Effects Of Proteasome Inhibitors Research Articles

Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction

Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the Gq/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of Gq/11-evoked Ca2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of Gq/11-coupled receptor signaling in the cardiovascular, immune, and nervous systems.

A Potential Asset of Proteasome Inhibitor Singly or Combined with Cisplatin Provides Inhibitory Effect on Human Gastric Cancer Cells

Backgrounds and Objective: Proteasome inhibitors are a kind of novel anti-tumor agent which can inhibit cell growth and induce apoptosis by inhibiting the function of proteasome system and impacting the degradation of proteins related to cell growth or apoptosis. The aim of the study is to explore the effect of proteasome inhibitor Z-Leu-Leu-Phe-CHO (ZLLFC) singly or combined with cisplatin on proliferation or apoptosis of humnan gastric cancer cell SGC-7901, as well as its influence on the expression level of excision repair cross complementing 1(ERCC1) mRNA. Methods: MTT assay was used to calculate the growth inhibitory rate of human gastric cancer cell SGC- 7901. The apoptotic cell morphology was observed with electron microscopy, the apoptotic rate was analyzed by flow cytometry and by expression level of ERCC1 mRNA detected by RT-PCR. Results: Proteasome inhibitor ZLLFC could inhibit the growth of gastric cancer cell SGC-7901. Combined application of proteasome inhibitor ZLLFC and cisplatin significantly increase the inhibitory effect on SGC-7901 cells, induced apoptosis and increased the apoptotic rate. While, the expression level of ERCC-1 mRNA in SGC-7901 cells in the combined group was significantly lower than that of the cisplatin group (p

Phase 2, open-label study of venetoclax in combination with carfilzomib and dexamethasone in patients with relapsed/refractory multiple myeloma.

TPS8056 Background: A significant unmet need for multiple myeloma (MM) therapy remains as many patients eventually relapse after or become refractory to current treatment options. Investigation of novel agents and combinations in relapsed/refractory (R/R) patients are therefore critical to advance therapy and improve patient outcomes. Venetoclax is a potent, selective, orally bioavailable inhibitor of BCL-2. Combination of venetoclax with dexamethasone and bortezomib, a proteasome inhibitor that can inhibit MCL-1 indirectly via stabilizing the MCL-1-neutralizing protein NOXA, showed high rates of clinical response in a Phase 1 study. The mechanism of MCL-1 inhibition is thought to be a class effect of proteasome inhibitors. Given the clinical data supporting the combination of venetoclax with a proteasome inhibitor, this study will evaluate whether venetoclax combined with carfilzomib and dexamethasone can provide a well-tolerated and efficacious treatment option for R/R MM patients. Methods: This Phase 2, open-label study will assess the combination of venetoclax, carfilzomib, and dexamethasone in patients with R/R MM (NCT02899052). Primary objectives are to assess the safety and tolerability of this combination. Secondary objectives include evaluation of the pharmacokinetics of venetoclax and carfilzomib, preliminary efficacy of the combination (including overall response rate, very good partial response or better rate, progression-free survival, time to progression, and duration of response), and minimal residual disease (MRD) in bone marrow by next generation sequencing. Exploratory objectives will assess pharmacodynamic and predictive biomarkers, MRD by PET, pharmacogenetics, and patient-reported outcomes. Safety and pharmacokinetic profiles of the combination will be evaluated in initial dose escalation cohorts to determine appropriate doses of venetoclax and carfilzomib to be used with dexamethasone; a dose expansion phase will evaluate the safety and efficacy profiles of the combination based on selected doses. Study recruitment began in January 2017, with target enrollment of ~40 patients from 10–15 sites in the United States. Clinical trial information: NCT02899052.

