All ETDs from UAB

Advisory Committee Chair

Selvarangan Ponnazhagan

Advisory Committee Members

Ralph D Sanderson

Susan L Bellis

Xu Feng

Richard D Lopez

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

ABSTRACT Multiple Myeloma (MM) remains the second most common hematologic malig-nancy occurring in adults, which primarily affects the skeletal system causing severe bone destruction, spinal cord compression and hypercalcemia. Current therapies, which include chemotherapy, radiotherapy, autologouos stem cell transplantation and in some cases surgery, have extended the median survival between 3 and 10 years. However, MM is still incurable and therefore improving current therapies or developing newer ones to reduce the burden of osteolytic damage and extend survival rate will be highly beneficial for patient management. The long-term goal of this study is to develop a novel therapeutic approach to the treatment of bone lesions in MM using genetically-engineered mesenchymal stem cells (MSC) for inhibiting osteoclast activity by stable expression of osteoprotegin (OPG). Studies have shown that osteoclast activity is enhanced in myeloma patients through in-creased expression of receptor activator of nuclear factor kappa B ligand (RANKL) lead-ing to RANKL/RANK signaling, resulting in osteoclast activation and ultimately bone resorption. Osteoprotegerin is a soluble decoy receptor for RANKL, and its expression is decreased in myeloma patients possibly because of a marked decrease in the number of osteoblasts and or MSC, which produce OPG. Thus, the central hypothesis of this study is cell therapy with MSC, engineered to overexpress OPG, will greatly decrease osteolytic damage and reduce morbidity. Despite its potential in inhibiting osteoclast activation, OPG also binds to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) making myeloma cells resistant to apoptosis. In order to eliminate TRAIL binding while still possessing RANKL binding, we sought to create OPG mutants based on interactive domain identification, and superim-posing structural models of RANKL and TRAIL for a representative OPG-RANKL-TRAIL complex. Five such OPG mutants were constructed and recombinant proteins produced in HEK-293 cells for characterization of their TRAIL binding ability. Among these, two mutants that greatly abolished TRAIL binding while maintaining RANKL binding were identified by TRAIL assay and osteoclast assay, respectively. When tested following cancer-induced osteolytic bone damage with a human epithelial cancer cell line, as xenograft in mouse tibia, these two OPG mutants provided significant therapy effects in bone remodeling in vivo. The therapeutic potential of these mutants for MM bone lesions was determined by developing a bone-disseminated myeloma disease in SCID mice using a human myeloma cell line CAG, overexpressing heparanase (CAGhep), and testing the bone-remodeling effects of mutant OPG by genetically-engineered adult stem cell approach. Results of this study indicated that when compared to the age-matched control, MSC-OPGmut therapy indicated an overall increase in trabecular density and number both in the tibia and spine of animals treated with OPG mutants. Finally, OPG mutants were also tested in vitro and confirmed that they do not inhibit TRAIL-induced myeloma cell death, suggesting the possibility of using the mutant OPG stem cell ap-proach in combination with TRAIL therapy for dual targeting of bone lesions and tumor cells, respectively.

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