Osteosclerosis is a major complication of primary myelofibrosis (PMF) and is characterized, in addition, by excess proliferation of bone marrow myeloid cells, abnormal connective tissue and fibrosis. Although mutant megakaryocytes (MKs) play a major role in driving connective tissue abnormalities in PMF, molecular mediators which develop between abundant MKs in PMF and increased bone formation from mesenchymal progenitors have not been established. Here, we propose to examine an innovative concept involving the multifunctional enzyme known as lysyl oxidase (LOX) and other MK-derived growth factors as agents which directly promote osteosclerosis in PMF by stimulating the proliferation and differentiation of bone marrow mesenchymal stem cells (MSCs). Human PMF is most often caused by clonal expansion promoted by a hyper- activating causative mutation in the JAK2 gene.
In Aim 1 we propose to use a mouse model of JAK2V617F? induced PMF to test the new contention that factors elaborated by JAK2V617F MKs impact bone marrow MSCs differentiation to osteoblasts. To this end, JAK2V617F MKs will be tested in co-cultures with wild type MSCs. Based on preliminary and other studies, levels of candidate factors (LOX, PDGF, TGF?, WNT3a) will first be assessed by ELISA assays, and functionality determined with blocking antibodies and proteins, and by LOX pharmacological inhibition. In the spirit of a relatively higher risk R21 grant mechanism, we also propose to measure 110 different cytokines via state-of-the-art Luminex assays, followed by blocking experiments of differentially regulated factors.
In Aim 2, we will examine the novel hypothesis that LOX contributes to the pathological bone phenotype in PMF in vivo. To assess this, we will cross breed JAK2V617F mice with LOX+/- mice available to us, and then characterize the bone phenotype of compound mutant mice JAK2V617F/LOX+/- in vivo and in vitro. Taken together, data obtained in this project will determine factors elaborated by associations between JAK2V617F MKs and wild type MSCs which drive osteosclerosis, and which, therefore, will become potential therapeutic targets to address osteosclerosis in PMF.

Public Health Relevance

Primary myelofibrosis (PMF) is a bone cancer that results in accumulation of too much bone (called osteosclerosis) and connective tissue in the bone marrow cavity which interferes with the function of bone marrow, weakens the structure of the mineralized bone, and leads to severe handicaps and even death. The research proposed here uses mice and bone marrow cells with a mutation in a bone marrow cell type (megakaryocytes or MKs) and that develop PMF, and a novel mouse strain produced by cross-breeding, to uncover mechanisms by which abnormalities in interactions between MKs with bone marrow stem cells lead to osteosclerosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AR072748-01
Application #
9428798
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Chen, Faye H
Project Start
2018-05-01
Project End
2020-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Boston University
Department
Dentistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118