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Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells.

ABSTRACT: Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade(®), also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines--RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.

SUBMITTER: Roy SS

PROVIDER: S-EPMC4036123 | biostudies-other | 2014 Jun

REPOSITORIES: biostudies-other

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Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells.

Roy Sudipa S SS Kirma Nameer B NB Santhamma Bindu B Tekmal Rajeshwar R RR Agyin Joseph K JK

Cancer chemotherapy and pharmacology 20140412 6

Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade(®), also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refr ...[more]

PMID: 24728817

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Project description:BackgroundMultiple myeloma (MM) remains an incurable malignancy, despite the advent of therapies such as proteosome inhibitors (PIs) that disrupt protein homeostasis and induce ER stress. We have pursued inhibition of geranylgeranyl diphosphate synthase (GGDPS) as a novel mechanism by which to target protein homeostasis in MM cells. GGDPS inhibitors (GGSI) disrupt Rab geranylgeranylation, which in turn results in perturbation of Rab-mediated protein trafficking, leading to accumulation of intracellular monoclonal protein, induction of ER stress and apoptosis. Our lead GGSI, RAM2061, has demonstrated favorable pharmacokinetic properties and in vivo efficacy. Here we sought to evaluate if combination therapy with GGSI and PI would result in enhanced disruption of the unfolded protein response (UPR) and increase anti-MM efficacy.MethodsMTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with bortezomib in human MM cells. The effects of RAM2061 and/or PI (bortezomib or carfilzomib) on markers of UPR and apoptosis were evaluated by a combination of immunoblot (ATF4, IRE1, p-eIF2a, cleaved caspases and PARP), RT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and flow cytometry (Annexin-V). Induction of immunogenic cell death (ICD) was assessed by immunoblot (HMGB1 release) and flow cytometry (calreticulin translocation). Cell assays were performed using both concurrent and sequential incubation with PIs. To evaluate the in vivo activity of GGSI/PI, a flank xenograft using MM.1S cells was performed.ResultsIsobologram analysis of cytotoxicity data revealed that sequential treatment of bortezomib with RAM2061 has a synergistic effect in MM cells, while concurrent treatment was primarily additive or mildly antagonistic. The effect of PIs on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was dependent on timing of the PI exposure. Combination treatment with RAM2061 and bortezomib enhanced activation of ICD pathway markers. Lastly, combination treatment slowed MM tumor growth and lengthened survival in a MM xenograft model without evidence of off-target toxicity.ConclusionWe demonstrate that GGSI/PI treatment can potentiate activation of the UPR and apoptotic pathway, as well as induce upregulation of markers associated with the ICD pathway. Collectively, these findings lay the groundwork for future clinical studies evaluating combination GGSI and PI therapy in patients with MM.

Dataset Information

Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells.

Publications

Effects of a novel proteasome inhibitor BU-32 on multiple myeloma cells.

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