Inhibition of Candida species via Proteosome Inhibitor MG-262 (ZL3B)

Backround: Candida species take the fourth place among the microorganisms which cause hospital infections. New therapeutic agents are needed because of rapidly increasing resistance rates. Boric acid preparations are preferred in local treatment especially in resistant cases while azole formulations are the first choice in oral treatment. The only boric acid analog that was approved by FDA (Food and Drug Administration) for systemic use is Bortezomib (Velcade). Bortezomib is a proteasome inhibitor and proteasome inhibitors cause apoptosis of eukaryotic cells. Although the yeast also has a well preserved 20S proteasome region, the studies on the effects of proteasome inhibitors on the yeast are limited. In this study, the effects of the one of the most potent proteasome inhibitor, MG-262, on Candida spp. were investigated. Methods: C. albicans ATCC 90028, C. krusei ATCC 6258 and C. glabrata (clinical strain) strains were used in our study. Antifungal susceptibilities of the strains against MG-262 and effects of MG-262 on oxidative metabolism, proteasome activity and apoptosis as well as virulence factors such as hyphal transformation, pseudohypha, germ tube and biofilm formation were investigated. CLSI (Clinical and Laboratory Standards Institute) guidelines were used for antifungal susceptibility tests. The effect on hypha and pseudohypha formation and germ tube were determined with conventional tests. Biofilm formation was detected according to the microplate method. Oxidative metabolism experiments, proteosome activity and apoptosis tests were performed with Alamarblue dye, fluorogenic peptides and DNA ladder assays, respectively. Findings: MG-262 exhibited minimum inhibitory activity at 25 μg/ml concentration for C. albicans ATCC 90028 and at 50 μg/ml concentration for C. krusei ATCC 6258 and C. glabrata. Minimum fungicidal concentrations for all species were 50 μg/ml. Hypha, pseudohypha, germ tube and biofilm formation, proteosome activity and oxidative metabolism were all inhibited at 50 μg/ml and higher concentrations. The apoptosis was detected at 200 μg/ml. Conclusion: MG-262 caused apoptosis by inhibiting oxidative metabolism and showed fungicidal activity. It also inhibited the virulence factors such as hypha, pseudohypha germ tube and biofilm formation. Inhibition of proteasome is promising in terms of discovery of a new generation antifungal agent.

The role of the proteasome in AML.

Acute myeloid leukemia (AML) is deadly hematologic malignancy. Despite a well-characterized genetic and molecular landscape, targeted therapies for AML have failed to significantly improve clinical outcomes. Over the past decade, proteasome inhibition has been demonstrated to be an effective therapeutic strategy in several hematologic malignancies. Proteasome inhibitors, such as bortezomib and carfilzomib, have become mainstays of treatment for multiple myeloma and mantle cell lymphoma. In light of this success, there has been a surge of literature exploring both the role of the proteasome and the effects of proteasome inhibition in AML. Pre-clinical studies have demonstrated that proteasome inhibition disrupts proliferative cell signaling pathways, exhibits cytotoxic synergism with other chemotherapeutics and induces autophagy of cancer-related proteins. Meanwhile, clinical trials incorporating bortezomib into combination chemotherapy regimens have reported a range of responses in AML patients, with complete remission rates >80% in some cases. Taken together, this preclinical and clinical evidence suggests that inhibition of the proteasome may be efficacious in this disease. In an effort to focus further investigation into this area, these recent studies and their findings are reviewed here.

The effects of proteasomal inhibition on synaptic proteostasis.

Synaptic function crucially depends on uninterrupted synthesis and degradation of synaptic proteins. While much has been learned on synaptic protein synthesis, little is known on the routes by which synaptic proteins are degraded. Here we systematically studied how inhibition of the ubiquitin‐proteasome system (UPS) affects the degradation rates of thousands of neuronal and synaptic proteins. We identified a group of proteins, including several proteins related to glutamate receptor trafficking, whose degradation rates were significantly slowed by UPS inhibition. Unexpectedly, however, degradation rates of most synaptic proteins were not significantly affected. Interestingly, many of the differential effects of UPS inhibition were readily explained by a quantitative framework that considered known metabolic turnover rates for the same proteins. In contrast to the limited effects on protein degradation, UPS inhibition profoundly and preferentially suppressed the synthesis of a large number of synaptic proteins. Our findings point to the importance of the UPS in the degradation of certain synaptic proteins, yet indicate that under basal conditions most synaptic proteins might be degraded through alternative pathways.

