were diminished by VP-16 exposure. Treatment resulted in BMSC with reduced ability to support migration and survival of hematopoietic progenitor cells and dysregulation of expression and activity of BMSC derived matrix metalloproteinase-2 (MMP-2) and TGF-21. Experiments proposed in the current application extend these studies to osteoblasts as additional, essential constituents of the hematopoietic stem cell niche. Osteoblasts contribute to the """"""""endosteal niche"""""""" as the unique anatomical location in which stem cells are maintained until recruited into the proliferative population available for subsequent differentiation. Pilot data relevant to the current application indicate that primary human osteoblasts exposed to melphalan or VP-16 have diminished CXCL12 expression and chemotactic support and altered support of CD34+ bone marrow cell differentiation. Furthermore, phosphorlyated-Smad-2 signaling in osteoblasts is enhanced by direct exposure to chemotherapy, suggesting autocrine effects of therapy-induced TGF-b. The influence of chemotherapy induced autocrine signaling via elevated TGF-2 was investigated by treatment of primary human osteoblasts to rTGF-b which resulted in elevated TGF-2 gene expression, suggesting potential for amplification of the effects of elevated TGF-2 signaling. Recombinant TGF-2 also reduced CXCL12 expression and blunted chemotactic support. Building on these preliminary data we will investigate the effects of dose escalated chemotherapy on osteoblasts with evaluation of the mechanisms that underlie changes in factors necessary for optimal chemotactic support of CD34+ human hematopoietic stem cell maintenance, with emphasis on the mechanisms that underlie diminished CXCL-12 gene expression and protein levels (Aim 1). We will extend our model to three dimensional matrices to include consideration of signaling that is influenced by architecture, an important aspect of models to investigate osteoblast function in a chemotherapy stressed bone marrow microenvironment (Aim 2) and will transition our studies to a murine model in which the osteoblast population can be specifically identified by virtue of constitutive expression of GFP (Col2.3-GFP mice) to allow isolation and characterization of ostoeblasts from mice exposed to chemotherapy in vivo. Successful completion of these aims we will test the hypothesis that TGF-b is a critical mediator of the effects of dose escalated chemotherapy on dysregulation of osteoblast function in the bone marrow microenvironment. Completion of this study will contribute to development of strategies in which neutralization of TGF-b can be optimized to enhance long-term hematopoietic recovery of bone marrow transplantation patients which relies, in part, on optimal function of the stem cell niche.

Public Health Relevance

Long term hematopoietic recovery following bone marrow transplantation relies on a functional stem cell niche in the bone marrow to which transplanted cells must efficiently migrate and are impacted on by diverse developmental cues. Osteoblasts of the endosteal niche define one essential population that supports stem cell development. In the current proposal we investigate their vulnerability to dose escalated chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056888-17
Application #
8680301
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Welniak, Lisbeth A
Project Start
1997-08-01
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
17
Fiscal Year
2014
Total Cost
Indirect Cost
Name
West Virginia University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Morgantown
State
WV
Country
United States
Zip Code
26505
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Moses, Blake S; Slone, William L; Thomas, Patrick et al. (2016) Bone marrow microenvironment modulation of acute lymphoblastic leukemia phenotype. Exp Hematol 44:50-59.e2
Slone, William L; Moses, Blake S; Evans, Rebecca et al. (2016) Modeling Chemotherapy Resistant Leukemia In Vitro. J Vis Exp :e53645
Moses, Blake S; Evans, Rebecca; Slone, William L et al. (2016) Bone Marrow Microenvironment Niche Regulates miR-221/222 in Acute Lymphoblastic Leukemia. Mol Cancer Res 14:909-919
Hare, Ian; Gencheva, Marieta; Evans, Rebecca et al. (2016) In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage. Stem Cells Int 2016:8270464
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Basu, Soumit K; Remick, Scot C; Monga, Manish et al. (2014) Breaking and entering into the CNS: clues from solid tumor and nonmalignant models with relevance to hematopoietic malignancies. Clin Exp Metastasis 31:257-67

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