Neurodegeneration Induced by Bortezomib Exposure in Vitro Occurs Via Proteasome Independent Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2859-2859
Author(s):  
Shirin Arastu-Kapur ◽  
Andrew J. Ball ◽  
Janet L. Anderl ◽  
Mark K Bennett ◽  
Christopher J Kirk
Keyword(s):  
Peripheral Nerve ◽  
Proteasome Inhibitor ◽  
Serine Proteases ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nerve Degeneration ◽  
Differentiated Cells ◽  
Favorable Safety ◽  
Neurite Degeneration

Abstract Abstract 2859 Poster Board II-835 BACKGROUND: The dipeptide boronate proteasome inhibitor bortezomib (BTZ; Velcade®) is approved for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Bortezomib-induced peripheral neuropathy (BIPN, Blood (2008)112:1593-1599) is seen in ∼30% of BTZ-treated patients and can result in dose reductions and discontinuations that may result in suboptimal levels of proteasome inhibition. Carfilzomib (CFZ), a tetrapeptide epoxyketone, is a selective and irreversible proteasome inhibitor that is structurally and mechanistically distinct from bortezomib. Single agent treatment with CFZ has demonstrated strong activity in relapsed and refractory myeloma and a favorable safety profile in Phase 2 trials (ASH2008:864 & 865). Importantly treatment-emergent PN was seen at low levels and did not result in dose modifications or discontinuations. The disparate safety data for these proteasome inhibitors suggest that non-proteasomal mechanisms may underlie BIPN. Using activity-based probes in peripheral blood mononuclear cell (PBMC) lysates, we previously demonstrated inhibition of non-proteasomal proteases by BTZ and other proteasome inhibitors with a boronate pharmacophore (EHA2009:0939). However, the involvement of the proteasome in the peripheral nerve degeneration and BIPN in BTZ-treated myeloma patients remains to be established. AIMS: To establish an in vitro model of peripheral nerve degeneration and to determine the effects of proteasome inhibition by BTZ and CFZ on neurite outgrowth and cell survival. METHODS: SH-SY5Y neuroblastoma cells were differentiated by long term culture in retinoic acid and brain derived nerve growth factor to induce neurite outgrowth. The effects of proteasome inhibitors were measured by high content image analysis of fluorescent images for cell survival (Hoechst nuclear counterstain) and neurite degeneration (FITC-mouse anti-beta-III-tubulin). Phase contrast images were also collected to observe morphological effects and gross cell death. Cell viability and proteasome inhibition was measured in undifferentiated and differentiated cells. The MEROPS (peptidase) database was mined for candidate serine proteases with a P1 selectivity of Leu/Phe/Tyr to identify candidate off-targets CFZ and BTZ and candidate proteases were validated by standard biochemical and cell biology techniques. RESULTS: In differentiated SH-SY5Y cells, the average neurite length decreased by 33% following 24 hr exposure to 10nM BTZ but was unaffected by the same concentration of CFZ. Proteasome inhibition as determined by a fluorescent substrate for the chymotrypsin-like activity was equivalent (∼70%) after a 24 hr exposure for both compounds in differentiated cells, suggesting that neurodegeneration involves non-proteasomal pathways. With 72 hrs continuous exposure, BTZ was 10-fold more potent than CFZ at inducing neurodegeneration. Furthermore, in both undifferentiated and differentiated SH-SY5Y cells, BTZ was 5-fold more cytotoxic than CFZ. Database mining for serine proteases with a selectivity for Leu/Phe/Tyr at P1 was used to identify other potential BTZ targets that might underlie neurotoxicity. One candidate is HtrA2 (also called Omi), an inducible mitochondrial serine protease whose activity protects neurons from stress induced apoptosis (Hum Mol Genet (2005) 14(5):2099-2111). HtrA2 levels increased 2-fold in SH-SY5Y cells treated with either BTZ or CFZ for 6 hrs at 40 nM. Using a gel based assay and purified enzyme preparations, BTZ inhibited HtrA2 activity with an IC50 ∼ 4 nM, equivalent to its activity against the proteasome. In contrast, Carfilzomib did not inhibit HtrA2 at the highest concentration tested (10 mM). CONCLUSIONS: These data demonstrate that BTZ induces neuronal cell death and neurite degeneration in vitro by proteasome-independent mechanisms. We propose that combined inhibition of the proteasome and HtrA2 by BTZ may underlie peripheral nerve toxicities in vitro and may be involved in BIPN in myeloma patients. In this model, CFZ, which mediates equivalent proteasome inhibition to BTZ in neurons, does not induce neurodegeneration due to inactivity against HtrA2. Future profiling of non-proteasomal targets of BTZ, including HtrA2 activity, in patient samples is merited. These results suggest that the favorable safety profile of CFZ in myeloma patients may be a result of its high selectivity for proteasomal proteases. Disclosures: Arastu-Kapur: Proteolix, Inc: Employment. Ball:Millipore Corp: Employment. Anderl:Millipore Corp: Employment. Bennett:Proteolix: Employment. Kirk:Proteolix, Inc: Employment.

Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant Chronic Lymphocytic Leukemic Lymphocytes

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4220-4229 ◽  
Author(s):  
Joya Chandra ◽  
Irina Niemer ◽  
Joyce Gilbreath ◽  
Kay-Oliver Kliche ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Proteasome Inhibitor ◽  
Transmembrane Potential ◽  
Proteasome Inhibitors ◽  
Nucleoside Analogs ◽  
Proteasome Inhibition ◽  
Conventional Chemotherapy ◽  
Partial Sequencing ◽  
Biochemical Mechanisms

Abstract Our previous work showed that the nuclear scaffold (NS) protease is required for apoptosis of both thymocytes and chronic lymphocytic leukemic (CLL) lymphocytes. Because partial sequencing of one of the subunits of the NS protease revealed homology to the proteasome, we tested the effects of classical proteasome inhibitors on apoptosis in CLL cells. Here we report that proteasome inhibition caused high levels of DNA fragmentation in all patients analyzed, including those resistant to glucocorticoids or nucleoside analogs, in vitro. Proteasome inhibitor-induced DNA fragmentation was associated with activation of caspase/ICE family cysteine protease(s) and was blocked by the caspase antagonist, zVADfmk. Analysis of the biochemical mechanisms involved showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the release of cytochrome c and a drop in mitochondrial transmembrane potential (▵Ψ). These changes were associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in other systems. Together, our results suggest that drugs that target the proteasome might be capable of bypassing resistance to conventional chemotherapy in CLL.

In Vitro and In Vivo Proteasome Activity Profiles of Bortezomib and a Novel Proteasome Inhibitor NPI-0052.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3363-3363 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ta-Hsiang Chao ◽  
Laurence Catley ◽  
Benjamin Nicholson ◽  
Mugdha Velanker ◽  
...  
Keyword(s):  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Catalytic Activities ◽  
Proteasome Subunits ◽  
20 Nm ◽  
Whole Blood Cells

