Caspase-like Proteasome Activities Research Articles

P-088: Indirubin-3’-monoxime acts as an alternative proteasome inhibitor and confers new regimens for the treatment of Multiple Myeloma

<h3>Background</h3> Multiple myeloma (MM) is still an incurable malignancy of plasma cells. Despite the therapeutic benefit of the proteasome inhibitor (PI) bortezomib, drug resistance still accounts for majority of tumor relapses and cancer-related death in MM patients. Thus, exploring the mechanism underlying BTZ resistance and investigating a novel treatment strategy are urgent requisites for clinical practise. Indirubin-3'-monoxime (Id-3) is an active ingredient of a traditional Chinese medicine. <h3>Method</h3> In the present study, we firstly investigated Id-3 effects on myeloma treatment in patient primary samples and myeloma cell lines. We also investigated the molecular mechanism of Id-3 treatment on MM cells via in vivo and in vitro study. <h3>Results</h3> We first reported that Id-3 is an effective cytotoxicity agentia against MM cells growth by inducing the MM cells apoptosis. Id-3 treatment also effectively induces the cell death in bortezomib-resistant MM cell lines and primary myeloma patient samples. Stringently, we found there is a synergistic anti-MM effect between Id-3 and bortezomib. The treatment with lower dosage combination of Id-3 and bortezomib exhibited a notably inhibition of the growth of myeloma cells both in xenograft mouse model and MM-PDX model. Mechanism study revealed that Id-3 treatment efficiently inhibited the chymotrypsin-like and caspase-like activity of proteasome in MM cells. The level of the proteasome 11s activator, PA200 (PSME4) and PA28y (PSME3) was significantly down-regulated both in mRNA and protein level. GEO datasets analysis showed that the high levels of PSME4 and PSME3 were over-expressed in relapsed patients and MM cell lines which correlated with the drug-resistance of MM cells. Kaplan-Meier analysis showed that patients with high-levels of PSME3 or PSME4 had the shortened survival. Knocking-down the level of PSME4 or PSME3 by shRNA noteworthy inhibited the cell growth and enhanced the sensitivity to bortezomib of MM cells. In vivo study also found that down-regulated the level of PSME3 or PSME4 can suppress the proliferation of MM cells and prolong the survival of MM-bearing mouse. <h3>Conclusions</h3> Taken together, our study supported that Id-3 is an effective agentia against the survival of MM cells both in bortezomib-sensitive and -resistance ones. We further identified that the 11s proteasome activators, PSME3 and PSME4, were down-regulated effectively by Id-3 treatment. High-levels of PSME3 or PSME4 were correlated with the inferior outcome of MM patients. Down-regulating PSME3 and PSME4 by Id-3 or shRNA could efficiently suppressed the growth of MM cells and improve the survival of MM-bearing mice. Our study provides the rational evidence for the clinical application of Id-3 in the treatment of MM.

LNA -Gapmer Antisense Oligonucleotides Targeting the Oncogenic Lncrna MALAT1 Trigger Anti-Multiple Myeloma Activity By Proteasome Blockade

