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.

Potentiation of Bortezomib-Induced Killing of Lymphoma Cells by Inhibition of Autophagy and Prevention of I-κBα Degradation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 116-116
Author(s):  
Li Jia ◽  
Ganga Gopinathan ◽  
Johanna T Sukumar ◽  
John G. Gribben
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Adaptor Protein ◽  
Proteasome Inhibition ◽  
Differential Sensitivity ◽  
Lymphoma Cells ◽  
Ubiquitinated Proteins ◽  
Apoptotic Proteins

Abstract Abstract 116 Previous studies have shown that germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) are resistant to proteasome inhibitors such as bortezomib. The mechanism by which GCB-DLBCL cells escape from proteasome inhibitor-induced apoptosis is unclear. To investigate this further, we examined the proteasomal pathway, expression of anti-apoptotic proteins and autophagy. Using bortezomib or MG-262, we show that DLBCL cells have differential sensitivity to proteasome inhibitor-mediated cell death, even though the effects on proteasome inhibition were similar. DLBCL cells that either over-express anti-apoptotic proteins such as Bcl-2, or lack pro-apoptotic proteins including Bax/Bak, are more resistant to proteasome inhibitor-induced reduction of mitochondrial membrane potential and activation of caspase-3. Treatment with bortezomib induced autophagy in both sensitive and resistant DLBCL cells, as demonstrated by an accumulation and aggregation of the autophagy marker protein LC3-II. Bortezomib induced accumulation of ubiquitinated proteins and a decrease in the adaptor protein p62, indicating activation of autophagic degradation. Fluorescent microscopy revealed that bortezomib induced p62 recruits both ubiquitinated proteins and LC3-II, suggesting that p62 leads ubiquitinated protein to autophagosomes. Treatment with bortezomib thereby promotes I-kBα degradation, demonstrating that the route of I-κBα degradation is not via the ubiquitin-proteasome degradation system. Bortezomib-induced I-kBα degradation was detected in both DLBCL cell lines and primary DLBCL and follicular lymphoma samples. Chloroquine (CQ), an inhibitor of autophagy, significantly increased bortezomib-induced accumulation of p62 and ubiquitinated proteins, including I-κBα, Bax and p53. CQ alone induced a dose-dependent increase in I-kBα protein levels, indicating that I-κBα protein can be degraded by autophagy even in the absence of proteasome inhibition. Importantly, the combination of proteasome and autophagy inhibitors shows great potential to kill apoptosis-resistant lymphoma cells. Proteasome inhibitor with or without CQ induced cell death in DLBCL cells cannot be blocked completely by either caspase inhibitors or knockdown of Bax/Bak proteins, suggesting that cell death occurs via a caspase-independent pathway. In summary, proteasome inhibitors induce autophagy and confer DLBCL cells resistance by eliminating I-κBα and possibly other pro-apoptotic proteins. Addition of autophagy inhibitors to bortezomib has the potential to induce increased killing in patients with resistant lymphoma. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

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.

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.

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.

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.

The Novel Proteasome Inhibitors Carfilzomib and Oprozomib Induce Milder Degenerative Effects Compared To Bortezomib When Administered Via Oral Feeding In An In Vivo Drosophila Experimental Model: A Biological Platform To Evaluate Safety/Efficacy Of Proteasome Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1930-1930
Author(s):  
Evangelos Terpos ◽  
Eleni N. Tsakiri ◽  
Efstathios Kastritis ◽  
Tina Bagratuni ◽  
Vassilis G. Gorgoulis ◽  
...  
Keyword(s):  
Oral Administration ◽  
Proteasome Inhibitor ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Somatic Tissue ◽  
Oral Feeding ◽  
Somatic Tissues

Abstract The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib. We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib. In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies’ culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies’ mortality rate, healthspan and overall longevity. Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Development of noninvasive biomarkers of response to proteasome inhibitor therapy (ixazomib) by imaging disrupted protein homeostasis in mouse models of solid tumors

