Expression Of Immunoproteasome Subunits Research Articles

Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes

BackgroundActivation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies.MethodsThis study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads.ResultsWe show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival.ConclusionsAltogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.

Immunoproteasome inhibition prevents progression of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment. This study aims to explore the effect and underlying mechanisms of immunoproteasome inhibition,a novel immunotherapy, on the progression of CRPC. The immunoproteasome subunit LMP7 was silenced by using gene knockout or inhibited by theepoxyketone inhibitor ONX 0914 in a mouse CRPC tumour graft model and in interferon-γ-pretreated human CRPC cell lines in vitro. CRPC tissues reveal a significant "tumour-elicited" Th17-type inflammatory response which induces immunoproteasome subunit expression. LMP7 deficiency in host mice or in CRPC tumour grafts had no effect on the "tumour-elicited" Th17-type inflammatory response and tumour progression. However, the selective LMP7 inhibitor ONX 0914 strongly suppressed the "tumour-elicited" Th17-type inflammatory response and CRPC tumour progression. Treatment of wild-type mice receiving LMP7-deficient CRPC tumour grafts with ONX 0914 further suggested that immunoproteasome inhibition prevents CRPC progression through suppressing IL-17-induced angiogenesis and epithelial-mesenchymal transition via inactivation of COX-2/VEGF-A signalling and β-catenin/Snail signalling. Treatment of LMP7-deficient mice receiving wild-type CRPC tumour grafts with ONX 0914 and inhibition of LMP7 in PC3 and 22Rv.1 cells with ONX 0914 showed that immunoproteasome inhibition also prevents CRPC progression through inducing CRPC cell apoptosis via activation of the unfolded protein response. We define a critical role of the immunoproteasome in CRPC and propose immunoproteasome inhibition as a promising therapeutic approach to suppress CRPC progression.

High Immunoproteasome Expression As Indicator for Sensitivity to Bortezomib-Containing Chemotherapy in Newly Diagnosed, Standard and Intermediate Risk, T-Cell Acute Lymphoblastic Leukemia Patients from Children's Oncology Group Trial AALL1231

Background: Proteasome inhibition by bortezomib (BTZ) has previously been shown to be beneficial for a subset of patients with relapsed/refractory acute leukemia. Several (pre)clinical studies showed that relatively high immunoproteasome expression (β1i and β5i) may serve as a biomarker for BTZ-sensitivity and aid to select patients that may respond well to BTZ-containing therapy (Cloos J et al. Cancer Metastasis Rev. 2017). In this study we investigated whether we could confirm these findings in newly diagnosed T-cell acute lymphoblastic leukemia patients (T-ALL) enrolled in the COG-AALL1231 phase 3 clinical trial (Teachey DT et al. J Clin Oncol 2022). In that trial, patients in treatment arm A received augmented Berlin-Frankfurt-Münster (aBFM) therapy, while for those in treatment arm B, BTZ was added to the aBFM regimen. Methods: Baseline immuno- and constitutive proteasome subunit expression was determined in bone marrow samples of 77 standard and intermediate risk T-ALL patients using Western blot. To correct for potential variation between blots, two cell lines were included as internal quantification controls, i.e. the human (BTZ-sensitive) T-ALL cell line CCRF-CEM (CEM-WT) and its subline (CEM-BTZ200) with acquired resistance to 200 nM BTZ. Protein bands were quantified and calculated relative to β-actin and the subunit expression of CEM-WT. Mann-Whitney U and log-rank tests were used for between group comparisons of proteasome subunit expressions and event free survival (EFS), respectively. Induction failure, relapse and death (induction and remission) were considered as EFS events. Results: In the bone marrow samples, a very strong correlation was observed between immunoproteasome β5i and β1i subunit expression (R=0.9, P<0.0001, Figure 1A). The correlation for constitutive subunits was R=0.7, P<0.0001. Interestingly, females had significantly higher β1i and β5i immunoproteasome levels compared to males with medians of 5.1 (range: 0.9-18.7) and 1.6 (0.3-17.9) respectively for the β1i subunit (P =0.05), and medians of 4.8 (1.9-16.1) and 1.8 (0.6-10.8) respectively for the β5i subunit (P =0.0084). Three year EFS was used as a measure for response to therapy. In patients treated with the BTZ-containing therapy (arm B), higher immunoproteasome expression levels (>0.8, based on Max-stat analysis) correlated with an improved EFS (P=0.034 for β1i and P=0.011 for β5i, Figure 1B). In contrast, in treatment arm A, higher immunoproteasome expression levels were associated with a relatively poor EFS, although this difference was not statistically significant likely due to low patient numbers in the low immunoproteasome subunit expression group in arm A (β1i N=5 and β5i N=1, for arm B β1i N=6 and β5i N=4). No other factors, including age, ethnicity, and gender, were found to be significantly associated with EFS. Discussion: These results support our hypothesis that patients with higher immunoproteasome expression levels are responsive to proteasome inhibitors such as BTZ, with better EFS after BTZ-containing therapy. When comparing the patients with high immunoproteasome expression of arm A versus arm B, the latter displays a better EFS (3 years EFS 96% for arm B vs 80% for arm A, P=0.08). Although not statistically significant, this factor may be indicative of a beneficial effect of BTZ-treatment in this subgroup of patients. The limitation of this study is the relatively low number of samples in each subgroup, in particular the patients with low expression of immunoproteasome subunits. Since most patients show a high level of immunoproteasome, only the few with low expression might not benefit from BTZ treatment and may be allocated for alternative strategies. The relevance of the immunoproteasome for BTZ sensitivity in these samples will be further examined by proteasome subunit catalytic activity measurements with subunit-specific substrates and other known factors contribution to diminished BTZ sensitivity (Niewerth et al. Drug Resist Updates 2015). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract P5-08-04: LMP7-specific inhibitor M3258 modulates the tumor microenvironment of aggressive breast cancer

