A humanized eNAMPT -neutralizing monoclonal antibody reduces cancer-induced bone pain and osteoclastogenesis in murine breast cancer model

Abstract

Cancer-induced bone pain (CIBP) is a common symptom among patients with metastasized cancer, however, health professionals often face challenges in managing patients’ pain. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a DAMP overexpressed in metastatic breast carcinoma that binds to Toll-Like Receptor 4 (TLR4) to potentially trigger a signaling cascade through Nuclear Factor kappa β (NFk- β ) inducing inflammation, osteoclastogenesis, and contributing to CIBP. To assess the impact of eNAMPT on CIBP and to identify alternative therapeutic options, ALT-100, an eNAMPT-neutralizing monoclonal antibody, was tested in murine and cell-based models. First, eNAMPT synthesis and secretion by EO771 breast adenocarcinoma cells and its modulation by ALT-100 were assessed using eNAMPT ELISA. Next, recombinant eNAMPT-induced osteoclastogenesis and its inhibition by ALT-100 were evaluated in RAW 264.7 osteoclast precursor cells using TRAP staining. In vivo , female C57BL/6J mice underwent intramedullary femoral injection of EO771 cells to establish CIBP or SHAM surgery. One week later, mice received ALT-100 (4 mg/kg, ip) or saline control, followed by longitudinal assessment of pain-related behaviors and femoral radiographs. The eNAMPT ELISA assay revealed that EO771 cells synthesize and secrete eNAMPT in a time-dependent manner maximal at 48 h, which were reduced by ALT-100 mAb. In RAW 264.7 cells, recombinant eNAMPT significantly increased TRAP activity, while co-administration of ALT-100 significantly reduced this effect. In the murine CIBP model, ALT-100 significantly reduced pain-related behaviors compared to saline control mice in the cancer group. Moreover, radiographic analysis demonstrated that ALT-100 mitigates cancer-induced bone loss. These data show that ALT-100 exhibits antinociceptive effects in a CIBP murine model and inhibits eNAMPT-induced osteoclast differentiation. Our results indicate that blockade of eNAMPT can potentially serve as an alternative clinical strategy for treating CIBP.