Jan Sterz

Bortezomib inhibits human osteoclastogenesis.

In multiple myeloma, the overexpression of receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) leads to the induction of NF-κB 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-κB inhibition using bortezomib (PS-341) and I-κB 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-κB 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 potential of proteasome inhibitors in cancer therapy

Background: The ubiquitin-proteasome system has become a promising novel molecular target in cancer due to its critical role in cellular protein degradation, its interaction with cell cycle and apoptosis regulation and its unique mechanism of action. Objective: This review focuses both on preclinical results and on data from clinical trials with proteasome inhibitors in cancer. Methods: Results in hematological malignancies and solid tumors were included, and important data presented in abstract form were considered in this review. Results/conclusion: Bortezomib as first-in-class proteasome inhibitor has proven to be highly effective in some hematological malignancies, overcomes conventional chemoresistance, directly induces cell cycle arrest and apoptosis, and also targets the tumor microenvironment. It has been granted approval by the FDA for relapsed multiple myeloma, and recently for relapsed mantle cell lymphoma. Combination chemotherapy regimens have been developed providing high remission rates and remission quality in frontline treatment or in the relapsed setting in multiple myeloma. The combination of proteasome inhibition with novel targeted therapies is an emerging field in oncology. Moreover, novel proteasome inhibitors, such as NPI-0052 and carfilzomib, have been developed. This review summarizes our knowledge of the ubiquitin-proteasome system and recent data from cancer clinical trials.

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in mantle cell lymphoma: Synergism of HDAC and bortezomib in MCL

Objectives:  Mantle cell lymphoma (MCL) is an incurable B cell lymphoma, and novel treatment strategies are urgently needed. We evaluated the effects of combined treatment with the proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) on MCL. Bortezomib acts by targeting the proteasome, and – among other mechanisms – results in a reduced nuclear factor‐kappa B (NF‐κB) activity. HDACi promote histone acetylation, and also interfere with NF‐κB signaling.

Histone deacetylase inhibitors reduce VEGF production and induce growth suppression and apoptosis in human mantle cell lymphoma.

Abstract:  Objectives: Mantle cell lymphoma (MCL) is an incurable disease with an aggressive course and novel treatment strategies are urgently needed. The purpose of this study was to evaluate the effects of histone deacetylase (HDAC) inhibitors, a new group of antiproliferative agents, on human MCL cells. Methods: Three MCL cell lines (JeKo‐1, Hbl‐2 and Granta‐519) were exposed to different concentrations of the HDAC inhibitors sodium butyrate (NaB) and suberoylanilide hydroxamic acid (SAHA) for 8–72 h. Their effects on cell viability, apoptosis induction and cell cycle proliferation were studied. Moreover, the influence of SAHA on the expression of cyclin D1, the cell cycle regulators p21 and p27 and the production of vascular endothelial growth factor (VEGF) were analyzed. Results: The HDAC inhibitors induced accumulation of acetylated histones in MCL cells. MTT assays and Annexin‐V staining showed that they potently inhibited viability in a dose‐dependent manner and induced apoptosis in all cell lines tested. Cell cycle analysis indicated that their exposure to SAHA or NaB decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases. Incubation with the two HDAC inhibitors resulted in downregulation of cyclin D1. SAHA lead to an upregulation of p21 in all cell lines and an upregulation of p27 in JeKo‐1 and Granta‐519 cells, while expression of p27 in Hbl‐2 was not altered. In addition, SAHA inhibited the production of the angiogenic cytokine VEGF. Treatment with NaB increased the expression of p21 in JeKo‐1 and Hbl‐2 cells, while in Granta 519 cells no effect was noted. The expression of p27 remained constant in all three cell lines after exposure to NaB. Conclusion: Based on these findings, we provide evidence that HDAC inhibitors have antiproliferative effects in MCL and may represent a promising therapeutic approach.

Proteasome as an Emerging Therapeutic Target in Cancer

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.

