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Bortezomib is highly effective in multiple myeloma and is widely used in this disease. Recently, an increased incidence of varicella zoster virus (VZV) reactivation was reported in patients with myeloma undergoing bortezomib treatment.We investigated the influence of bortezomib on T-cell subpopulations in 53 patients with myeloma before initiation of bortezomib and during therapy.A decrease of CD4+ T cells was seen in 41 of 53 patients (77%). The median CD3+/CD4+ lymphocyte counts declined from 494/microL (range, 130-2187/microL) to 274/microL (range, 41-1404/microL) during bortezomib treatment (P.001). In the majority of patients (40 of 53 patients, 75%), CD4+ lymphocytes dropped to400/microL during bortezomib treatment, and in 18 of 53 patients (33.9%) the CD4+ T cells fell below 200/microL. The minimum CD4S+ cell count was observed at a median of 6 weeks (range, 2-22 weeks) after initiation of treatment. The incidence of herpes zoster reactivation was 5.7% in the whole population of patients with myeloma receiving bortezomib. Nineteen of 53 patients received acyclovir at a dose of 400 mg daily as prophylaxis against VZV reactivation. In this group, none of the patients developed herpes zoster. The incidence of VZV reactivation in patients not receiving acyclovir was 3 of 34 (8.8%). Importantly, occurrence of herpes zoster was associated with reduced CD4+ T-cell subpopulation: all patients who developed herpes zoster had CD4+ lymphocytes400/microL.Our results show that bortezomib leads to a transient decrease in CD4+ lymphocytes, accompanied by an increased incidence of VZV infections. The antiviral prophylaxis with acyclovir is effective in patients with myeloma treated with bortezomib.

Objectives: Lytic bone disease is a hallmark of multiple myeloma (MM) and is caused by osteoclast activation and osteoblast inhibition. Secretion of Dickkopf (DKK)-1 by myeloma cells is a major factor which causes inhibition of osteoblast precursors. So far, there is no study showing a significant difference in serum DKK-1 levels in MM patients with or without lytic bone lesions. Methods: DKK-1 serum levels were quantified in 184 untreated MM patients and 33 monoclonal gammopathy of undetermined significance (MGUS) patients by ELISA, using a monoclonal anti-DKK-1 antibody. Results: Serum DKK-1 was elevated in MM as compared with MGUS (mean 11 963 pg/mL vs. 1993 pg/mL; P 3 lesions: 3114 pg/mL vs. 3559 pg/mL vs. 24 068 pg/mL; P = 0.002). Conclusion: Using a large series of myeloma patients, we could show for the first time a correlation between DKK-1 serum concentration and the amount of lytic bone disease, indicating that DKK-1 is an important factor for the extent of bone disease and supporting the hypothesis of DKK-1 as a therapeutic target in myeloma bone disease.

In multiple myeloma, the overexpression of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL) leads to the induction of NF-kappaB and activator protein-1 (AP-1)-related osteoclast activation and enhanced bone resorption. The purpose of this study was to examine the molecular and functional effects of proteasome inhibition in RANKL-induced osteoclastogenesis. Furthermore, we aimed to compare the outcome of proteasome versus selective NF-kappaB inhibition using bortezomib (PS-341) and I-kappaB kinase inhibitor PS-1145. Primary human osteoclasts were derived from CD14+ precursors in presence of RANKL and macrophage colony-stimulating factor (M-CSF). Both bortezomib and PS-1145 inhibited osteoclast differentiation in a dose- and time-dependent manner and furthermore, the bone resorption activity of osteoclasts. The mechanisms of action involved in early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase pathways, whereas the later phase of differentiation and activation occurred due to inhibition of p38, AP-1 and NF-kappaB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutic option of cancer-induced lytic bone disease.

The proteasome is a proteolytic complex for intracellular degradation of ubiquitinated proteins which are involved in cell-cycle regulation and apoptosis. A constitutively increased proteasome activity has been found in myeloma cells. We studied circulating proteasome levels and their prognostic significance in sera of 50 control subjects, 20 persons with monoclonal gammopathies of undetermined significance (MGUS), and 141 previously untreated patients with multiple myeloma (MM) by an anti-20S proteasome enzyme-linked immunoabsorbent assay (ELISA). Serum proteasome concentrations were significantly elevated in MM compared with controls (P < .001), in MM versus MGUS (P = .03), and in active (n = 101) versus smoldering (n = 40) MM (P < .001). In patients with active MM, there was a significant (P < .001) decrease from pretreatment to post-treatment proteasome concentrations in responders to chemotherapy, but not in nonresponders. Circulating proteasome levels were identified as a prognostic factor for overall survival in the univariate (P < .001 log-rank test) and in the multivariate (hazard ratio, 4.38) survival analysis in patients with active MM. We demonstrate for the first time that increased serum proteasome concentrations correlate with advanced disease and are an independent prognostic factor in MM.

