Myeloma (MM) is the most frequent cancer to involve the skeleton. Up to 90% of patients develop bone lesions that can result in severe bone pain and frequent pathologic fractures. Unfortunately, these bone lesions rarely heal even when patients are in long-term remission because of the permanent MM-induced suppression of osteoblast precursor (OBP) differentiation into functional bone-forming osteoblasts. MM induces an intrinsic and persistent change in the OBP differentiation potential, the basis of which is unknown. Preliminary studies with a murine in vivo model of MM-induced OB suppression using 5TGM1 MM cells (which we modified to express GFP for detection and thymidine kinase, which allows them to be selectively killed by ganciclovir) demonstrate a persistent inhibition of OB differentiation even in the absence of MM cells. The OBP from these mice maintained low levels of the critical OB transcription factor, Runx2 even when induced to differentiate and had elevated expression of the transcriptional repressor Gfi-1, which can mediate chromatin remodeling. Importantly, Gfi-1 levels in OBP from 7/7 MM patients were elevated compared to 3 normals. 5TGM1 MM cells inhibited OB differentiation in vitro by producing TNF-1 and IL-7, which increased Gfi-1 in a mouse OBP line (MC4). Further, mouse Runx2 promoter analysis identified a 1003 bp region (-992/+111) that is responsible for suppression of Runx2 expression by MM cells and contains 29 putative Gfi-1 binding sites. This region is also repressed by TNF-1 and by co-transfection with a Gfi-1 expression plasmid. Importantly, knockdown of Gfi-1 expression, using a specific siRNA, significantly restored expression of Runx2 as well as the expression of several OB markers in both MC4 cells pretreated with MM cells and in MSC from 2 MM patients. These results suggest the hypothesis that MM cells secrete soluble factors (TNF-1 and IL-7) that increase Gfi-1 expression in OBPs. Gfi-1 then suppresses Runx2 production and thereby inhibits osteoblastogenesis. Further, since Gfi-1 can recruit to genes histone-modifying enzymes that create epigenetic changes, this results in long-term suppression of Runx2 that is maintained in the absence of MM cells. However, the role of Gfi-1 plays in OBP differentiation and in MM in particular is currently unknown and is the focus of this proposal. The following specific aims will be pursued to test this hypothesis: (1) Determine if Gfi-1 up-regulation in MSC is necessary for MM or TNF-1/IL-7 suppression of OB differentiation. (2) Determine if elevated Gfi-1 is sufficient for OB suppression and/or for increased IL-6 and RANKL production by MSC and does it act by direct binding to the Runx2 gene. (3) Determine if MM cells induce epigenetic changes in the Runx2 gene in MSC via Gfi-1, and if they are responsible for long-term OB suppression by assessing if altering the epigenetic status of the Runx2 gene relieves the differentiation block. (4) Determine if Gfi-1 deficiency in MSC in vivo prevents MM-induced suppression of OB differentiation.

Public Health Relevance

Multiple Myeloma (MM) is the most frequent cancer to involve the skeleton, with up to 90% of patients developing bone lesions that can result in severe bone pain and frequent pathologic fractures. Unfortunately, these lytic bone lesions rarely heal even when patients are in long-term complete remission because of the permanent MM-induced block of osteoblast precursor differentiation into functional bone-forming osteoblasts. This application proposes to investigate what intrinsic changes to the osteoblast precursor are triggered by MM cells and are responsible for the selective differentiation block.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Skeletal Biology Structure and Regeneration Study Section (SBSR)
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Sharrock, William J
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University of Pittsburgh
Internal Medicine/Medicine
Schools of Medicine
United States
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Adamik, Juraj; Silbermann, Rebecca; Marino, Silvia et al. (2018) XRK3F2 Inhibition of p62-ZZ Domain Signaling Rescues Myeloma-Induced GFI1-Driven Epigenetic Repression of the Runx2 Gene in Pre-osteoblasts to Overcome Differentiation Suppression. Front Endocrinol (Lausanne) 9:344
Lontos, Konstantinos; Adamik, Juraj; Tsagianni, Anastasia et al. (2018) The Role of Semaphorin 4D in Bone Remodeling and Cancer Metastasis. Front Endocrinol (Lausanne) 9:322
Adamik, Juraj; Jin, Shunqian; Sun, Quanhong et al. (2017) EZH2 or HDAC1 Inhibition Reverses Multiple Myeloma-Induced Epigenetic Suppression of Osteoblast Differentiation. Mol Cancer Res 15:405-417
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Fu, Jing; Li, Shirong; Feng, Rentian et al. (2016) Multiple myeloma-derived MMP-13 mediates osteoclast fusogenesis and osteolytic disease. J Clin Invest 126:1759-72
Teramachi, J; Silbermann, R; Yang, P et al. (2016) Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo. Leukemia 30:390-8
Delgado-Calle, Jesus; Bellido, Teresita; Roodman, G David (2014) Role of osteocytes in multiple myeloma bone disease. Curr Opin Support Palliat Care 8:407-13
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