A dominant-negative F-box deleted mutant of E3 ubiquitin ligase, ß-TrCP1/FWD1, markedly reduces myeloma cell growth and survival in mice.

Treatment of multiple myeloma with bortezomib can result in severe adverse effects, necessitating the development of targeted inhibitors of the proteasome. We show that stable expression of a dominant-negative F-box deleted (∆F) mutant of the E3 ubiquitin ligase, SCFß-TrCP/FWD1, in murine 5TGM1 myeloma cells dramatically attenuated their skeletal engraftment and survival when inoculated into immunocompetent C57BL/KaLwRij mice. Similar results were obtained in immunodeficient bg-nu-xid mice, suggesting that the observed effects were independent of host recipient immune status. Bone marrow stroma offered no protection for 5TGM1-∆F cells in cocultures treated with tumor necrosis factor (TNF), indicating a cell-autonomous anti-myeloma effect. Levels of p100, IκBα, Mcl-1, ATF4, total and cleaved caspase-3, and phospho-ß-catenin were elevated in 5TGM1-∆F cells whereas cIAP was down-regulated. TNF also activated caspase-3 and downregulated Bcl-2, correlating with the enhanced susceptibility of 5TGM1-∆F cells to apoptosis. Treatment of 5TGM1 tumor-bearing mice with a ß-TrCP1/FWD1 inhibitor, pyrrolidine dithiocarbamate (PDTC), significantly reduced tumor burden in bone. PDTC also increased levels of cleaved Mcl-1 and caspase-3 in U266 human myeloma cells, correlating with our murine data and validating the development of specific ß-TrCP inhibitors as an alternative therapy to nonspecific proteasome inhibitors for myeloma patients.

CCR1 blockade reduces tumor burden and osteolysis in vivo in a mouse model of myeloma bone disease.

The chemokine CCL3/MIP-1α is a risk factor in the outcome of multiple myeloma (MM), particularly in the development of osteolytic bone disease. This chemokine, highly overexpressed by MM cells, can signal mainly through 2 receptors, CCR1 and CCR5, only 1 of which (CCR1) is responsive to CCL3 in human and mouse osteoclast precursors. CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. Here we show that formation of mature osteoclasts is blocked by the highly potent and selective CCR1 antagonist CCX721, an analog of the clinical compound CCX354. We also show that doses of CCX721 selected to completely inhibit CCR1 produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease. Similar effects were observed when the antagonist was used prophylactically or therapeutically, with comparable efficacy to that of zoledronic acid. 5TGM1 cells were shown to express minimal levels of CCR1 while secreting high levels of CCL3, suggesting that the therapeutic effects of CCX721 result from CCR1 inhibition on non-MM cells, most likely osteoclasts and osteoclast precursors. These results provide a strong rationale for further development of CCR1 antagonists for the treatment of MM and associated osteolytic bone disease.

Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma.

PURPOSE: Eradication of post-treatment residual myeloma cells is needed to prevent relapses, and immunostimulatory monoclonal antibodies (mAb) such as anti-CD137, CTLA-4, CD40, etc., which enhance the immune response against malignancies, represent a means of achieving this purpose. This study explores anti-CD137 mAbs for multiple myeloma treatment in preclinical models of the disease because they safely augment tumor immunity and are in clinical trials for other cancers. EXPERIMENTAL DESIGN: The antitumor effect of anti-CD137 mAb on mouse plasmacytomas derived from HOPC and NS0 cell lines was studied and compared with that of anti-CTLA-4, anti-CD40, and anti-ICAM-2 mAbs. The antitumor effect of anti-CD137 mAb was also examined in a mouse syngeneic disseminated myeloma (5TGM1) model, which more closely resembles human multiple myeloma. Depletions of specific cell populations and gene-targeted mice were used to unravel the requirements for tumor rejection. RESULTS: Agonistic mAb against CD137 and blocking anti-CTLA-4 mAb showed activity against i.p. HOPC tumors, resulting in extended survival of mice that also became immune to rechallenge. Anti-CD137 mAbs induced complete eradications of established s.c. NS0-derived tumors that were dependent on IFN-gamma, natural killer cells, and CD8(+) T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-gamma production. Antitumor efficacy of anti-CD137 mAb was preserved in CD28-deficient mice despite the fact that CD28 signaling increases the expression of CD137 on CD8(+) T cells. Importantly, anti-CD137 mAb treatment significantly decreased systemic tumor burden in the disseminated 5TGM1 model. CONCLUSIONS: The immune-mediated antitumor activity of anti-CD137 mAb in mouse models holds promise for myeloma treatment in humans.

Transdermal lovastatin enhances fracture repair in rats.

Statins have been shown to stimulate BMP2 transcription and bone formation. This raises the possibility that they could be useful for enhancing rates of fracture repair. Observational studies in patients treated with oral statins for lipid-lowering have been controversial. The likely reason for their inconsistent effects is that the statin concentration reaching the periphery was too low after oral administration to produce a reproducible biologic effect. Thus, we examined the effects of lovastatin (LV) given transdermally in a well-described preclinical model of fracture repair. Effects on the healing fracture callus were assessed by biomechanical strength, radiographs, and quantitative morphology. LV was administered transdermally (TD) for 5 days after fracture in several doses (0.1-5 mg/kg/d) and compared with vehicle-treated control rats and rats treated with LV by oral gavage (PO) at 5-25 mg/kg/d for 5 days from the day of fracture. Radiological evaluation of bones treated with TD LV showed enhanced fracture repair at 2 and 6 wk. BMD in the callus area at 6 wk was also increased in the TD group compared with vehicle-treated controls (p < 0.05). The force required to break TD-treated bones (0.1 mg/kg/d for 5 days) was 42% greater than vehicle-treated controls (p < 0.02), and there was a 90% increase in stiffness (p < 0.01). PO LV at much higher doses (10 and 25 mg/kg/d) showed increased stiffness but no change in other biomechanical properties. By histological examination, a significant increase was also observed in the size of the callus, surrounding proliferating cell nuclear antigen-positive cells, and osteoblast and osteoclast number in TD-treated rats compared with controls at day 8 after fracture (n = 6). In summary, we found that TD LV in low doses accelerates fracture healing, whereas 10-fold the lipid-lowering dose was required to produce any effect when it was administered orally. These studies provide valuable information on the potential of statins and TD delivery as a new and effective therapeutic modality in fracture repair.

Increasing Wnt signaling in the bone marrow microenvironment inhibits the development of myeloma bone disease and reduces tumor burden in bone in vivo.

There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of beta-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.