Dysregulation of non-coding RNAs is emerging as a common feature of human cancer. While short non-coding RNAs have been extensively investigated in multiple myeloma (MM), the biological role and the therapeutic significance of aberrantly expressed long non-coding RNAs (lncRNAs) are yet undetermined. A proprietary custom annotation pipeline of microarray data on lncRNA expression in different plasma cell (PC) dyscrasias, including 20 MGUS, 33 smoldering MM, 170 MM, 36 PC leukemia patients and 9 healthy donors, revealed high expression of MALAT1 in MM PCs. On this finding, we investigated the functional significance of MALAT1 by using novel 16-mer LNA gapmeR synthetic ASOs, that trigger selective RNAse-H dependent degradation of MALAT1. LNA-gapmeR ASOs targeting MALAT1 inhibited proliferation and clonogenicity of MM cell lines (n=7) and patient PCs (n=5), triggering ER-stress response and apoptosis, even in the presence of bone marrow stromal cells, while sparing healthy peripheral blood mononuclear cells. Conversely, MALAT1-enforced expression by lentiviral vectors enhanced cell proliferation and migration, reverted the effect of anti-MALAT1 ASOs and activated major oncogenic signal transduction pathways, such as AKT and MAPK. In vivo, i.p. administration of anti-MALAT1 gapmeRs inhibited the growth of human MM xenografts, with no evidence of organ toxicity. Microarray gene expression profiling indicated that MALAT1 depletion downregulated genes encoding for 20S proteasome β-subunits, as evidenced by gene set enrichment analysis (GSEA).Down-modulation of PSMβ2/4/5/7 mRNA and protein by anti-MALAT1 ASOs was confirmed by qPCR and Western blot respectively, and was associated to reduced trypsine-, chymotrypsine- and caspase-like proteasome activities and to the accumulation of polyubiquitylated proteins. Mechanistically, we demonstrated that proteasome inhibition upon MALAT1 knock-down was due to downmodulation of the major transcriptional activators of proteasome gene expression NRF1 and NRF2. At the same time, NRF1 was found to bind to and transactivate MALAT1 promoter, as demonstrated by chromatin immunoprecipitation and reporter assays, thus establishing a positive feedback-loop with MALAT1. Intriguingly, this loop was overactivated in bortezomib-resistant MM cells. Collectively, our findings highlight an unprecedented role of the lncRNA MALAT1 in the regulation of the proteasome machinery, and provide the first preclinical rationale for using ASO therapeutics targeting dysregulated lncRNAs for MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Effects of the selective inhibition of proteasome caspase-like activity by CLi a derivative of nor-cerpegin in dystrophic mdx mice.

Previous studies have shown that proteasome inhibition can have beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of a new selective proteasome inhibitor, CLi, a strong caspase-like inhibitor of the 20S proteasome, on skeletal and cardiac muscle functions of mdx mice. In the first series of experiments, five-month-old male mdx mice (n = 34) were treated with 2 different doses (20 and 100 μg/kg) of CLi and in the second series of experiments, five-month-old female mdx (n = 19) and wild-type (n = 24) mice were treated with 20 μg/kg CLi and Velcade (1 mg/kg) for 1-month. All animals were treadmill exercised twice a week to worsen the dystrophic features. In the first series of experiments, our results demonstrated that 20 μg/kg CLi did not significantly increase absolute and specific maximal forces in skeletal muscle from male mdx mice. Moreover, the higher susceptibility to contraction induced skeletal muscle injury was worsened by 100 μg/kg CLi since the force drop following lengthening contractions was increased with this high dose. Furthermore, we found no differences in the mRNA levels of the molecular markers implicated in dystrophic features. Concerning cardiac function, CLi had no effect on left ventricular function since ejection and shortening fractions were unchanged in male mdx mice. Similarly, CLi did not modify the expression of genes implicated in cardiac remodeling. In the second series of experiments, our results demonstrated an improvement in absolute and specific maximal forces by CLi, whereas Velcade only increased specific maximal force in female mdx mice. In addition, exercise tolerance was not improved by CLi. Taken together, our results show that CLi treatment can only improve maximal force production in exercised female mdx mice without affecting either exercice tolerance capacity or cardiac function. In conclusion, selective inhibition of caspase-like activity of proteasome with CLi has no compelling beneficial effect in dystrophic mdx mice.

Drugging the lncRNA MALAT1 via LNA gapmeR ASO inhibits gene expression of proteasome subunits and triggers anti-multiple myeloma activity

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still to be investigated. Here, we studied the functional significance and the druggability of the oncogenic lncRNA MALAT1 in MM. Targeting MALAT1 by novel LNA-gapmeR antisense oligonucleotide antagonized MM cell proliferation and triggered apoptosis both in vitro and in vivo in a murine xenograft model of human MM. Of note, antagonism of MALAT1 downmodulated the two major transcriptional activators of proteasome subunit genes, namely NRF1 and NRF2, and resulted in reduced trypsin, chymotrypsin and caspase-like proteasome activities and in accumulation of polyubiquitinated proteins. NRF1 and NRF2 decrease upon MALAT1 targeting was due to transcriptional activation of their negative regulator KEAP1, and resulted in reduced expression of anti-oxidant genes and increased ROS levels. In turn, NRF1 promoted MALAT1 expression thus establishing a positive feedback loop. Our findings demonstrate a crucial role of MALAT1 in the regulation of the proteasome machinery, and provide proof-of-concept that its targeting is a novel powerful option for the treatment of MM.