Abstract 3038: Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL)

Abstract Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. Previously, our retrospective review from single institution series revealed that 30 out of 611 DLBCL patients had aberrations in c-MYC and BCL2 or BCL6 by FISH. These patients exhibited inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS), suggesting that newer therapies are in dire need. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL. Materials & Methods: At the pre-clinical level, we studied 3 novel therapeutic agents targeting BCL2 (ABT-199), c-MYC (JQ-1), and various cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT-199 activity (irreversible proteasome inhibitor carfilzomib(CFZ)) using DH lymphoma (DHL) cell lines (Val, DOHH-2, ROS-50). DHL cell lines were exposed to ABT-199 (0-10 uM), JQ-1 (0-100 uM) and carfilzomib (CFZ) (0-50 nM) at 24, 48 and 72 hours. Changes in cell viability were evaluated using Presto Blue assay. Subsequently, DHL cells were exposed to doublet combinations of ABT-199, JQ-1 and CFZ for 48 hours. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Conclusion: ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. Citation Format: Priyank P. Patel, Alison Zeccola, Juan Gu, Cory Mavis, Sheila N. J. Sait, Vishala Neppalli, Francisco J. Hernandez-Ilizaliturri. Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3038.

Interaction of Gcn4 with target gene chromatin is modulated by proteasome function.

The ubiquitin-proteasome system (UPS) influences gene transcription in multiple ways. One way in which the UPS affects transcription centers on transcriptional activators, the function of which can be stimulated by components of the UPS that also trigger their destruction. Activation of transcription by the yeast activator Gcn4, for example, is attenuated by mutations in the ubiquitin ligase that mediates Gcn4 ubiquitylation or by inhibition of the proteasome, leading to the idea that ubiquitin-mediated proteolysis of Gcn4 is required for its activity. Here we probe the steps in Gcn4 activity that are perturbed by disruption of the UPS. We show that the ubiquitylation machinery and the proteasome control different steps in Gcn4 function and that proteasome activity is required for the ability of Gcn4 to bind to its target genes in the context of chromatin. Curiously, the effect of proteasome inhibition on Gcn4 activity is suppressed by mutations in the ubiquitin-selective chaperone Cdc48, revealing that proteolysis per se is not required for Gcn4 activity. Our data highlight the role of Cdc48 in controlling promoter occupancy by Gcn4 and support a model in which ubiquitylation of activators-not their destruction-is important for function.

Proteasome or immunoproteasome inhibitors cause apoptosis in human renal tubular epithelial cells under normoxic and hypoxic conditions.

Ischemic acute kidney injury is characterized by apoptosis of tubular epithelial cells. Proteasome plays a key role in cellular processes such as proliferation, apoptosis and inflammation. The results of animal studies about the effect of proteasome inhibitors on the course of ischemic acute kidney injury are controversial. Primary human renal tubular epithelial cells were cultured with or without the hypoxia mimetic CoCl2 and with or without the proteasome inhibitor CEP-18770 and/or the immunoproteasome inhibitor ONX-0914. The level of the proteasome subunit β5, the immunoproteasome subunits LMP7 and LMP2, the function of these proteolytic machines, HIF-1α and its transcriptional target lactate dehydrogenase-A, p53 and its transcriptional targets TP53-inducible glycolysis and apoptosis regulator and p21, and finally of activated cleaved caspase-3 were assessed by means of western blotting. CoCl2 decreased the expression of β5, LMP7 and LMP2, as well as the activity of proteasome and immunoproteasome. It increased HIF-1α and its function, along with p53 and its function and induced apoptosis. CEP-18770 and ONX-0914 induced the above alterations toward the same directions as CoCl2 does. In CoCl2-treated cells, pretreatment with CEP-18770 and/or ONX-0914 potentiates the changes induced by CoCl2 alone. CoCl2, CEP-18770 and ONX-0914 induce apoptosis in human renal tubular epithelial cells. Importantly, proteasome or immunoproteasome inhibitors are rather toxic than beneficial in human renal tubular epithelial cells treated with the hypoxia mimetic CoCl2.

Abstract P3-06-12: Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?

