Thymidine Phosphorylase Exerts Complex Effects on Bone Resorption and Formation in Myeloma

Thymidine phosphorylase (TP), an enzyme that can reversibly catalyze the conversion of thymidine to thymine and 2-deoxy-D-ribose (2DDR), has been shown to participate in tumor angiogenesis and proliferation. Yet little is known regarding its function in bone. The goal of this study is to elucidate the role and mechanism of myeloma-expressed TP in the activation of osteoclast-mediated bone resorption and the suppression of osteoblast-mediated bone formation. We hypothesized that myeloma-expressed TP plays an important role in the pathogenesis of myeloma bone disease. We observed that TP is highly expressed in myeloma cells but not in normal plasma cells. To examine the role of myeloma-expressed TP in lytic bone lesions, we categorized all tested patient-derived myeloma cells and human myeloma cell lines into two groups: TP-high and TP-low expressing cells. These myeloma cells, as well as human myeloma cells with overexpressed or knocked downed levels of TP, were injected into the implanted human bone chips of SCID-hu mice or the femurs of SCID mice. Analysis of radiography and histomorphometry were used for assessing lytic lesions. Our results showed that injection of TP-high expressing myeloma cells into mice caused more lytic lesions than injection of TP-low cells. To examine its role in osteoclast and osteoblast differentiation, the progenitors were co-cultured with the myeloma cells, and analyzed with staining of TRAP and Alizarin red S. We observed that co-culture with TP-high expressing myeloma cells induced more osteoclast differentiation and less osteoblast formation than those co-cultured with TP-low cells. Mechanistic studies further showed that TP-high expressing myeloma cells secreted more 2DDR than TP-low cells. The secreted 2DDR bound to the integrin a_Vb₃ in osteoclast progenitors, activated the PI3K/Akt signaling, and enhanced DNMT3A expression and methylation of IRF8, leading to increased NFATc1 expression and osteoclast differentiation. The secreted 2DDR could also bind to the integrins a_Vb₃ and a₅b₁ in osteoblast progenitors, activated the PI3K/Akt signaling, and enhanced DNMT3A expression and methylation of RUNX2 and osterix, leading to decreased osteoblast differentiation. We further examined the patient bone marrow samples, and demonstrated a positive correlation between TP expression in myeloma cells and osteolytic bone lesions in myeloma patients. Thus, our study not only elucidates a novel mechanism of myeloma-induced increased osteoclast-mediated bone resorption and suppressed osteoblast-mediated bone formation, but also implicates a potential therapeutic approach for myeloma bone disease.