2017 ◽  
Author(s):  
Yanan Zhu ◽  
Rajiv Ramasawmy ◽  
Sean Peter Johnson ◽  
Valerie Taylor ◽  
Alasdair Gibb ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Solid Tumors ◽  
Proteasome Inhibitor ◽  
Chemical Exchange ◽  
Proteasome Inhibitors ◽  
Surrogate Marker ◽  
Standard Of Care ◽  
Proteasome Inhibition ◽  
Protein Homeostasis

AbstractWith clinically-approved proteasome inhibitors now a standard of care for multiple myeloma, and increasing interest in their use in solid tumors, methods for monitoring therapeutic response in vivo are critically required. Here, we show that tumor protein homeostasis can be noninvasively monitored, using chemical exchange (CEST) magnetic resonance imaging (MRI) as a surrogate marker for proteasome inhibition, alongside diffusion MRI and relaxometry. We show that the in vivo CEST signal associated with amides and amines increases in proportion to proteasome inhibitor dose (ixazomib) and the magnitude of therapeutic effect in colorectal cancer xenografts. Moreover, we show that SW1222 and LS174T human colorectal cancer cell lines demonstrate differing sensitivities to ixazomib, which was reflected in our MRI measurements. We also found evidence of a mild stimulation in tumor growth at low ixazomib doses. Our results therefore identify CEST MRI as a promising method for safely and noninvasively monitoring changes in tumor protein homeostasis.

S-2209, a Novel, Non-Boronic Proteasome Inhibitor, Induces Apoptosis and Cell Growth Arrest in Multiple Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1513-1513
Author(s):  
Philipp Baumann ◽  
Karin Mueller ◽  
Sonja Mandl-Weber ◽  
Helmut Ostermann ◽  
Ralf Schmidmaier ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Wistar Rats ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Inhibition Assay ◽  
Induction Of Apoptosis

Abstract Purpose: Multiple Myeloma (MM) is still an incurable disease. Patients become resistant to cytotoxic drugs and die of disease progression. Bortezomib is the first approved member of a new class of antineoplastic agents, the proteasome inhibitors. It has synergistic effects with genotoxic drugs and steroids in vitro and in vivo. However, single agent activity in humans is only moderate and specific toxicity (e.g. neurotoxicity) often limits its clinical use. Further proteasome inhibitors need to be developed to optimize this promising treatment option. Methods: The new proteasome inhibitor S-2209 was characterized by several assays. Inhibition of the chymotryptic activity of the human 20S proteasome was determined with the in-vivo protease inhibition assay. Additionally, proteasome inhibition was determined in isolated PBMCs from S2209-pretreated wistar rats. Inhibition of NFκB activity was determined using a NFκB reporter gene assay. Cell growth rates of MM cells (OPM-2, U266, RPMI-8226 and NCI-H929) were measured with the WST-1 assay. Induction of apoptosis was shown by flow cytometry after staining with annexin-V-FITC and propidium iodide. Intracellular signal modulation was demonstrated by western blotting. Toxicity of the substance was tested in male wistar rats. Results: The proteasome inhibition assay revealed an IC50 at ∼220nM. The NFκB inhibition assay using an A549-NFκB-SEAP transfected cell line showed an EC50 of 0.9μM. Upon incubation with S-2209, cell growth as well as cell proliferation in MM cell lines was significantly inhibited (IC50 100nM – 600nM). Furthermore, the incubation with S-2209 resulted in strong induction of apoptosis in all four MM cell lines even at nanomolar concentrations (IC50 at ∼300nm) as well as primary cells. Western blotting revealed caspase-3 cleavage and upregulon of p53 and increased phosphorylation of IκB. No induction of apoptosis was detected in PBMCs from healthy humans. Despite the administration of 5, 10 or 15mg/kg/day in wistar rats, no toxicity with respect to body weight, hepatic enzymes (ALAT ASAT, ALP), creatinin or hemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals than in control animals treated with 0.1mg/kg/d bortezomib (89% vs. 70% respectively). Conclusions: The proteasome inhibitor S-2209 inhibitis MM cell growth and induces apoptosis. This is accompanied by a strong inhibition of proteasome and of the NFκB activity. Because S-2209 shows a favourable toxicity profile in vivo, further clinical development of this promising drug is urgently needed.