Abstract 20950: An Intrinsic Mechanism for a Gain in Proteasome Activity Preserving Cardiac Function

Cardiac remodeling and heart failure are characterized by misaligned adaptations in protein synthesis and degradation, harboring the therapeutic potential for interventional harmonization. Partial inhibition of protein degradation via the ubiquitin-proteasome system was reported to reduce acute hypertrophic remodeling as well as recover systolic function. The studies are suggestive of an inadequate gain in proteasome activity, contributing to cardiac remodeling and heart failure. Aim was to identify a common mechanism promoting proteasome activity during cardiac remodeling and heart failure and to test whether it contributes to the pathogenesis. Incorporation of the inducible proteasome subunit Lmp2 has been described to increase proteasome activity. In fact, it is increasingly expressed and incorporated in proteasome complexes during isoproterenol-induced hypertrophic remodeling, which is associated with a gain in proteasome activity. We found that Lmp2 is also increasingly expressed upon transverse aortic constriction and in a murine model of familial hypertrophic cardiomyopathy. Knock-out (KO) of Lmp2 in mice increased the caspase-like proteasome activity by 19% (n=5, p&lt;0.05), but had no apparent influence on cardiac morphology or function. As predicted, lack of Lmp2 confined the regulatory capacity of proteasomes to gain activity during hypertrophic remodeling induced by isoproterenol. Unexpectedly, the restriction in additional activity did not reduce cardiac remodeling as previously described for proteasome inhibition. In contrast, lack of Lmp2 exacerbated hypertrophic remodeling and caused loss of systolic function (fractional shortening: -32% vs. wildtype, n≥8, p&lt;0.01). Cardiomyocyte-specific expression of Lmp2 via gene transfer into adult Lmp2 KO mice completely rescued the isoproterenol-induced deterioration of cardiac remodeling and function. Interestingly, the deprived gain in proteasome activity in Lmp2 KO hearts rather affected a specific subset than the general pool of ubiquitinated proteins. In conclusion, a gain in proteasome activity during acute cardiac remodeling is not entirely maladaptive. At least in part, it is dependent on Lmp2, controlling remodeling and preserving cardiac function.

Wedelolactone Acts as Proteasome Inhibitor in Breast Cancer Cells.

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.

Proteasome and HDAC Inhibitor Therapy in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL): Nuclear Factor Erythroid 2 like 2 (NRF2)-Dependent Cell Death and Function