Incorporation of the bone marker carboxy-terminal telopeptide of type-1 collagen improves prognostic information of the International Staging System in newly diagnosed symptomatic multiple myeloma

Several prognostic markers, including parameters of tumor burden and cytogenetics, were adopted to identify high-risk patients in multiple myeloma (MM). Recently, the International Staging System (ISS), including β2-microglobulin (β2M) and albumin, was introduced for patients with symptomatic MM. As bone disease is a hallmark of MM, we investigated the prognostic impact of the bone resorption marker carboxy-terminal telopeptide of type-1 collagen (ICTP) in combination with ISS, β2M, albumin, deletion of chromosome 13 and high-dose therapy (HDT) in 100 patients with newly diagnosed symptomatic MM. β2M alone, albumin alone, ISS, HDT, del(13q14) and ICTP were significant prognostic factors for overall survival (OS). In a multivariate analysis, ICTP was the most powerful prognostic factor (log-rank P<0.001, hazard ratio: ninefold increase). ICTP clearly separated two subgroups with a good and a worse prognosis within each of the three ISS stages (ISS I: P=0.027, ISS II: P=0.022, ISS III: P=0.013). Incorporation of ICTP in a combined ICTP-ISS score significantly (P<0.001) separated four risk groups with a 5-year OS rate of 95, 65, 46 and 32%, respectively. These data demonstrate for the first time that the inclusion of the collagen-I degradation product ICTP, as a biomarker of bone resorption, adds to the prognostic value of ISS.

Osteoblasts promote migration and invasion of myeloma cells through upregulation of matrix metalloproteinases, urokinase plasminogen activator, hepatocyte growth factor and activation of p38 MAPK

Formation of osteolytic lesions is a key pathophysiological feature in multiple myeloma and results from the interaction of myeloma cells with the bone marrow microenvironment. Matrix metalloproteinases (MMPs) and plasmin may be involved in bone destruction, but their precise roles have not been clarified. Furthermore, the impact of osteoblast‐related alterations on myeloma bone disease is not well understood. We addressed this complex phenomenon by applying a coculture system between myeloma cells and osteoblasts. Osteoblasts induced expression of MMP‐1 and upregulated the expression of MMP‐2, urokinase plasminogen activator (uPA) and hepatocyte growth factor (HGF) in myeloma cells. In turn, interaction with myeloma cells led to abundant MMP‐1 expression in osteoblasts. Because MMP‐1 degrades collagen, its upregulation might represent an essential mechanism contributing to bone destruction. Cocultures using primary myeloma cells confirmed the results obtained with cell lines. The mechanisms responsible for MMP‐1 upregulation are mediated by both membrane‐bound and soluble factors, and involve the p38 mitogen‐activated protein kinase (MAPK) pathway. The interaction with osteoblasts enhances the capability of myeloma cells to transmigrate and invade through Matrigel or type I collagen. Using appropriate inhibitors, we provide evidence that these processes involve MMPs, uPA, HGF and activation of p38 MAPK.

Serum levels of total-RANKL in multiple myeloma.

BACKGROUND 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). PATIENTS AND METHODS 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). RESULTS 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 a > 50% decrease in tRANKL levels after 3 months of combined chemotherapy and bisphosphonate treatment. CONCLUSION Our study demonstrates for the first time that serum tRANKL reflects advanced disease, lytic bone destruction, and poor prognosis in MM.

The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases.

Heat shock protein 90 (HSP90) binds and stabilizes numerous proteins and kinases essential for myeloma cell survival and proliferation. We and others have recently demonstrated that inhibition of HSP90 by small molecular mass inhibitors induces cell death in multiple myeloma (MM). However, some of the HSP90 inhibitors involved in early clinical trials have shown limited antitumor activity and unfavorable toxicity profiles. Here, we analyzed the effects of the novel, orally bioavailable HSP90 inhibitor NVP‐HSP990 on MM cell proliferation and survival. The inhibitor led to a significant reduction in myeloma cell viability and induced G2 cell cycle arrest, degradation of caspase‐8 and caspase‐3, and induction of apoptosis. Inhibition of the HSP90 ATPase activity was accompanied by the degradation of MM phospho‐Akt and phospho‐ERK1/2 and upregulation of Hsp70. Exposure of MM cells to a combination of NVP‐HSP990 and either melphalan or histone deacetylase (HDAC) inhibitors caused synergistic inhibition of viability, increased induction of apoptosis, and was able to overcome the primary resistance of the cell line RPMI‐8226 to HSP90 inhibition. Combined incubation with melphalan and NVP‐HSP990 led to synergistically increased cleavage of caspase‐2, caspase‐9, and caspase‐3. These data demonstrate promising activity for NVP‐HSP990 as single agent or combination treatment in MM and provide a rationale for clinical trials.

BSc2118 is a novel proteasome inhibitor with activity against multiple myeloma.

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).