Variants of Osteoprotegerin Lacking TRAIL Binding for Therapeutic Bone Remodeling in Osteolytic Malignancies

Abstract

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of Receptor Activator of Nuclear Factor Kappa B Ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remains the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to tumor necrosis factor related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Towards uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage.

A conserved aromatic moiety in the ectodomain is a key determinant for structural integrity and protein trafficking of TNFR superfamily

“…The functionality of mutant OPG proteins in terms of inhibiting osteoclastogenesis was determined, as previously described. 24 Briefly, RAW 264.7 cells were cultured in 24well plates (1 × 10 4 cells/well) for 10 days in the presence of 100 ng/mL recombinant RANKL (a kind gift from Dr Xu Feng, UAB), either alone or in combination with conditioned medium from plasmid transfected cells with either OPG WT or OPG Y49R . The medium was changed every 48 hours for 10 days, following which tartrate-resistant acid phosphatase (TRAP) staining was performed using Leukocyte Acid Phosphatase kit (MilliporeSigma), as per manufacturer's instruction to enumerate multinucleated TRAP-positive osteoclasts.…”

“…14 Similar to clinical reports with molecular alteration in TNFRSFassociated pathologies, we previously identified that mutations on key TRAIL-binding residues in OPG result in similar delay in subcellular protein trafficking. 24 In our quest to mitigate the limitations of a clinical use of OPG in skeletal remodeling for osteolytic malignancies, we endeavored uncoupling the bispecific function of OPG from sequestering tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL). During this process, we have identified key domains involved in TRAIL binding and generated OPG variants lacking the structural determinant for TRAIL binding.…”

Mesenchymal stem cells expressing osteoprotegerin variants inhibit osteolysis in a murine model of multiple myeloma

“…17 The recombinant OPG used comprises the ligand-binding domain of human OPG (1-201 amino acids), fused to the Fc domain of human immunoglobulin G in pcDNA3.1 vector, under the control of cytomegalovirus promoter. Variants of OPG containing mutations Y49R and F107A were created by sitedirected mutagenesis as described earlier.…”

“…Variants of OPG containing mutations Y49R and F107A were created by sitedirected mutagenesis as described earlier. 17 To generate recombinant adeno-associated virus (rAAV) plasmids containing wild-type (WT) or mutant OPG, the open reading frame, encoding the transgene was excised from pcDNA3.1 and subcloned into the multicloning site (MCS) of rAAV plasmid. Packaging and production of rAAV-OPG WT/mut were performed by transient transfection in 293 cells with the helper plasmid pDP6 using an established protocol as described earlier.…”

“…To overcome this limitation of OPG therapy, we recently identified domains on OPG that are important for TRAIL binding, developed and demonstrated functional ability of such OPG mutants, abolished in TRAIL binding, but retained in RANKL binding. 17 The last decade has seen increasing therapeutic use of mesenchymal stem cells (MSCs). 18,19 Along with their potential of self-renewal, MSCs can differentiate into adipocytes, chondrocytes, and osteoblasts.…”