Chronic myeloid leukemia developing after transplantation of solid organs and concomitant immunosuppression is a rare but still significant clinical phenomenon. We here describe an additional case of a 62-year-old male patient developing CML after orthotopic heart transplantation and medication with cyclosporine A, mofetil-mycophenolate, and steroids. Initial antileukemic therapy was imatinib at a standard dose and within 15 months of therapy a complete cytogenetic response was noted. In this report we discuss the clinical implications of these rare but biologically important cases.

Objectives: The ubiquitin–proteasome system emerged as a new therapeutic target in cancer treatment. The purpose of this study was to elucidate the effects of the novel proteasome inhibitor BSc2118 on t(4;14) positive and negative multiple myeloma (MM) cells and normal peripheral blood mononuclear cells (PBMNC). Methods: Human MM cell lines OPM-2, RPMI-8226, and U266 and primary MM cells from bone marrow aspirates were exposed to BSc2118. Cytotoxicity levels were evaluated using the MTT-test. BSc2118-induced apoptosis was analyzed by annexin-V assay. Further methods used included proteasomal activity determination, cell cycle analysis, western blot, and transcription factor assays. Results: In OPM-2, RPMI-8226, U266 cell lines and primary MM cells, BSc2118 caused dose-dependent growth inhibitory effects. After 48 h, dose-dependent apoptosis occurred both in cell lines and primary myeloma cells irrespective of t(4;14). A significant G2-M cell cycle arrest occurred after 24 h. Furthermore, we observed a marked inhibition of intracellular proteasome activity, an increase in intracellular p21 levels, and an inhibition of NF-κB activation. The toxicity against PBMNC remained low, suggesting a broad therapeutic range of this agent. Conclusion: Taken together, BSc2118 shows significant antimyeloma activity and may be considered as a promising agent in cancer drug development.

Receptor activator of nuclear factor-kappaB ligand (RANKL) plays a key role in osteoclast activation in myeloma bone disease. The increased expression of RANKL in the bone marrow microenvironment was demonstrated in several studies, but there are only rare data on circulating RANKL levels in patients with multiple myeloma (MM).In the current study, we investigated the clinical significance of serum RANKL levels, using an enzyme-linked immunosorbent assay test that detects both free and osteoprotegerin (OPG)-bound RANKL (total-RANKL, tRANKL) in patients with newly diagnosed MM (n = 93) and monoclonal gammopathy of undetermined significance (MGUS; n = 20) compared with healthy controls (n = 20).Circulating serum tRANKL was significantly elevated in patients with MM compared with controls (P.001) or MGUS (P.001). Furthermore, tRANKL levels were higher in smoldering MM versus MGUS (P = .031) and in symptomatic versus smoldering MM (P.001). Serum tRANKL increased parallel to International Staging System stages I to III (P = .004) and correlated with the presence of lytic bone lesions (P.001). Total-RANKL was a prognostic factor for overall survival in symptomatic MM (P = .043). A significantly longer progression-free survival was observed in patients with a50% decrease in tRANKL levels after 3 months of combined chemotherapy and bisphosphonate treatment.Our study demonstrates for the first time that serum tRANKL reflects advanced disease, lytic bone destruction, and poor prognosis in MM.

The 26S proteasome is a multicatalytic intracellular protease expressed in eukaryotic cells. It is responsible for selective degradation of intracellular proteins that are responsible for cell proliferation, growth, regulation of apoptosis and transcription of genes involved in execution of key cellular functions. Thus proteasome inhibition is a potential treatment option for cancer and diseases due to aberrant inflammation condition. Treatment with proteasome inhibitors results in stabilization and accumulation proteasome substrates, a phenomenon that may result in confounding signals in cells, cell cycle arrest and activation of apoptotic programs. The inhibition of the transcriptional factor nuclear factor kappaB (NF-kappaB) activation was found as one of crucial mechanisms in induction of apoptosis, overcoming resistance mechanisms and inhibition of immune response and inflammation mechanisms. Bortezomib (PS-341) and PS-519 are the first proteasome inhibitors that have entered clinical trials. In multiple myeloma, both the FDA (United States Food and Drug Administration) and EMEA (European Medicine Evaluation Agency) granted an approval for the use of bortezomib (Velcade) for the treatment of relapsed multiple myeloma. At present, several phase II and phase III trials in hematological malignancies and solid tumors are ongoing. PS-519 that focuses on inflammation, reperfusion injury and ischemia is currently under evaluation for the indication of acute stroke.

