2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

Novel therapy for humoral hypercalcemia of malignancy in adult T-cell leukemiaEffects of proteasome inhibitor and bisphosphonate on tumor growth and bone formationin vivo and in vitroSherry Shu, Murali VP Nadella, Wessel Dirksen, and Thomas J RosolDepartment of Veterinary Biosciences, The Ohio State University,1925 Coffey Road, Columbus, OH, USAAdult T-cell lymphoma/leukemia (ATLL) is an aggressive and often fatalmalignancy of CD4+ T-lymphocytes caused by infection with a complex retrovirus, human T-celllymphotropic virus type 1 (HTLV-1). More than 80% of ATLL patients develop HHM resulting fromincreased osteoclastic bone resorption and renal reabsorption of calcium. Factors secreted by tumorscells that can contribute to HHM include, interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α),TNF-β, and parathyroid hormone-related protein (PTHrP). PTHrP has been shown to play an essentialrole in HHM and the expression of PTHrP and cytokines, including TNF-β, can be activated by NF-кB.Proteasome inhibitor, PS-341, is able to disrupt NF-кB in vitro and in vivo. In addition, bisphosphonates,such as zoledronic acid (Zol), have been used for treatment of tumor-induced hypercalcemia; however,little is known about the effects of these two drugs on tumor burden and hypercalcemia in ATLLpatients. Our goal is to establish an in vivo model to evaluate the effects of NF-кB and osteoclastinhibition on the development of T-cell malignancy and HHM. To monitor tumor burden in vivo bynoninvasive bioluminescent imaging (BLI), ATLL cells derived from a patient (RV-ATL cells) werestably transduced with luciferase reporter gene (luc) in a lentivirus construct in vitro. Twenty millioncells (RV-ATL-luc) were injected into NOD/SCID mice and BLI was performed every week. Signalwas detected as early as week one and continued to increase until sacrifice (Figure 1). The ex vivo BLIfor each organ indicated that tumor cells infiltrated mainly the mesenteric lymph nodes and spleen(Figure 2). Solid tumors formed at the site of injection have also been observed. Histological evaluationconfirmed the ex vivo findings. To examine the in vitro effects of PS-341 and Zol on proliferation, cellviability, and apoptosis in ATLL cells, MTT, trypan blue exclusion and TUNEL assays were performed,respectively. PS-341 significantly inhibited proliferation (IC50≈10 nM), decreased viability (50%reduction at 25 nM), and induced apoptosis (43% at 20 nM) in RV-ATL cells; however, the effects ofZol on proliferation, cell viability and apoptosis were not significant, as expected (Figure 3). We havealso found that PS-341 inhibited PTHrP expression by 80% in RV-ATL cells at 10 nM using real-timeRT-PCR (Figure 4). For the in vivo assay, 3x107 RV-ATL-luc cells were injected into 5-week-oldNOD/SCID mice. Mice were treated twice a week with vehicle alone, PS-341 alone, Zol alone, andcombined PS-341 and Zol. BLI was performed at weeks 4 and 5. The signals in PS-341 alone and thecombined groups were lower than the vehicle control group and Zol alone group (Figure 5). Thetrabecular bone density in Zol alone group was higher than the vehicle group, which indicated theinhibition of osteoclastic activity (Figure 6). Our results indicate that a combination of PS-341 and Zolwas an effective treatment for ATLL.34