Background: Activation of NF-kB pathways are a hallmark feature of TCL and HL, making proteasome inhibition an attractive therapeutic target. Previous studies have demonstrated prominent in vivo efficacy of ixazomib, an oral proteasome inhibitor for both TCL and HL. Among the common significantly regulated genes identified via systems biology approach include upregulation of genes encoding for ubiquitin proteasome subunits (Ravi et al. Cancer Res. 2016). Further combination studies with HDACi belinostat were synergistic in Jurkat, HH and L428 cell lines, and NRF2 was discovered as mediator of proteasome gene expression. We hypothesized that HDACi abrogates NRF2 mediated proteasome recovery leading to synergistic effects on cell viability in combination with ixazomib.Methods: Global transcriptome analysis was performed on RNA isolated from multiple cell lines include TCL (Jurkat) and HL (L540, L428) treated with ixazomib and control, as well as Jurkat cell lines treated with single agent ixazomib, belinostat and combination. Significant genes were determined by applying a one-way ANOVA with an adjusted Bonferroni correction for a false discovery rate (FDR) < 0.05. Further pathway analysis from significant genes was performed by using a fold change greater than ±1.2 comparing all samples to each other and observing pathway relationships using Ingenuity Pathway Analysis. Gene Set Enrichment Analysis was performed with FDR <0.05 for functional analysis. Proteasome-Glo cell based assay was used to evaluate caspase-, chymotrypsin, and trypsin-like activity. Proteasome activity was measured at 24 and 72 hours after Jurkat cell lines were treated with control, ixazomib or belinostat as single agents, and in combination. SiRNA knockdown experiments were performed in Jurkat cell line with NRF2 and non-targeting (NT) SiRNA transfection. Real-time quantitative PCR (qPCR) for proteasome subunit and NRF2 genes was performed on RNA isolated from treated cells.Results: Transcriptome analysis revealed upregulation of proteasome genes in ixazomib treated cell lines Jurkat, L540 and L428 at 24 hours. In Jurkat TCL, ixazomib caused decreased caspase-like and chymotrypsin-like proteasome activity at 24 hours that was followed by recovery of these activities at 72 hours. The combination of ixazomib and belinostat significantly decreased proteasome activity for chymotrypsin-like, caspase-like and trypsin-like activity at 72 hours compared to single agent ixazomib or belinostat. In Jurkat cells, NRF2 was identified as a transcriptional regulator involved in proteasome gene regulation, showing upregulation of proteasomal genes and NRF2 with ixazomib single agent, downregulation with belinostat single agent and in combination with ixazomib. These results were confirmed with qPCR for NRF2 and proteasome genes in Jurkat and L428. SiRNA knockdown for NRF2 in Jurkat cells resulted in decreased cell viability, NRF2 and proteasome gene expression compared with NT SiRNA following ixazomib treatment.Conclusions: Treatment with single agent ixazomib induced prominent proteasome gene expression in all TCL and HL cell lines. In Jurkat, recovery of chymotrypsin and caspase-like proteasome activity occurred by 72 hours suggesting that transcriptional changes induced by proteasome inhibition contributed to proteasome function recovery. Combination therapy with belinostat resulted in downregulation of proteasome genes in Jurkat and L428 and prevented functional recovery of the proteasome observed in Jurkat. Our results suggest that targeting the proteasome itself with ixazomib and preventing the induced recovery of proteasome genes with belinostat contributes to synergistic effects observed on proteasome function and cell viability in TCL and HL. Further studies with CRISPR/Cas to confirm the effect of NRF2 on proteasome gene and functional recovery in the context of proteasome inhibition are ongoing and will be reported. DisclosuresEvens:Takeda: Other: Advisory board.

Pmd-107: Marizomib, Pomalidomide and Low Dose-Dexamethasone Combination Study in Relapsed/Refractory Multiple Myeloma (NCT02103335): Full Enrollment Results from a Phase-1 Multicenter, Open Label Study