Abstract Proteasome inhibition is an effective anti-cancer therapy. Proteasome function is mediated by three catalytic activities: chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L). Kinetics of inhibition of catalytic activities may define the pharmacologic utility of proteasome inhibitors. Here we utilized two structurally distinct proteasome inhibitors Bortezomib, a dipeptide boronic acid; and a non-peptide proteasome inhibitor NPI-0052 to determine their effect on proteasome activities in vitro and in animal model. Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates shows that NPI-0052 and Bortezomib inhibit all three proteasome activities, albeit at different concentrations: NPI-0052 inhibits CT-L and T-L activities at lower concentrations than Bortezomib (NPI-0052: EC50 = 3.5 ± 0.3 nM versus Bortezomib: 7.9 ± 0.5 nM for CT-L activity; and NPI-0052: EC50 = 28 ± 2 nM versus Bortezomib: EC50 = 590 ± 67 nM for T-L activity); in contrast, higher concentrations of NPI-0052 than Bortezomib are required to inhibit C-L activity (NPI-0052 EC50 = 430 ± 34 nM versus Bortezomib: EC50 = 53 ± 10 nM for C-L activity). We next compared the effects of NPI-0052 and Bortezomib on all three proteasome activities in vivo. Mice were treated with a single MTD dose of NPI-0052 (0.15 mg/kg i.v) or Bortezomib (1 mg/kg i.v); blood samples were collected at 90 mins, 24h, 48h, 72h, or 168h; and whole blood cells were then analyzed for proteasome activity. NPI-0052 completely inhibited CT-L activity by 90 mins, which was recoverable by 168h; whereas Bortezomib-inhibited CT-L activity is recoverable at 24h. T-L activity is significantly inhibited by NPI-0052 at 90 mins, 24h, 48h, and 72h; and is recoverable by 168h; in contrast, Bortezomib enhances T-L activity. Finally, NPI-0052 inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and this activity recovered at 168h, whereas Bortezomib significantly inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and is similarly recoverable at 168h. We next utilized a novel methodology to measure proteasome activity by immunoblotting using dansylAhx3L3VS as a probe (Berkers et al., Nature Methods, 2005), which also allow for determining subunit specificity of a proteasome inhibitor. Multiple myeloma (MM) cells were cultured in the presence or absence of various concentrations of either NPI-0052 (2 nM; 7 nM: IC50; or 20 nM) or Bortezomib (2 nM; 5 nM: IC50; or 20 nM). Competition experiments between either NPI-0052 or Bortezomib and dansylAhx3L3VS revealed that NPI-0052 (7 nM) markedly inhibits the CT-L activity represented by beta-5 subunit of the proteasome and decreased the dansylAhx3L3VS-labeling of the beta-1 (C-L activity) and -2 (T-L activity) subunits. Slightly higher concentrations of Bortezomib are necessary to markedly inhibit beta-5 and -1 subunits, whereas beta-2 subunits are not inhibited. Importantly, both agents trigger apoptosis in MM cells; however, NPI-0052 is remarkably less toxic to normal lymphocytes than Bortezomib. Our data show that NPI-0052, like Bortezomib, targets the proteasome, but triggers a proteasome activity profile distinct from Bortezomib. The mechanistic insights gained from these studies will allow for improved drug design based on targeting specific proteasome subunits.

Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant Chronic Lymphocytic Leukemic Lymphocytes

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4220-4229 ◽  
Author(s):  
Joya Chandra ◽  
Irina Niemer ◽  
Joyce Gilbreath ◽  
Kay-Oliver Kliche ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Proteasome Inhibitor ◽  
Transmembrane Potential ◽  
Proteasome Inhibitors ◽  
Nucleoside Analogs ◽  
Proteasome Inhibition ◽  
Conventional Chemotherapy ◽  
Partial Sequencing ◽  
Biochemical Mechanisms

Our previous work showed that the nuclear scaffold (NS) protease is required for apoptosis of both thymocytes and chronic lymphocytic leukemic (CLL) lymphocytes. Because partial sequencing of one of the subunits of the NS protease revealed homology to the proteasome, we tested the effects of classical proteasome inhibitors on apoptosis in CLL cells. Here we report that proteasome inhibition caused high levels of DNA fragmentation in all patients analyzed, including those resistant to glucocorticoids or nucleoside analogs, in vitro. Proteasome inhibitor-induced DNA fragmentation was associated with activation of caspase/ICE family cysteine protease(s) and was blocked by the caspase antagonist, zVADfmk. Analysis of the biochemical mechanisms involved showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the release of cytochrome c and a drop in mitochondrial transmembrane potential (▵Ψ). These changes were associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in other systems. Together, our results suggest that drugs that target the proteasome might be capable of bypassing resistance to conventional chemotherapy in CLL.

scholarly journals Isoginkgetin, a Natural Biflavonoid Proteasome Inhibitor, Sensitizes Cancer Cells to Apoptosis via Disruption of Lysosomal Homeostasis and Impaired Protein Clearance