Modeling Proteasome Inhibition in Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4946-4946
Author(s):  
Linda B. Baughn ◽  
Holly Stessman ◽  
Aatif Mansoor ◽  
Brian Van Ness
Keyword(s):  
Drug Resistance ◽  
Genomic Instability ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Proteasome Inhibitors ◽  
Fold Increase ◽  
Proteasome Inhibition ◽  
Burkitt’S Lymphoma ◽  
Model System ◽  
Burkitt's Lymphoma

Abstract Abstract 4946 The proteasome inhibitor Bortezomib (Bz) has been widely used to treat multiple myeloma, relapsed mantle cell lymphoma and is undergoing clinical evaluation for other B cell malignancies including non-Hodgkin lymphoma. Despite its initial success, patients treated with Bz eventually relapse due to the development of drug resistance. Therefore, understanding the basis of drug resistance is a critical component for improved therapy. The acquisition of Bz resistance in lymphomas, particularly those with constitutive expression of the B cell-specific DNA mutator, activation-induced cytidine deaminase (AID), has not been previously characterized. We have utilized the AID-expressing human non-Hodgkin Burkitt's lymphoma as a model system for this study. Burkitt's lymphoma lines (Ramos and BL-2) are suitable because they are highly sensitive to Bz induced apoptosis with an IC50 of approximately 11 nM after 48 hrs of treatment. In order to generate Bz resistant Burkitt's lymphoma lines, Ramos cells were treated weekly with increasing concentrations of Bz for 3 months. Compared to the parental line, this newly formed line displayed an approximately 2.5–3-fold increase in IC50 to Bz as well as to three other proteasome inhibitors (next-generation proteasome inhibitor, MLN 2238, epoxomicin and carfilzomib), while maintaining sensitivity to different chemotherapeutic agents (PD 0332991 cyclin 4/6 dependent kinase inhibitor and melphalan). In this model system, resistance to Bz conferred a general cross-resistance to other proteasome inhibitors, a phenotype that has been stably maintained for 5 months. We next asked whether AID plays a role in the acquisition of Bz resistance in Ramos cells by promoting hypermutation and genomic instability. In support of this hypothesis, the G322A and C326T mutations in the gene encoding the proteasome subunit and target of Bz, psmb5, occur within AID hotspots raising the possibility that AID could directly mutate psmb5. Furthermore, like many proteins, AID is degraded by the proteasome arguing that proteasome inhibition further stabilizes AID protein resulting in aberrant hypermutation. Consistent with this, we detected by flow cytometry (intracellular staining) a 2-fold increase in AID protein following a 24-hour, 20 nM Bz treatment of Ramos cells. AID normally mutates immunoglobulin (Ig) genes and these mutations are necessary for the production of protective antibodies, while aberrant AID activity leads to mutations in non-Ig genes. Surprisingly, despite the increase in AID protein, we observe reduced mutation frequency within the functional IgH gene following 3-month Bz treatment compared to untreated controls. Despite this reduction, array comparative genomic hybridization (a-CGH) studies indicate copy number abnormalities in Bz resistant cells and details of the chromosomal abnormalities and target genes deregulated will be presented. These data demonstrate that Burkitt's lymphoma cells are sensitive to Bz and drug resistance can be readily achieved in vitro. Furthermore, Bz treatment stabilizes AID protein and promotes increased genomic instability. Disclosures: Stessman: Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Pre-Clinical Activity of the Novel, First-in-Class p97 Inhibitor, CB-5083, in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4701-4701
Author(s):  
Blake T. Aftab ◽  
Daniel J Anderson ◽  
Ronan Le Moigne ◽  
Stevan Djakovic ◽  
Eugen Dhimolea ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Stromal Cell ◽  
Hematological Malignancies ◽  
Proteasome Inhibitors ◽  
Protein Homeostasis ◽  
Ubiquitinated Proteins