Marizomib (MRZ) is a novel, irreversible, pan subunit proteasome inhibitor (PI) with preclinical evidence demonstrating in vitro and in vivo activity in multiple myeloma (MM). This study was designed to evaluate the safety and antimyeloma activity of pomalidomide (POM), MRZ and low dose dexamethasone (Lo-DEX) (PMD) in patients with relapsed and refractory multiple myeloma (RRMM).Thirty-eight heavily pretreated patients with RRMM were enrolled [dose-escalation cohort (n=14); recommended Phase 2 dose (RP2D) cohort (n=24)]. IV MRZ (0.3 to 0.5 mg/m2) was administered on Days (D) 1, 4, 8, 11; POM (3 or 4 mg) on D1 through 21; and Lo-DEX (5 or 10 mg) on D1, 2, 4, 5, 8, 9, 11, 12, 15, 16, 22, 23 of every 28-D cycle. Patients received a median of 4 (range 1-9) prior lines of therapy; 100% received prior lenalidomide (LEN) and bortezomib (BTZ), 34% carfilzomib (CFZ), and 50% thalidomide. 53% of patients were refractory to both LEN and BTZ and 21% were refractory to LEN, BTZ, and CFZ.There were no dose limiting toxicities during the study. The most common study treatment related ≥Grade 3 adverse events (AEs) were neutropenia (11/38 pts: 29%), pneumonia (4/38 pts 11%), anemia (4/38 pts; 11%), thrombocytopenia (4/38 pts; 11%), and febrile neutropenia (2/38 pts; 5%), with two grade 4 AEs (neutropenia related to POM and viral infection related to DEX), and one grade 5 AE (cardio-respiratory arrest from a suspected PE related to POM). Overall, MRZ was well tolerated, did not add to the incidence or severity of POM/Lo-DEX AEs and the regimen may have fewer hematological and infectious AEs compared to that observed with POM/Lo-DEX.MRZ pharmacokinetic analysis revealed that it was rapidly cleared with a short T1/2 (6.2-11mins) and a large volume of distribution (41-86L) suggesting extensive tissue distribution. Pharmacodynamic analysis demonstrated rapid and robust inhibition of chymotrypsin-like activity in both packed whole blood (PWB) and peripheral blood mononuclear cells (PMBCs), reflecting the irreversible binding nature of MRZ. Evolving inhibition of trypsin-like and caspase-like proteasome activity was also observed in PWB and PBMC with continued dosing.The overall response rate (ORR) and clinical benefit rate (CBR) for the 36 response evaluable patients was 53% (19/36) and 64% (23/36), respectively (Table 1). Subpopulation analysis demonstrated an ORR of 50% (5/10) in high risk cytogenetic patients, 56% (10/18) in LEN/BTZ refractory patients, 71% (5/7) in LEN/BTZ/CFZ refractory patients and 80% (8/10) in CFZ refractory patients. These data compare favorably against POM/Lo-Dex with a near doubling of ORR in both the total patient population and the double refractory patients. Substantial activity in high-risk patients that are triple refractory and in patients that are refractory to CFZ in prior last regimen was observed. MRZ activity in RRMM patients exposed and/or refractory to multiple PIs is likely a consequence of its unique pan proteasome subunit inhibitory actions. In conclusion, MRZ in combination with POM and Lo-DEX was well tolerated and demonstrated promising activity in heavily pretreated, high-risk RRMM patients. [Display omitted] DisclosuresHarrison:Janssen-Cilag: Research Funding, Speakers Bureau; Celgene: Honoraria. Zonder:Prothena: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Pharmacyclics: Other: DSMC membership. Khot:Amgen: Honoraria; Janssen: Consultancy; Pfizer: Speakers Bureau. Anderson:C4 Therapeutics: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; C4 Therapeutics: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. MacLaren:Triphase Accelerator: Employment, Equity Ownership. Reich:Triphase Accelerator: Consultancy. Trikha:Encycle Therapeutics: Consultancy, Equity Ownership; Triphase Accelerator: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Interferon-gamma activity is potentiated by an intracellular peptide derived from the human 19S ATPase regulatory subunit 4 of the proteasome

Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines. Here, the effect of immuneproteasome expression on the intracellular peptide profile was evaluated, and its functional significance was investigated. The expression of the immuneproteasome in HeLa cells was induced by interferon gamma treatment, and the relative concentrations of the intracellular peptides were compared to those of the control cells using isotope labeling and electron spray mass spectrometry. One of the peptides identified, VGSELIQKY (EL28), corresponds to amino acids 251–259 of the human 19S ATPase regulatory subunit 4. This peptide was increased in the extracts of HeLa cells that had been treated with interferon gamma compared to those of control cells. In vitro, EL28 increased the chymotrypsin, trypsin and caspase-like proteasome activities. In vivo, when covalently linked to a cell-penetrating peptide, EL28 potentiated the ability of interferon gamma to stimulate the expression of the immuneproteasome β5i subunit and to increase the proliferation of CD8+ T-cells. The EL28/cell-penetrating peptide construct also improved and positively modulated the secondary IgG anti-bovine serum albumin immune responsiveness elicited in high antibody-responder mice. Together, these results suggest that EL28 is a functional intracellular peptide that can potentiate interferon gamma activity. Biological significanceThe functional identification of EL28 advances our understanding regarding the bioactive peptides generated by limited proteolysis within cells.

Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells.

Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit.

Proteasome function is not impaired in healthy aging of the lung.

Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging.