Abstract Proteasome, the essential giant protease, is the hub of the ubiquitin-proteasome pathway critical for maintaining intracellular proteostasis. Inhibitors of the proteasome cause overload of cells with non-degraded protein debris and activate proapoptotic signaling. The effects are especially dramatic in fast metabolizing cancer cells; hence the proteasome inhibitors are used as anti-cancer drugs. There are two FDA-approved drugs directly blocking the proteasome activity, bortezomib and carfilzomib, and several others are in trials. So far, the high effectiveness of these drugs is limited to multiple myeloma and other hematological malignancies. Unfortunately, breast and other solid cancers are much more resistant to apoptosis triggered by attenuation of the ubiquitin-proteasome pathway than majority of blood cancers. The relatively low effectiveness of proteasome inhibitors has been noted even for cancers that have been identified in genome-wide screens as addicted to the proteasome activity, such as triple negative breast cancers. The source of the resistance, noted in cell culture, animal studies and clinical trials, remains unknown. Here we propose that, at least in part, the resistance can be tracked down to protein-protein interactions between the drug target, the proteasome, and a cytosolic heat shock protein, Hsp72 (inducible Hsp70). Indeed, high levels of Hsp72 have been observed in many cancers, including breast, and generally correlate with poor prognosis. The intracellular roles of the ubiquitous chaperone are undoubtedly very diverse, however we postulate a novel molecular action: direct protection of the proteasome from competitive inhibitors of its enzymatic activity. The hypothesis has been inspired by our recent work exploring the very effective ubiquitin-proteasome pathway in the naked mole rats. These small African rodents do not develop cancers and maintain unusually long lifespan and health-span. Interestingly, the proteasome in naked mole rats is exceptionally resistant to inhibition. We discovered that the resistance is bestowed by a cytosolic protein factor containing Hsp72 and Hsp40 chaperones (Biochim. Biophys. Acta-MBD 1842; 2060-2072; 2014). We partially purified the factor and tested its in vitro effectiveness with human proteasomes. The molecular mechanism beneficial for naked mole rats may be also protective for human cancer cells. We used pifithrin μ, a specific small molecule inhibitor blocking the interactions of Hsp72 with other proteins, to test the hypothesis. We prepared lysates from cultured triple negative breast cancer cells MDA-MB-231. Such lysates are rich in proteasome activity and preserve interactions of the proteasomes with natural protein ligands. Interestingly, upon the treatment with pifithrin μ proteasomes in lysates maintained their high activity while becoming significantly more sensitive to inhibition with bortezomib. The studies are in progress to assess the identity of a putative chaperone-based proteasome resistance factor in breast cancer cells. Citation Format: Gaczynska M, Osmulski PA. Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-12.

Proteomic Profiling of Radiation-Induced Skin Fibrosis in Rats: Targeting the Ubiquitin-Proteasome System

To investigate the molecular changes underlying the pathogenesis of radiation-induced skin fibrosis. Rat skin was irradiated to 30 or 45Gy with an electron beam. Protein expression in fibrotic rat skin and adjacent normal tissues was quantified by label-free protein quantitation. Human skin cells HaCaT and WS-1 were treated by x-ray irradiation, and the proteasome activity was determined with a fluorescent probe. The effect of proteasome inhibitors on Transforming growth factor Beta (TGF-B) signaling was measured by Western blot and immunofluorescence. The efficacy of bortezomib in wound healing of rat skin was assessed by the skin injury scale. We found that irradiation induced epidermal and dermal hyperplasia in rat and human skin. One hundred ninety-six preferentially expressed and 80 unique proteins in the irradiated fibrotic skin were identified. Through bioinformatic analysis, the ubiquitin-proteasome pathway showed a significant fold change and was investigated in greater detail. Invitro experiments demonstrated that irradiation resulted in a decline in the activity of the proteasome in human skin cells. The proteasome inhibitor bortezomib suppressed profibrotic TGF-β downstream signaling but not TGF-β secretion stimulated by irradiation in HaCaT and WS-1cells. Moreover, bortezomib ameliorated radiation-induced skin injury and attenuated epidermal hyperplasia. Our findings illustrate the molecular changes during radiation-induced skin fibrosis and suggest that targeting the ubiquitin-proteasome system would be an effective countermeasure.

Abstract A81: Investigation of possible cell state dependent differential protein homeostasis mechanism(s)