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Jessica Tsalikis ◽  
Mena Abdel-Nour ◽  
Armin Farahvash ◽  
Matthew T. Sorbara ◽  
Stephanie Poon ◽  
...  
Keyword(s):  
Cell Death ◽  
Proteasome Inhibitor ◽  
Adaptor Protein ◽  
Proteasome Inhibition ◽  
20S Proteasome ◽  
Protein Homeostasis ◽  
Cancer Cell Death ◽  
Protein Clearance ◽  
Lysosome Biogenesis

ABSTRACTProtein degradation pathways are critical for maintaining proper protein dynamics within the cell, and considerable efforts have been made toward the development of therapeutics targeting these catabolic processes. We report here that isoginkgetin, a naturally derived biflavonoid, sensitized cells undergoing nutrient starvation to apoptosis, induced lysosomal stress, and activated the lysosome biogenesis geneTFEB. Isoginkgetin treatment led to the accumulation of aggregates of polyubiquitinated proteins that colocalized strongly with the adaptor protein p62, the 20S proteasome, and the endoplasmic reticulum-associated degradation (ERAD) protein UFD1L. Isoginkgetin directly inhibited the chymotrypsin-like, trypsin-like, and caspase-like activities of the 20S proteasome and impaired NF-κB signaling, suggesting that the molecule may display its biological activity in part through proteasome inhibition. Importantly, isoginkgetin was effective at killing multiple myeloma (MM) cell linesin vitroand displayed a higher rate of cell death induction than the clinically approved proteasome inhibitor bortezomib. We propose that isoginkgetin disturbs protein homeostasis, leading to an excess of protein cargo that places a burden on the lysosomes/autophagic machinery, eventually leading to cancer cell death.

scholarly journals Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation

2018 ◽  
Vol 217 (5) ◽  
pp. 1757-1776 ◽  
Author(s):  
Zhe Sha ◽  
Helena M. Schnell ◽  
Kerstin Ruoff ◽  
Alfred Goldberg
Keyword(s):  
Proteasome Inhibitor ◽  
Neuroblastoma Cells ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nuclear Factor ◽  
Ubiquitinated Proteins ◽  
Rapid Induction ◽  
Independent Mechanism ◽  
Transcription Factor Nuclear Factor ◽  
Inhibitor Treatment

Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.

scholarly journals Proteasome inhibition as a therapeutic approach in atypical teratoid/rhabdoid tumors

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Andrew Morin ◽  
Caroline Soane ◽  
Angela Pierce ◽  
Bridget Sanford ◽  
Kenneth L Jones ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
High Grade Glioma ◽  
Proteasome Inhibition ◽  
Atypical Teratoid ◽  
Rhabdoid Tumors ◽  
Approved Drugs

Abstract Background Atypical teratoid/thabdoid tumor (AT/RT) remains a difficult-to-treat tumor with a 5-year overall survival rate of 15%–45%. Proteasome inhibition has recently been opened as an avenue for cancer treatment with the FDA approval of bortezomib (BTZ) in 2003 and carfilzomib (CFZ) in 2012. The aim of this study was to identify and characterize a pre-approved targeted therapy with potential for clinical trials in AT/RT. Methods We performed a drug screen using a panel of 134 FDA-approved drugs in 3 AT/RT cell lines. Follow-on in vitro studies used 6 cell lines and patient-derived short-term cultures to characterize selected drug interactions with AT/RT. In vivo efficacy was evaluated using patient derived xenografts in an intracranial murine model. Results BTZ and CFZ are highly effective in vitro, producing some of the strongest growth-inhibition responses of the evaluated 134-drug panel. Marizomib (MRZ), a proteasome inhibitor known to pass the blood–brain barrier (BBB), also strongly inhibits AT/RT proteasomes and generates rapid cell death at clinically achievable doses in established cell lines and freshly patient-derived tumor lines. MRZ also significantly extends survival in an intracranial mouse model of AT/RT. Conclusions MRZ is a newer proteasome inhibitor that has been shown to cross the BBB and is already in phase II clinical trials for adult high-grade glioma (NCT NCT02330562 and NCT02903069). MRZ strongly inhibits AT/RT cell growth both in vitro and in vivo via a moderately well-characterized mechanism and has direct translational potential for patients with AT/RT.

scholarly journals Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

2013 ◽  
Vol 87 (23) ◽  
pp. 13035-13041 ◽  
Author(s):  
Angela M. Mitchell ◽  
R. Jude Samulski
Keyword(s):  
Cysteine Protease ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Protease Inhibition ◽  
Adeno Associated Virus

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.

