Project Summary Multiple myeloma (MM) grows in and attacks the skeleton, causing pain and bone-related pathologies, such as fracture, as well as contributing to death. Many interactions between the tumor and the bone microenvironment are understood, but none involves pathways unique to bone, and myeloma bone disease (MBD) remains largely incurable. We asked whether factors made only by bone acted specifically on myeloma cells. MM cells, both laboratory cell lines and from patients, expressed the receptor for the hormone fibroblast growth factor (FGF)-23, which is made by osteocytes, abundant cells in bone. In newly diagnosed patients, intact serum FGF23 was 3X higher in MM than in controls. Recombinant FGF23 increased heparanase mRNA 5-18x in 2 MM cell lines at 24hrs. Heparanase is a secreted enzyme that enhances tumor homing and growth in bone and is a marker of poor MM prognosis. It increases bone destruction via the factor RANK ligand and suppresses bone formation, releases growth factors from bone matrix and modifies the MM protein syndecan-1. We propose a new vicious cycle of myeloma bone disease: A) MM cells stimulate osteocytes to secrete FGF23; B) FGF23 increases heparanase expression by MM cells; C) Heparanase alters the bone microenvironment and increases RANK ligand, Dkk1, osteolysis & tumor growth; D) Heparanase increases hepatocyte growth factor (HGF) in bone, which additionally stimulates MM cells; E) FGF23 may enhance MM adhesion to bone. The two Investigators combine extensive individual interests in cancer:bone molecular interactions (JMC) and the biology of clinical myeloma (AS) with a shared interest in the etiology and improved treatment of myeloma bone disease. We propose Four Specific Aims: 1: Determine the cellular pathways by which MM increases FGF23, including testing if known factors made by MM cells regulate FGF23 production, or if it is regulated by heparanase, HGF or the approved MM treatment bortezomib. 2: Determine the role of circulating FGF23 in MM patients, asking if FGF23 & heparanase concentrations are correlated in MM patients and testing if MM cells from patients increase bone release of FGF23. 3: Determine the importance of FGF23-increased heparanase in MBD. 4. Determine molecular mechanisms that control heparanase expression in the bone microenvironment. The project uses a new experimental model of myeloma:bone interactions. In EVOCA (ex vivo organ culture assay), human MM cells are cultured on mouse bone, which supports growth of cell lines and patient samples (which dramatically increase FGF23 in bone). We will make MM cell lines with promoters driving secreted luciferases to analyze changes in gene activity over time in the tumor:bone microenvironment. Experiments with live animal are avoided, lowering costs & increasing efficiency, along with elimination of animal pain & suffering. EVOCA enables new studies in Aim 2 with primary samples to test the importance of the FGF23:heparanase vicious cycle in MBD in individual patients. Inhibitors of FGF23 & heparanase are in clinical trials. Our studies will guide their future application to the treatmentof myeloma bone disease and other cancer bone metastases.