Abstract Proteasome inhibition has been shown to be an important strategy for treating many types of cancer. Although proteasome inhibitors are effective at eradicating rapidly proliferating cancer cells, a subset of cancer cells enter quiescence and survive, contributing to tumor recurrence. Using primary human fibroblasts as a model system we have shown that quiescent cells have decreased reactive oxygen species levels and protein aggregates, and undergo apoptosis less frequently than proliferating cells in response to proteasome inhibition. However, the precise mechanism by which quiescent cells become resistant to proteasome inhibition remains largely unknown. Using a GFP-tagged proteasome reporter, we investigated mechanisms protecting quiescent cells from proteasome inhibition-induced apoptosis. We found that, in proliferating cells, proteasome inhibition by MG132 led to high levels of a GFP-tagged proteasome reporter with a visible ubiquitination pattern. In contrast, MG132 treatment of quiescent cells resulted in reduced levels of the ubiquitinated form of this reporter as observed by Western blot analysis. Interestingly, preliminary immunoprecipitation followed by mass-spectrometry analysis revealed none canonical ubiquitin modification on lysine 27 of the reporter in quiescent cells. These findings suggested that quiescent cells may have one or more alternative pathways preventing specific proteasome substrates accumulation in response to proteasome inhibition. Moreover, our data has shown that, upon proteasome inhibition, a GFP-tagged proteasome reporter was secreted into the condition medium. Secretion of cytoplasmic proteins in response to proteasome inhibition is one possible mechanism for reducing protein aggregation and cytotoxicity. Identification of regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in cancer treatment. Citation Format: Gordon-Victor Fon, Chandler R. Keller, David H. Perlman, Aster Legesse-Miller. Investigation of possible cell state dependent differential protein homeostasis mechanism(s). [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A81.

Abstract A80: p53 protects against MG132-induced apoptosis in a cell state-dependent manner

Abstract Since the approval of proteasome inhibitor, Bortezomib, for use in multiple myeloma in 2003, pharmacological proteasome inhibitors has been shown to be an important strategy for treating many types of cancer. While proteasome inhibitors induce apoptosis in certain rapidly proliferating cancer cells, some cells enters quiescence and survives. The mechanism by which quiescent cells become resistant to proteasome inhibitors is largely unknown. The tumor suppressor protein p53 plays an essential role in establishing and maintain quiescence in human cells. It also regulates several cell survival mechanisms, including cell cycle arrest, autophagy, and activation of reactive oxygen detoxifying enzymes. Here, we investigated the role of p53 in establishing resistance against proteasome inhibitors in quiescent cells. We found that MG132 (a know proteasome inhibitor) treatment induces the accumulation of p53 and its downstream target p21 more in quiescent cells than in proliferating primary human fibroblasts. shRNA mediated down-regulation of p53 sensitizes both proliferating and quiescent fibroblasts to MG132-mediated apoptosis and cell death, suggesting that p53 plays a protective role in all cell states. Additional experiments demonstrated that proliferating fibroblasts are susceptible to inhibition of cytoplasmic p53 activity, whereas quiescent fibroblasts are sensitive to inhibition of nuclear p53 activity. These findings suggest that p53 induces different protective mechanisms against MG132-mediated cell death, depending of the proliferative state of the cells. Autophagy, an alternative degradation pathway regulated by p53, is also induced in both cell states upon MG132 treatment. Identification of p53-mediated regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in treating cancer. Citation Format: Aster Legesse-Miller, Jacky Ho Wa Cheng. p53 protects against MG132-induced apoptosis in a cell state-dependent manner. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A80.

Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

Abstract A18: MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib.

Abstract Purpose: The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin+proteasome system (UPS) and also disrupt the aggresome+autophagy pathway for anti-myeloma benefit. Background: Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Drug resistance accounts for the majority of tumor relapses and cancer-related deaths. Functional blockade of the UPS using targeted therapeutics has translated into clinical success and transformed the management of MM. However, drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism leading to treatment failure, disease relapse and ultimately fatal outcome. Experimental Procedures: Myeloma cells were exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naïve cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly reduced in drug-resistant cells. To determine the effect of individual miRNAs, synthetic miRNA replacements were transfected into myeloma cells. Individual miRNA replacements were also transfected into human embryonic kidney (HEK) cells that expressed a short-lived green fluorescent protein that is degraded by the proteasome. The effect of individual miRNAs on the UPS was then quantitated in HEK cells by confocal microscopy and fluorescent imaging. Results: MiRNA-29b was significantly downregulated in the myeloma cells generated with acquired resistance to bortezomib, carfilzomib and ixazomib relative to the parental cells. MiRNA-29b was also downregulated in MM patient tumor cells relative to plasma cells form healthy individuals. MiRNA-29b targeted PSME4 which encodes the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29b replacements reduced the viability of myeloma cells and patient tumor cells and synergistically enhanced the cytotoxic effect of proteasome inhibitors. The miRNA-29b replacement also reduced the growth of myeloma xenografts in mouse models in vivo. MiRNA-29b replacements decreased expression of PSME4 and the protein product PA200, a proteasome activator. Reduction in PA200 reduced the proteasome's chymotrypsin-like peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble ubiquitinated proteins, termed aggresomes, accumulate and are degraded by the autophagy pathway. Importantly, in contrast to bortezomib, miRNA-29b replacement inhibited proteasome activity but did not induce the formation of perinuclear aggresomes or autophagosomes. Rather, immunofluorescence studies indicated that miRNA-29b replacement promoted the accumulation of small, ubiquitinated protein aggregates that were dispersed throughout the cytoplasm but were not transported by microtubules and were not coalesced into mature aggresomes. The ubiquitinated proteins also were not detected in autophagosomes. Conclusions: MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and uncouple the UPS from the aggresome-autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma effect of PIs. Citation Format: Sajjeev Jagannathan, Nikhil Vad, Mohamed Abdel-Malek, Ehsan Malek, James Driscoll. MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A18.