Abstract Hematological malignancies such as multiple myeloma (MM) have an increased reliance on the ubiquitin proteasome system (UPS) presumably as a consequence of their high protein synthetic and secretory burden. Chemical agents that target the proteasome, such as bortezomib and carfilzomib, have been successful in treating multiple myeloma; however patients treated with these drugs ultimately relapse. The AAA-ATPase p97/VCP (p97) facilitates ATP-dependent extraction and degradation of ubiquitinated proteins destined for proteasomal elimination. In addition to ubiquitin-dependent protein degradation, p97 is also closely involved in other aspects of protein homeostasis, including endoplasmic reticulum-associated degradation (ERAD) and autophagy. Pharmacologic inhibition of p97 provides a compelling therapeutic approach for hematological malignancies that rely on tight regulation of protein homeostasis as a component of their survival. CB-5083 is a novel small molecule inhibitor of p97 ATPase activity with nanomolar enzymatic and cellular potency. Treatment of cancer cells with CB-5083 causes a dramatic increase in poly-ubiquitinated proteins as well as an accumulation of substrates of the UPS and ERAD. CB-5083 causes a profound induction of the unfolded protein response (UPR) with consequent activation of the DR5 death receptor, caspase 8, caspase 3/7 and ultimately cell death. Induction of the UPR occurs to a greater magnitude with CB-5083 when compared to the proteasome inhibitor, bortezomib, suggesting the potential for increased efficacy in cancers with sensitivity to UPR-mediated cell death. In addition, activation of apoptosis and cell death occur more rapidly with CB-5083 than with bortezomib. Sequencing of cell lines made resistant to CB-5083 reveals missense mutations mapping to the D2 ATPase site in p97, supporting on-target association with cytotoxicity. In an expanded panel of MM cell lines there is no correlation between the cytotoxic sensitivity to CB-5083 and the cytotoxic sensitivity to proteasome inhibitors, suggesting differential mechanisms of cytotoxicity and potential activity of CB-5083 in proteasome inhibitor resistant settings. Compared to myeloma cell lines, CB-5083 has reduced cytotoxic potency in immortalized stromal cell lines and in patient-derived CD138-negative bone marrow mononuclear cells. Furthermore, unlike the reduced potency demonstrated by carfilzomib in the context of MM cell-bone marrow stromal cell (BMSC) interactions, the cyto-reductive potential of CB-5083 is unaffected in co-cultures of MM cells with patient-derived BMSCs or immortalized BMSCs from healthy donors. In vivo, CB-5083 is orally bioavailable, shows a pharmacodynamic effect in tumor tissue (as measured by poly-ubiquitin accumulation) and demonstrates robust anti-tumor activity across several MM models. CB-5083 treatment of mice bearing subcutaneous xenografts leads to tumor stasis and regression in RPMI8226 and AMO1 MM models, respectively. In advanced models of disseminated, ortho-metastatic disease, intermittent oral administration of CB-5083 demonstrates significant inhibition of myeloma burden and improves survival, with an overall efficacy profile that compares favorably to that of clinically approved proteasome inhibitors. Furthermore, in the Vk*Myc genetically engineered mouse model of MM, treatment with CB-5083 results in a significant reduction in M-spike by 55%. Combination treatment of mice bearing the RPMI8226 subcutaneous xenograft model with CB-5083, dexamethasone and lenalidomide results in tumor regression. Taken together, these data demonstrate that CB-5083 is a potent and selective inhibitor of the p97 ATPase with robust activity in vitro and in vivo in numerous MM models and strongly support clinical evaluation. Based on these observations, a phase 1 dose-escalation trial has recently been initiated and is currently underway in patients with relapsed/refractory multiple myeloma. Disclosures Anderson: Cleave Biosciences: Employment. Le Moigne:Cleave Biosciences: Employment. Djakovic:Cleave Biosciences: Employment. Rice:Cleave Biosciences: Employment. Wong:Cleave Biosciences: Employment. Kumar:Cleave Biosciences: Employment. Valle:Cleave Biosciences: Employment. Menon:Cleave Biosciences: Employment. Kiss von Soly:Cleave Biosciences: Employment. Wang:Cleave Biosciences: Employment. Yao:Cleave Biosciences: Employment. Soriano:Cleave Biosciences: Employment. Bergsagel:ONYX: Consultancy; Janssen: Consultancy; BMS: Consultancy; Novartis: Research Funding. Yakes:Cleave Biosciences: Employment. Zhou:Cleave Biosciences: Employment. Wustrow:Cleave Biosciences: Employment. Rolfe:Cleave Biosciences: Employment.

Sign in / Sign up

Export Citation Format

Share Document