Multiple myeloma (MM) was the first haematological malignancy in which a prognostic relevance of bone marrow microvessel density (MVD) was shown. Myeloma-induced angiogenesis involves either the direct production of angiogenic molecules by myeloma cells or their induction in bone marrow stromal cells or endothelial cells (EC). Recent data demonstrate an increased angiogenic potential and a paracrine stimulatory effect of bone marrow EC on plasma cells (PC) in MM. Soluble angiogenic factors are elevated in bone marrow (BM) and in peripheral blood samples from myeloma patients. Furthermore, correlation with disease stage and prognosis was shown for serum levels of the angiogenic factors basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF). In this review we summarize recent data which give strong evidence for an increased angiogenic activity in bone marrow microenvironment and support the hypothesis that angiogenesis is not only an epiphenomenon of tumour growth but may also promote PC growth in MM.

Multiple myeloma (MM) is a malignancy with excessive production of monoclonal proteins. At disease presentation 30% of MM patients have significant renal impairment which may progress to renal failure requiring dialysis. Besides chemotherapy extracorporeal elimination procedures such as plasma exchange have been applied as adjuvant strategies to eliminate free light chains from circulating blood, however the efficacy was poor with older techniques. We report about a highly efficient method to eliminate serum free light chain (sFLC) using a newly designed protein leaking membrane in patients suffering from sFLC induced acute renal failure. The protein leaking membrane (HCO 1100) is characterized by increased pore size facilitating elimination of middle molecules such as sFLC kappa (22.5 kD). The HCO 1100 membrane was applied in a hemodialysis and hemodiafiltration mode and compared to standard procedures (high flux hemodialysis, hemodiafiltration and plasma exchange). Hemodiafiltration with the protein leaking membrane HCO 1100 was superior to all other extracorporeal replacement strategies in eliminating sFLC-kappa from circulating blood. A median blood reduction rate of 40.8% (range 13.9% - 66.4%) was achieved during hemodiafiltration. The corresponding peak clearance rate was 25 ml/min. Importantly, the poorest elimination rate was achieved by plasma exchange followed by standard high flux hemodialysis. Extracorporeal elimination strategies with the protein leaking membrane HCO 1100 may be a promising adjuvant treatment strategy for patients with sFLC nephropathy requiring dialysis. Hemodiafiltration and to lesser extend also hemodialysis with the HCO 1100 hemofilter are able to eliminate substantial amounts of sFLC kappa in MM patients.

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and novel treatment strategies are urgently needed. Proteasome inhibitors, e.g. bortezomib, act by targeting the catalytic 20S core of the proteasome and induce apoptosis in tumor cells. Among other mechanisms, they lead to cytoplasmic accumulation of the IκBa protein, resulting in a reduced NF-κB activity. Histone deacetylase inhibitors (HDAI), e.g. SAHA, promote histone acetylation, chromatin uncoiling, and transcription of a variety of genes. Previous studies have indicated that HDAIs also interfere with NF-κB signaling. Since NF-κB is constitutively activated MCL cells and plays a major role in a variety of cellular processes, we hypothesized synergist effects of bortezomib and HDAI in MCL cells. Human mantle cell lymphoma cell lines (JeKo-1 and Granta-519) were exposed to bortezomib and/ or SAHA for 4 to 48 hours. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. The effect of the combination of both agents was analyzed using the median effect method of Chou and Talalay. Reactive oxygen species (ROS) were quantified by the fluorophore H2DCFDA. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine (LNAC). In addition, activated caspases, proteasome- and NF-κB activity were quantified. After 48 hours of incubation, IC50 of SAHA and bortezomib were noted at 0.8μM and 7.7nm in JeKo-1 cells and at 3.9μM and 5.7nm in Granta-519 cells, respectively. Combined incubation resulted in synergistic cytotoxic effects, as indicated by CI values