Abstract A44: p53 protects against MG132-induced apoptosis in a cell state-dependent manner.

Abstract Proteasome inhibition is an important strategy for treating many types of cancer. While proteasome inhibitors induce apoptosis in certain rapidly proliferating cancer cells, some cells enters quiescence and survives. The mechanism by which quiescent cells become resistant to proteasome inhibitors is unknown. The tumor suppressor protein p53 plays an essential role in establishing and maintaining quiescence in human cells. It also regulates several cell survival mechanisms, such as cell cycle arrest, autophagy, and activation of detoxifying enzymes. Here, we investigate the role of p53 in establishing resistance against proteasome inhibitors in quiescent cells. We found that MG132 (a known proteasome inhibitor) treatment induces the accumulation of p53 and its downstream target p21 more in quiescent than in proliferating primary human fibroblasts. shRNA-mediated down-regulation of p53 sensitizes both proliferating and quiescent fibroblasts to MG132-mediated apoptosis and cell death, suggesting that p53 plays a protective role in all cell states. Additional experiments demonstrated that proliferating fibroblasts are susceptible to inhibition of cytoplasmic p53 activity, whereas quiescent fibroblasts are sensitive to inhibition of either nuclear or cytoplasmic p53 activity. These findings suggest that proliferating and quiescent cells depend on different p53-induced survival mechanisms to be protected from proteasome inhibition. Autophagy, an alternative degradation pathway regulated by p53, is also induced in both cell states upon MG132 treatment. Identification of p53-mediated regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in treating cancer. Citation Format: Aster Legesse-Miller, Jacky Ho Wa Cheng. p53 protects against MG132-induced apoptosis in a cell state-dependent manner. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A44.

Abstract 1008: Cytoplasmic p53 protects against MG132-induced apoptosis in a cell state-dependent manner

Abstract Since the approval of proteasome inhibitor, Bortezomib, for use in multiple myeloma in 2003, pharmacological proteasome inhibitors has been shown to be an important strategy for treating many types of cancer. While proteasome inhibitors induce apoptosis in certain rapidly proliferating cancer cells, some cells enters quiescence and survives. The mechanism by which quiescent cells become resistant to proteasome inhibitors is largely unknown. The tumor suppressor protein p53 plays an essential role in establishing and maintain quiescence in human cells. It also regulates several cell survival mechanisms, including cell cycle arrest, autophagy, and activation of reactive oxygen detoxifying enzymes. Here, we investigated the role of p53 in establishing resistance against proteasome inhibitors in quiescent cells. We found that MG132 (a know proteasome inhibitor) treatment induces the accumulation of p53 and its downstream target p21 more in quiescent cells than in proliferating primary human fibroblasts. shRNA mediated down-regulation of p53 sensitizes both proliferating and quiescent fibroblasts to MG132-mediated apoptosis and cell death, suggesting that p53 plays a protective role in all cell states. Additional experiments demonstrated that proliferating fibroblasts are susceptible to inhibition of cytoplasmic p53 activity, whereas quiescent fibroblasts are sensitive to inhibition of nuclear p53 activity. These findings suggest that p53 induces different protective mechanisms against MG132-mediated cell death, depending of the proliferative state of the cells. Autophagy, an alternative degradation pathway regulated by p53, is also induced in both cell states upon MG132 treatment. Identification of p53-mediated regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in treating cancer. This work was supported by NCI K01 CA128887 to Aster Legesse-Miller. Citation Format: Ho Wa (Jacky) Cheng, Aster Legesse-Miller. Cytoplasmic p53 protects against MG132-induced apoptosis in a cell state-dependent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1008. doi:10.1158/1538-7445.AM2015-1008