Phase 1 Clinical Trial of the Novel Structure Proteasome Inhibitor NPI-0052.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2693-2693 ◽  
Author(s):  
Andrew Spencer ◽  
Michael Millward ◽  
Paul Mainwaring ◽  
Simon Harrison ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Whole Blood ◽  
Proteasome Inhibitor ◽  
Mononuclear Cells ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Pharmacokinetic Data ◽  
Employment Equity ◽  
Cognitive Changes ◽  
Equity Ownership

Abstract Abstract 2693 Poster Board II-669 Background: NPI-0052 is a proteasome inhibitor with a novel bicyclic structure (other proteasome inhibitors in clinical use are peptide based). Preclinical studies indicate rapid, broad and prolonged inhibition of all 3 catalytic sites of the proteasome, and subsequently unique proteasome inhibition, signal transduction, toxicology and efficacy profiles. Taken together these suggest the potential for improvements in therapeutic ratio and activity in hematologic and solid tumor malignancies. Materials and Methods: Patients with solid tumor, lymphoma, leukemia or myeloma diagnoses without standard treatment options have been treated with IV NPI-0052 on one of two arms (weekly or twice weekly) in this 3+3 design dose escalation study. This is followed by 10 patient Recommended Phase 2 dose Cohorts of patients with lymphomas, CLL and myeloma respectively. Proteasome inhibition (pharmacodynamics) and pharmacokinetics are also assayed in whole blood, and proteasome inhibition in peripheral blood mononuclear cells (PBMC). Results: 44 patients have been treated with NPI-0052 at doses ranging from 0.075 mg/m2 to 0.9 mg/m2. Common adverse events include fatigue, parosmia/dysgeusia, transient peri-infusion site pain, lymphopenia, headaches, dizziness / unsteady gait, closed-eye visuals, cognitive changes. Incidence and grade of these events correlate with dose, being quite tolerable at the MTD of 0.7 mg/m2 on the weekly dosing arm. An MTD has not yet been determined for the twice weekly dosing arm. Pharmacokinetic data has demonstrated a rapid elimination half-life (<20 minutes) and relatively large volume of distribution. Assessment of proteasome inhibition has demonstrated increasing inhibition of chymotrypsin-like activity of up to 88% Day 1 and 100% Day 15. Inhibition of caspase-like and trypsin-like activity of up to 52% and 71% respectively has also been seen. Inhibition remains between doses in whole blood (principally RBC), but recovers between doses in PBMC. Clinical benefit, including stable disease, regression or response, was reported in patients with mantle cell lymphoma, myeloma, Hodgkin's lymphoma, cutaneous marginal zone lymphoma, follicular lymphoma, sarcoma, prostate carcinoma and melanoma. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range, while producing a toxicity profile that is dissimilar to what is reported with other proteasome inhibitors (notably deficient in peripheral neuropathy, neutropenia and thrombocytopenia) in spite of producing equal or greater proteasome inhibition. These data indicate a broad range of potential uses, and led to additional studies in hematologic malignancies and solid tumors alone and in combination. Disclosures: Longenecker: Nereus Pharmaceuticals: Employment. Palladino:Nereus Pharmaceuticals: Employment, Equity Ownership. Lloyd:Nereus Pharmaceuticals: Employment, Equity Ownership. Neuteboom:Nereus Pharmaceuticals: Employment, Equity Ownership. Spear:Nereus Pharmaceuticals: Employment, Equity Ownership.