Neuroprotective Effect of Demethylsuberosin, a Proteasome Activator, against MPP+-induced Cell Death in Human Neuroblastoma SH-SY5Y Cells

Demethylsuberosin isolated from the roots of Cudrania tricuspidata demonstrated a potent proteasome activator by enhancing all three chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in a 20S proteasome activity assay. It also attenuated the 1-methyl-4-phenylpyridinium-induced dysfunction of the chymotrypsin-like and caspase-like activities of proteasome in SH-SY5Y cells with EC 50 values of 0.76 µM and 0.82 µM, respectively. Additionally, demethylsuberosin protected neuronal cells against 1-methyl-4-phenylpyridinium-induced cell death with an EC 50 value of 0.17 µM, while the EC 50 value of betulinic acid was 4.29 µM. We are reporting that demethylsuberosin is a potent proteasome activator with a neuroprotective effect, suggesting a possible candidate for the protection or treatment of neurodegenerative diseases such as Parkinsonʼs disease.

Photosensitisation facilitates cross-priming of adjuvant-free protein vaccines and stimulation of tumour-suppressing CD8 T cells

Cancer vaccines aim to induce CD8 T cells infiltrating the tumour. For protein-based vaccines, the main biological barrier to overcome is the default MHC class-II-pathway, with activation of CD4 T cells rather than CD8 T cells. The latter requires antigens to access the cytosol and MHC class I antigen presentation. We applied photosensitiser and light to trigger disruption of antigen-containing endosomes and thereby MHC class I cross-presentation of a model cancer vaccine. This “photochemical internalisation” resulted in activation, proliferation, and IFN-γ production of cytotoxic CD8 T cells, which suppressed tumour growth by infiltrating CD8 T cells and caspase-3-dependent apoptosis. The process was independent of MHC class II, MyD88, and TLR4 signalling, but dependent on trypsin- and caspase-like proteasome activity and partly also on chloroquine. This novel method of vaccination may find applications in cancer immunotherapy where the activation of CD8 T cells is important.

Homogeneous, bioluminescent proteasome assays.

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits in embryonic and early postnatal development of rat spleen and liver is investigated in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The immune subunits LMP7 and LMP2 distribution in spleen and liver cells in the development process is also studied. A mutual for both organs tendency to the increase of the expression of both LMP7 subunit and LMP2 one on P21 (the 21st postnatal day) as compared to the embryonic period is discovered. However, the total proteasome level is shown to be constant. At definite development stages, the dynamics of immune subunits expression in the spleen and liver was different. In the spleen gradual enhancement of both immune subunits level being detected on P1, P18 and P21, in the liver gradual enhancement periods on E16 (the 16th embryonic day) and E18 changed to the stage of the shrink of immune subunits level on P5. This level did not reliably change till P18 and was augmented on P21. The alterations revealed were accompanied by chymotrypsin-like activity raise and caspase-like activity drop in spleen by P21 as compared with the embryonic period, which proves the enlargement of proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased by P21 in comparison with the embryonic period. Such dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Besides, it is discovered that the increment of immune subunits expression in the early spleen development is connected with the process of successive forming the white pulp by B- and T-lymphocytes enriched by immune subunits. In the liver, the growth of immune subunits level by P21 was accompanied by their expression expansion in hepatocytes, while their plunge by P5 may be related to the loss of liver function of a primary lymphoid organ of the immune system by this stage and disappearance of B-lymphocytes enriched by immune proteasomes in it. In the spleen and liver, MHC class I molecules were revealed at the periods of the raise of proteasome immune subunits level. On E21 , the liver was enriched by neuronal NO-synthase, its level decreased after birth and enhanced to P18. This fact indicates the possibility of the induction of the immune subunits LMP7 [character: see text] LMP2 expression in hepatocytes in signal way with neuronal NO-synthase participation. The results obtained prove that T-cell immune response with spleen participation as regards rat liver cells is possible starting with P19-P21 stage. First, at this period, white pulp T-area is formed in the spleen. Second, enhanced immune proteasomes and MHC class I molecules levels in hepatocytes can procure antigenic epitopes formation from foreign proteins and their delivery to cell surface for their subsequent presentation for cytotoxic T-lymphocytes.

