Project 1: p62^ZIP, a novel target for myeloma bone disease

Osteoclastic bone destruction and tumor growth in myeloma are mediated by factors that activate the NF-kB signaling pathway. In particular, RANK ligand (RANKL), a potent inducer of osteoclast (OCL) formation, is increased in the myeloma bone microenvironment as are IL-6, TNF-a, and IL-1, which further enhance tumor cell growth and survival, adhesion to stromal cells, chemoresistance, and bone destruction. Recently, we demonstrated that p62^ZIP was a critical factor in NF-kB activation by RANKL, as well as TNF-a and IL-1. In this project, we will test the hypothesis that inhibiting p62^ZIP will profoundly diminish osteolytic bone destruction and myeloma growth in vivo. The following specific aims will be pursued: 1) characterize the effects of overexpressing or deleting p62^ZIP in human OCL precursors. 2) Determine the effects of overexpressing or deleting p62^ZIP in stromal cells on their capacity to support OCL formation and growth of myeloma cells. 3) Use p62^ZIP knock-out mice to: a) determine the effects of deleting p62^ZIP on OCL formation in vivo and in vitro; b) assess the capacity of p62^ZIP +/- stromal cells to support OCL formation by normal spleen cells and support the growth of 5TGM1 murine myeloma cells and MM.1S human myeloma cells. 4) Breed p62^ZIP knock-out mice to the NOD-SCID background and transplant the mice with 5TGM1 myeloma cells to determine if deleting p62^ZIP in vivo decreases myeloma cell growth and bone destruction.

Project 2: Bone microenvironmental factors and myeloma bone disease

Multiple factors have been identified in vitro that may play an important role in myeloma bone disease, but their role in vivo is unclear. It is our hypothesis that there are only very limited number of factors produced in the microenvironment that are critical to the development of bone destruction and the absent new bone formation in myeloma. In this project, a new model of myeloma bone disease will be developed using the Òossicle modelÓ in which stromal cells from murine or human sources are combined with Gelfoam and implanted subcutaneously in SCID mice to form marrow containing ossicles. Dr. McCauley has recently shown that prostate cancer cells can home to these ossicles and induce new bone formation. The ossicles can be composed of stromal cells that differ genetically and removed at different time points for histologic evaluation without sacrificing the animals, providing a distinct advantage over current models.

The specific aims of this project will be to: 1) Adapt the ossicle model for studying myeloma bone disease. 2) Use stromal cells from animals either lacking or overexpressing TNF-a, Il-6, Il-1, or B3 integrins or their cognate receptors to test their capacity to support myeloma growth and bone destruction. 3) Once critical factors are identified, examine the effects of agents that specifically inhibit these factors on the growth of the myeloma cells, osteoclast formation and osteoblast growth in vivo in murine models of myeloma.

Project 3: Pure anabolic skeletal therapy with PTH-related protein for myeloma bone disease: Preclinical and Clinical Studies

The PI identified and structurally defined parathyroid hormone-related protein (PTHrP), and demonstrated that PTHrP signals via the common PTH/PTHrP receptor with identical potency to PTH. PTHrP, like PTH, increases osteoblast numbers, osteoid synthesis, and accelerates mineral apposition rates, the hallmarks of anabolic skeletal activity. Most recently, we demonstrated that PTHrP can safely be administered to postmenopausal women, and in contrast to PTH, is a pure anabolic agent, dramatically increasing bone mineral density (BMD) by 5% in only 3 months.

Myeloma bone disease results from increased osteoclastic bone resorption, and also reflects a failure of osteoblastic bone formation. Moreover, the multiple agents currently available that inhibit bone resorption do not increase the already low bone mass in patients with myeloma. An anabolic skeletal agent is needed.

We therefore propose three specific aims: 1) To determine whether PTHrP and PTH increase bone formation in a mouse model of myeloma, and to define whether this effect is enhanced or hindered by the potent anti-resorptive agent, zoledronate. 2) To identify doses of PTHrP and PTH, in a one-month study, which stimulate biochemical markers of bone formation in patients with myeloma, again, in the presence or absence of zoledronate. 3) To determine in a three-month clinical trial whether PTHrP or PTH can cause marked increases in BMD in early and late stages of myeloma, as it does in postmenopausal osteoporosis.