Investigational Agent MLN2238/MLN9708, a Specific, Orally Available, Small Molecule Proteasome Inhibitor, Shows Promising In Vitro Activity Against Multiple Myeloma Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3014-3014
Author(s):  
Giada Bianchi ◽  
Vijay G. Ramakrishnan ◽  
Teresa Kimlinger ◽  
Jessica Haug ◽  
S. Vincent Rajkumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Cell Lines ◽  
Small Molecule ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Proteasome Inhibitors ◽  
Biologically Active ◽  
Investigational Agent

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

Selective Inhibition of the proteasome's β2 Catalytic Subunit Alone Does Not Induce Cytotoxicity, but Resensitizes Bortezomib-Refractory Myeloma Cells for Bortezomib Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2915-2915
Author(s):  
Marianne Kraus ◽  
Bobby Florea ◽  
Jürgen Bader ◽  
Nan Li ◽  
Paul Geurink ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Active Site ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Selective Inhibition ◽  
Catalytic Site ◽  
Myeloma Cells ◽  
Bortezomib Treatment

Abstract Abstract 2915 Bortezomib is a reversible first-generation proteasome inhibitor that inhibits the β5 and to a lesser extent the β1 catalytic site of the proteasome. However, bortezomib does not inhibit the β2 catalytic proteasomal site at clinically relevant concentrations, and bortezomib-resistance is accompanied by upregulation of the β2 subunit, suggesting that increased β2 activity may compensate for the loss of β1/ β5 activity during bortezomib-treatment. The second generation proteasome inhibitor carfilzomib, due to the chemistry of its epoxyketone warhead, has a higher substrate specificity and functions as an irreversible proteasome inhibitor, but is still a β1/ β5 inhibitor that does not affect the β2 active site. We investigated the effect of β2-specific proteasome inhibition on myeloma and acute myeloid leukemia (AML) cells and tested the hypothesis that β2-selective proteasome inhibition may overcome bortezomib-resistance. To this end we have developed a set of epoxyketone- and vinylsulfone-based, cell permeable proteasome inhibitors of which we selected the compounds PR523A and PR671A for further testing in cell-based assays. PR671A is a peptide-vinylsulfone that selectively inhibits the proteasome's β2/ β2i subunit in an irreversible fashion in human cell lines and primary cells at low micromolar concentrations without inhibition of other protease species. PR523A is a β5-selective peptide-epoxyketone with otherwise similar properties. Treatment of myeloma and AML cell lines (AMO-1, U-266, HL-60, THP-1) with PR523A induced ER-stress mediated apoptosis, very similar to bortezomib. The combination of bortezomib with PR523A led to additive, but not synergistic induction of apoptosis, as expected. Selective β2 inhibition by PR671A resulted in the induction of ER stress and the accumulation of poly-ubiquitinated protein, however, this was not effectively translated into apoptotic cell death. This indicates that selective inhibition of the β2 proteasome subunit alone has only a poor cytotoxic effect on myeloma and AML cell lines, suggesting that the function of β2 is largely redundant and can be compensated when the remaining proteasome catalytic subunits (β1 and β5) remain active. However, when the β2 inhibitor PR671A was combined with agents that target the proteasome's β5 active site (PR523A) or the β5 and the β1 site (bortezomib), the combination of either inhibitor with the β2 inhibitor PR671A was highly synergistic for both activation of ER stress and the induction of apoptotic death. Importantly, the bortezomib-resistance in bortezomib-adapted myeloma and AML cell lines could be overcome by combining PR671A with either bortezomib or PR523A, while β2 inhibition by PR671A alone had no effect on the viability of bortezomib-adapted cells. We conclude that PR671A is a β2 selective proteasome inhibitor. Selective Inhibition of the proteasome's β2 subunit has little effect on viability or ER stress both in normal and bortezomib-resistant myeloma and leukemia cells, suggesting that the function of the β2 catalytic site is largely redundant. However, when β1/ β5 proteasome activity is inhibited by drugs like bortezomib or carfilzomib, proper function of the β2 proteasome active site is crucial for cell survival, also in bortezomib-resistant myeloma cells. The use of specific β2 inhibitors like PR671A in combination with β1/ β5 inhibitors like bortezomib or carfilzomib is therefore a promising strategy to overcome resistance against β1/ β5-selective proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

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