Age-related and sex-specific differences in proteasome activity in individual Drosophila flies from wild type, longevity-selected and stress resistant strains

We have measured the caspase-like proteasome activity in individual male and female Drosophila flies by using a non-denaturing lysing technique that consistently extracts total protein. The mean proteasome activity in control C1 females was more than two times higher as compared with that in C1 males. However, in longevity-selected LS1 flies the proteasome activity was significantly lower compared to C1 flies, but the sex differences were maintained to some extent. Five other stress resistant lines also had significantly reduced proteasome activity in both sexes. During ageing, there was a progressive decrease in proteasome activity in C1 females, but not in C1 males. This age-related decline in proteasome activity observed in C1 females was not observed in LS1 flies. We conclude that the proteasome activity in control male and female flies is significantly different from each other and that increased lifespan and stress resistance lead to a reduction in proteasome activity and recession of the age-related decline observed in control females.

Proteasome-based mechanisms of intrinsic and acquired bortezomib resistance in non-small cell lung cancer

The proteasome inhibitor bortezomib, registered for Multiple Myeloma treatment, is currently explored for activity in solid tumors including non-small cell lung cancer (NSCLC). Here we studied the proteasome-based mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. Various NSCLC cell lines displayed differential intrinsic sensitivities to bortezomib. High basal chymotrypsin- and caspase-like proteasome activities correlated with bortezomib resistance in these cells. Next, via stepwise selection, acquired bortezomib resistant cells were obtained with 8–70-fold increased resistance. Cross-resistance was found to proteasome inhibitors specifically targeting β-subunits, but not to the novel α-subunit-specific proteasome inhibitor (5AHQ). Consistently, bortezomib-resistant cells required higher bortezomib concentrations to induce G2/M arrest and apoptosis. Interestingly, bortezomib concentration-dependent caspase cleavage, Mcl-1 and NOXA accumulation remained intact in resistant H460 and SW1573 cells, while A549 resistant cells displayed different expression profiles suggesting additional and more protein specific adaptations. Furthermore, bortezomib-resistant cells exhibited increased levels of both constitutive and immuno-β-subunits. Sequence analysis of the bortezomib-binding pocket in the β5-subunit revealed Ala49Thr, Met45Val and Cys52Phe substitutions that were not previously described in solid tumors. Bortezomib-resistant cells displayed reduced catalytic proteasome activities and required higher bortezomib concentrations to achieve comparable inhibition of proteasome activity. Taken together, these findings establish that high basal levels of proteasome activity correlate with intrinsic bortezomib resistance. Furthermore, acquired bortezomib resistance in NSCLC is associated with proteasome subunit overexpression and emergence of mutant β5-subunits that likely compromise bortezomib binding. α-Subunit-specific proteasome inhibitors, however, can efficiently bypass this resistance modality.

Bortezomib Targets the Caspase-Like Proteasome Activity in Cervical Cancer Cells, Triggering Apoptosis That Can be Enhanced by Nelfinavir

The occurrence of chemoresistance is a serious problem in the treatment of cancer, urging the need for second and third-line treatment options that rely on different cell death pathways to overcome previously acquired resistance mechanisms. The inhibition of proteasomal activity by specific proteasome inhibitors or cross-reactivity of certain protease inhibitors with proteasomal enzymes recently became of interest because of the anti-tumoral properties of these agents. We tested the proteasome inhibitor bortezomib and the HIV protease inhibitor nelfinavir on human cervical cancer cells. Both drugs induced cell cycle arrest in cervical cancer cells, as reflected by marked changes in the expression of cell cycle-regulatory cyclins and ensuing mitochondrial-independent apoptosis. Upregulation of the molecular chaperone BiP and the cell stress marker ATF3 indicated induction of the unfolded protein response (UPR) as the main cause of apoptosis induced by these drugs in cervical cancer cells. Unlike in leukemia cells, bortezomib mainly inhibited the caspase-like activity of the proteasome in cervical cancer cells. Nelfinavir exhibited no effects on proteasomal activity in cervical cancer cells and leukemia cells. Although both bortezomib and nelfinavir acted on cisplatin-resistant cervical cancer cells (SiHa), neither of the drugs induced a sensitization to cisplatin treatment. Instead, both drugs could effectively be combined with each other, and enhanced the efficacy of an apoptosis-inducing TRAIL receptor antibody. These results suggest that both bortezomib and nelfinavir are effective agents against chemoresistant cervical cancer cells and might be of interest for clinical studies on cervical cancer patients with recurrent or metastatic cancer.

