Ivana von Metzler

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in cutaneous T cell lymphoma

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors are novel targeted therapies being evaluated in clinical trials for cutaneous T‐cell lymphoma (CTCL). However, data in regard to tumor biology are limited with these agents. In the present study we analyzed the effects of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the proteasome inhibitor bortezomib on human CTCL cells. Four CTCL cell lines (SeAx, Hut‐78, MyLa, and HH) were exposed to bortezomib and/ or SAHA at different concentrations. Cell viability was quantified using the MTT assay. In addition, apoptosis and generation of reactive oxygen species were analyzed. Both agents potently inhibited cell viability and induced apoptosis. After 48 h of incubation, IC50 of bortezomib was noted at 8.3 nm, 7.9 nm, 6.3 nm, and 22.5 nm in SeAx, Hut‐78, HH, and MyLa cells, respectively. For SAHA, the IC50 values were at 0.6 μm in SeAx cells, 0.75 μm in Hut‐78 cells, 0.9 μm in HH cells, and 4.4 μm in MyLa cells. Importantly, combined treatment resulted in synergistic cytotoxic effects, as indicated by Combination indices values <1 using the median effect method of Chou and Talalay. We furthermore found that combined treatment with both agents lead to a decreased proteasome activity, an upregulation of the cell regulators p21 and p27 and increased expression of phosphorylated p38. In addition, we showed that SAHA reduced the vascular endothelial growth factor production of CTCL cells. Our results demonstrate that bortezomib and SAHA synergistically induce apoptosis in CTCL cells and thus provide a rationale for clinical trials of combined proteasome and histone deacetylase inhibition in the treatment of CTCL.

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.

Synergistic action of the novel HSP90 inhibitor NVP-AUY922 with histone deacetylase inhibitors, melphalan, or doxorubicin in multiple myeloma

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP‐AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM‐2, RPMI‐8226, U‐266, LP‐1, MM1.S, and primary myeloma cells were exposed to NVP‐AUY922 and one of the combination partners histone deacetylase inhibitor NVP‐LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP‐AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP‐AUY922 with SAHA, NVP‐LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI‐8226 cell line and relative resistance of some primary myeloma cells against NVP‐AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with melphalan, doxorubicin, NVP‐LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.

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.

The proteasome inhibitor bortezomib stimulates osteoblastic differentiation of human osteoblast precursors via upregulation of vitamin D receptor signalling

Interactions of myeloma cells with the bone marrow microenvironment lead to enhanced osteoclast recruitment and impaired osteoblast activity. Recent evidence revealed that the proteasome inhibitor bortezomib stimulates osteoblast differentiation, but the mechanisms are not fully elucidated. We hypothesised that bortezomib could influence osteoblastic differentiation via alteration of vitamin D signalling by blocking the proteasomal degradation of the vitamin D receptor (VDR). This is of clinical importance, as a high rate of vitamin D deficiency was reported in patients with myeloma. We performed cocultures of primary human mesenchymal stem cells (hMSCs) and human osteoblasts (hOBs) with myeloma cells, which resulted in an inhibition of the vitamin D‐dependent differentiation of osteoblast precursors. Treatment with bortezomib led to a moderate increase in osteoblastic differentiation markers in hMSCs and hOBs. Importantly, this effect could be strikingly increased when vitamin D was added. Bortezomib led to enhanced nuclear VDR protein levels in hMSCs. Primary hMSCs transfected with a VDR luciferase reporter construct showed a strong increase in VDR signalling with bortezomib. In summary, stimulation of VDR signalling is a mechanism for the bortezomib‐induced stimulation of osteoblastic differentiation. The data suggest that supplementation of vitamin D in patients with myeloma treated with bortezomib is crucial for optimal bone formation.

Decrease in CD4+ T-Cell Counts in Patients With Multiple Myeloma Treated With Bortezomib

BACKGROUND 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. PATIENTS AND METHODS We investigated the influence of bortezomib on T-cell subpopulations in 53 patients with myeloma before initiation of bortezomib and during therapy. RESULTS 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 to < 400/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+ lymphocytes < 400/microL. CONCLUSION 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.

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).