The ubiquitin-proteasome pathway was recently identified as a promising new therapeutic target in cancer treatment. An increased proteasome activity was described in certain cancer types, especially in multiple myeloma (MM) and also in mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has been approved for the treatment of refractory MM and has also shown reproducible activity in MCL. Recently we described a novel tripeptide compound, BSc2118, with inhibitory activity against all three proteolytic activities (post-glutamyl peptide hydrolase-like, trypsin-like and chymotrypsin-like activities) of the 20S proteasome (Cancer Res2006;66:7598–605). We investigated the in vitro effects of BSc2118 in MM and MCL cell lines by MTT cell viability and AnnexinV apoptosis-assays. Furthermore, the intracellular chymotrypsin-like proteasome activity and NF-κB activity were detected in MM and MCL cells by detection of proteasome-degraded peptides or NF-κB p65 subunit. In MM cell lines OPM-2 and U266 we could show a significant time and dose-dependent reduction of cell viability by BSc2118 with an IC50 at 48hrs of 52nM and 65nM, respectively, whereas the MM cell line RPMI-S was less sensitive with an IC50 of 287nM. Using AnnexinV assay, a dose-dependent induction of apoptosis by BSc2118 was shown after 48hrs incubation. Comparably, in MCL we also found a time and dose-dependent reduction of cell viability in the cell lines HBL-2, JeKo-1 and Granta-519 with an IC50 of 82nM, 130nM and 262nM, respectively. Furthermore, BSc2118 induced apoptosis in all three MCL cell lines. Additionally, we detected a significant dose-dependent inhibition of intracellular chymotrypsin-like proteasome activity in MM and MCL cells, and a dose-dependent inhibition of TNFα-induced NF-κB activation in the MM cell lines OPM-2 and RPMI-S. This is the first report of anti-tumor effects of the novel proteasome inhibitor BSc2118 in MM and MCL cells. The compound effectively reduces the cell survival and shows a high pro-apoptotic activity in the MM cell lines OPM-2 and U266 and a significant activity in MCL cell lines HBL-2 and JeKo-1. Mechanisms of action are the inhibition of proteasome and NF-κB activity. Since previous clinical trials have shown an activity of the proteasome inhibitor bortezomib in MM and MCL, and adverse effects of other proteasome inhibitors may differ, our preclinical data support the idea to consider BSc2118 as a promising new agent in anti-tumor drug development.

Myeloma bone disease is caused by an enhanced osteoclast activation and impaired osteoblast function. Until now, there is no specific treatment to restore osteoblast activity, and anti-myeloma therapies that lead to a disease remission are usually not associated with an increase of osteoblast markers. Recently, preclinical data suggested that proteasome inhibitors may enhance osteoblast function. Bortezomib (Velcade) represents the first substance from this group which is clinically used in relapsed multiple myeloma. To evaluate whether there is clinical evidence for an osteoblast stimulation under bortezomib treatment, we analyzed serum levels of two specific osteoblast markers, i.e. bone-specific alkaline phosphatase (BAP) and osteocalcin, in 25 multiple myeloma patients treated with bortezomib alone or in combination with dexamethasone. 56 percent of patients achieved a complete or partial remission. In the whole group of patients, mean serum levels of osteocalcin significantly increased from 6.3 μg/l before treatment to 10.8 μg/l after three months of therapy (P=0.024). In parallel, mean levels of BAP increased from 19.7 U/l to 30.2 U/l (P

Enhanced osteolytic bone resorption is a hallmark of multiple myeloma. Several studies demonstrated an elevation of RANKL levels in the bone marrow microenvironment in multiple myeloma, but serum levels of soluble RANKL (sRANKL) revealed controversial results. One study reported elevated sRANKL in serum in myeloma patients, but using the same test, the levels in the majority of the patients were below the detection limit in the hand of other groups. Thus, a novel test was developed which measures both soluble and OPG-bound RANKL (total-RANKL, tRANKL). The objective of the present study was to investigate the clinical significance of circulating levels of tRANKL in monoclonal gammopathies of undetermined significance (MGUS) or multiple myeloma (MM). Serum levels of tRANKL were analyzed by ELISA in 128 individuals: 20 healthy donors, 20 with MGUS and 88 newly diagnosed patients with multiple myeloma. Multiple myeloma patients had significantly (P