Abstract 746: Acquired bortezomib resistance in non-small cell lung cancer is associated with overexpression of the mutated β5 proteasome subunit

Abstract The proteasome inhibitor bortezomib (Velcade®) is registered for the treatment of multiple myeloma, but has limited activity in solid tumors such as non-small cell lung cancer (NSCLC). Bortezomib predominantly inhibits proteasome subunit β5 and mutations in this subunit were recently associated with bortezomib resistance in leukemic cell line models. Herein we studied the molecular mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. H460, A549 and SW1573 NSCLC cells demonstrated differential intrinsic sensitivities towards bortezomib with IC50 values of 12.6, 9.0 and 2.1 nM, respectively. Basal proteasome activities in these cell lines measured by an intact cell proteasome activity assay revealed that high basal levels of chymotrypsin- and caspase-like proteasome activities strongly correlate with intrinsic bortezomib resistance (R2=0.99, P&amp;lt;0.05 and R2=0.99, P&amp;lt;0.06, respectively).We established a model for acquired bortezomib resistance in H460, A549 and SW1573 cells by exposing cells to gradually increasing concentrations of bortezomib starting from 5nM to 80 or 200 nM for H460, 40 or 100 nM for A549 as well as 30 or 150 nM for SW1573. These bortezomib-resistant cell lines, named H460BTZR80, H460BTZR200, A549BTZR40, A549BTZR100, SW1573BTZR30 and SW1573BTZR150 displayed 14 to 57-fold bortezomib resistance. Consistantly, bortezomib-resistant cells required higher bortezomib concentrations to induce a G2/M cell cycle arrest and activation of apoptosis. Overall activation of apoptosis was reduced even at higher bortezomib concentrations when compared to parental cells treated with equitoxic drug doses. Furthermore, bortezomib-resistant cells exhibited increased levels of both constitutive β-subunits and immuno-β-subunits. Moreover, sequence analyses of the bortezomib binding pocket encoded by exon 2 of the PSMB5 gene revealed a previously described Ala49Thr mutation and two newly identified Met45Val and Cys52Phe mutations. Nevertheless, alterations in the proteasome subunits did not affect basal catalytic proteasome activities, although higher bortezomib concentrations were required in bortezomib-resistant cells to achieve comparable inhibition levels of proteasome activity. Cross-resistance was also observed to other proteasome inhibitors that specifically target the β-subunits of the proteasome. Interestingly, no cross-resistance was found to 5AHQ, a novel non-competitive proteasome inhibitor that targets the α7-subunit. Taken together, these findings establish a correlation between bortezomib-sensitivity and low basal levels of proteasome activity. In addition, acquired resistance to bortezomib in NSCLC was associated with expression of mutant β5-subunits that may compromise bortezomib binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2011-746

Combined chemical and genetic approach to inhibit proteolysis by the proteasome

Regulated protein destruction by the proteasome is crucial for the maintenance of normal cellular homeostasis. Much of our understanding of proteasome function stems from the use of drugs that inhibit its activity. Curiously, despite the importance of proteasomal proteolysis, previous studies have found that proliferation of the yeast Saccharomyces cerevisiae is relatively resistant to the effects of proteasome inhibitors such as MG132, even in the presence of mutations that increase inhibitor levels in cells. We reasoned that part of the resistance of S. cerevisiae to proteasome inhibitors stems from the fact that most proteasome inhibitors preferentially target the chymotryptic activity of the proteasome, and that the caspase-like and tryptic sites within the 20S core could compensate for proteasome function under these conditions. To test this hypothesis, we generated a strain of yeast in which the gene encoding the drug efflux pump Pdr5 is deleted, and the tryptic and caspase-like proteasome activities are inactivated by mutation. We find that this strain has dramatically increased sensitivity to the proteasome inhibitor MG132. Under these conditions, treatment of yeast with MG132 blocks progression through the cell cycle, increases the accumulation of polyubiquitylated proteins and decreases the ability to induce transcription of certain genes. These results highlight the contribution of the caspase-like and tryptic activities of the proteasome to its function, and provide a strategy to potently block proteasomal proteolysis in yeast that has practical applications.