The human ubiquitin conjugating enzyme UBE2J2 (Ubc6) is a substrate for proteasomal degradation

The human Ube2J2 enzyme functions in the ubiquitination of proteins at the ER. Here we demonstrate that it, and a second ubiquitin conjugating (Ubc) enzyme Ube2G2, are unstable, and incubation of transfected cells with proteasome inhibitors increased steady-state protein levels. For Ube2J2, pharmacological induction of the unfolded protein response (UPR) did not significantly alter ectopic protein levels, however the effect of proteasomal inhibition was abolished if the enzyme was inactivated or truncated to disrupt its ER-localization. These results suggest for the first time that the steady state expression of Ubcs’ may be important in regulating the degradation of ER proteins in mammalian cells.

Effect of bortezomib on the efficacy of AAV9.SERCA2a treatment to preserve cardiac function in a rat pressure-overload model of heart failure

Adeno-associated virus (AAV)-based vectors are promising vehicles for therapeutic gene delivery, including for the treatment of heart failure. It has been demonstrated for each of the AAV serotypes 1 through 8 that inhibition of the proteasome results in increased transduction efficiencies. For AAV9, however, the effect of proteasome inhibitors on in vivo transduction has until now not been evaluated. Here we demonstrate, in a well-established rodent heart failure model, that concurrent treatment with the proteasome inhibitor bortezomib does not enhance the efficacy of AAV9.SERCA2a to improve cardiac function as examined by echocardiography and pressure volume analysis. Western blot analysis of SERCA2a protein and RT-PCR of SERCA2a mRNA demonstrated that bortezomib had no effect on either endogenous rat SERCA2a levels nor on expression levels of human SERCA2a delivered by AAV9.SERCA2a. Similarly, the number of AAV9 genomes in heart samples was unaffected by bortezomib treatment. Interestingly, whereas transduction of HeLa cells and neonatal rat cardiomyocytes by AAV9 was stimulated, transduction of adult rat cardiomyocytes was inhibited. These results indicate an organ/cell-type specific effect of proteasome inhibition on AAV9 transduction. A future detailed analysis of the underlying molecular mechanisms promises to facilitate the development of improved AAV vectors.

Evaluation of tubulin polymerization and chronic inhibition of proteasome as citotoxicity mechanisms in bortezomib-induced peripheral neuropathy

Bortezomib (BTZ) is the first proteasome inhibitor entered in clinical practice. Peripheral neuropathy is likely to be a class side effect of these drugs, although its severity is largely variable, and it deserves to be further investigated, since the mechanisms of BTZ-induced peripheral neurotoxicity (BiPN) are still unknown. In our study, we investigated in vivo and in vitro possible pathogenic events relevant to BiPN using a well-established rat model, with particular reference to the extent of proteasome inhibition and the effects on α-tubulin polymerization in sciatic nerves and dorsal root ganglia specimens obtained from animals treated with chronic regimens at a dose of 0.2 mg/kg intravenously. The same assessments were also performed after a single injection. Moreover, these studies were replicated in vitro using embryonic DRG neurons exposed to 100 nM BTZ and adult DRG neurons exposed to 10–50 nM BTZ for 24 h and 48 h. A significant increase in the polymerized fraction of α-tubulin and prolonged proteasome inhibition were observed after the chronic BTZ treatment in vivo. Recovery to physiological levels was observed after a 4-week follow-up post-treatment period. Proteasome inhibition and increased α-tubulin polymerization were also observed following BTZ treatment of both embryonic and adult DRG neurons in vitro. Our in vivo results suggest that proteasome inhibition and alteration of tubulin dynamics contribute to BiPN. The in vitro systems here described reliably replicate the in vivo results, and might therefore be used for further mechanistic studies on the effects of proteasome inhibitors on neurons.