Abstract Background: LMP7 (beta5i/PSMB8) is a proteolytic subunit of the immunoproteasome that is predominantly expressed in leukocytes and inflamed tissues. The immunoproteasome degrades ubiquitinated proteins for maintenance of cell homeostasis and generation of peptides for MHC class I presentation. LMP7 has been implicated in multiple myeloma, autoimmune and inflammatory diseases, and inflammation-related cancers. Here, we evaluated the effect of M3258, an orally bioavailable, highly-selective, and reversible inhibitor of LMP7, on the tumor microenvironment (TME) using in vitro and in vivo models of triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC). Methods: The proteolytic activity of M3258 was determined using the fluorogenic LMP7-specific peptidic substrate (Ac-ANW)2R110. Its anti-proliferation activity was determined using the CellTiter-Blue cell viability and sulforhodamine B staining assays. Expression of immunoproteasome subunits under different conditions was examined by Western blotting. All in vitro assessments were done with or without IFNγ, which is known to increase immunoproteasome expression. The in vivo anti-tumor efficacy of M3258 was evaluated using a SUM-149 PT TN-IBC xenograft mouse model implanted in humanized mice. Single-cell RNA sequencing (scRNA-seq) was used in this same study to examine the impact of M3258 on the TME. Results: M3258 inhibited LMP7 activity (IC50 = 6.5-212.8 nM) and had no effects on LMP7 protein expression in a panel of TNBC cells (DU4475, HCC1187), IBC cells (SUM190, IBC3, KPL4), and TN-IBC cells (SUM-149 PT, BCX-010, FC-IBC02). M3258 inhibited proliferation of TNBC and IBC cell lines in vitro (IC50 = 1-20 µM). In keeping with previous reports, IFNγ treatment strongly induced LMP7 expression and enhanced its proteolytic activity in these cell lines in vitro and increased M3258 sensitivity (2- to 5-fold). In the humanized SUM-149 PT xenograft mouse model, M3258 orally administered at 10 mg/kg significantly inhibited tumor growth versus vehicle (31.4%, P &amp;lt; 0.01) and was tolerated. ScRNA-seq analysis of the terminal tumor samples from this study revealed that M3258 reduced the abundance of CD4+ and CD8+ T cells, Tregs, M1 and M2 macrophages, and dendritic cells. Gene expression profiling indicated a particularly pronounced inhibitory effect of M3258 on M1 and M2 macrophages. Furthermore, M3258 treatment activated CD8+ T cells and suppressed expression of genes involved in inflammatory response, IFNα signaling, IFNγ signaling, and TNFα signaling in immune cells. In light of the scRNA-seq findings, we assessed the impact of M1 and M2 macrophages on the invasiveness of TN-IBC cells in vitro. Co-cultures with M1 or M2 macrophages enhanced invasiveness of SUM-149 PT cells (4.6-fold by M1, P &amp;lt; 0.001; 6.3-fold by M2, P &amp;lt; 0.01) and BCX-010 cells (2.1-fold by M1, P &amp;lt; 0.05; 4.0-fold by M2, P &amp;lt; 0.01). Since M2 macrophages showed a superior effect than M1 macrophages on TN-IBC cell invasiveness, we further assessed M3258’s ability to inhibit such activity. M3258 at 500 nM significantly inhibited M2 macrophage-induced invasion of SUM-149 PT cells (29.6%, P &amp;lt; 0.05) and BCX-010 (49.5%, P &amp;lt; 0.01). Conclusion: Using the exquisitely selective LMP7 inhibitor M3258, we were able to demonstrate for the first time that LMP7 plays an important role in the inflammatory microenvironment, proliferation and invasiveness of TNBC and IBC cells, in particular by modulating the pathogenic role of M2 macrophages. These data warrant future in vivo studies into the antitumor efficacy of M3258 when used with immunotherapy agents. Citation Format: Xuemei Xie, Jangsoon Lee, Ganiraju C Manyam, Troy Pearson, Gina Walter-Bausch, Manja Friese-Hamim, Samantha M Goodstal, Debu Tripathy, Wang Jing, Michael Sanderson, Naoto T Ueno. LMP7-specific inhibitor M3258 modulates the tumor microenvironment of aggressive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-04.

Expression of immunoproteasome subunits in the brains of Toxoplasma gondii-infected mice

The immunoproteasomes are specific proteasomes that clear oxidant-damaged proteins under inflammatory conditions in various diseases. Toxoplasma gondii (T. gondii) infects the central nervous system and causeencephalitis. However, the relationship between the immunoproteasomes and brain inflammation during T. gondii infection is not well characterized. In this study, we established an in vivo mouse model of T. gondii PLK strain infection via intraperitoneal injection and evaluated the expression of immunoproteasome subunits in the brains of infected mice. The results demonstrated that first, pathological changes in the brains of infected mice increase in severity over time. Second, following T. gondii infection, activated microglia and astrocytes undergo a series of functional alterations and morphological transformations, including proliferation and migration. Third, T. gondii infection induces expression of inflammatory cytokines, including IFN-γ, IL-1β, TNF-α, and IL-6. Fourth, the immunoproteasome subunits low-molecular-weight polypeptide 2 (LMP2), LMP7, and LMP10 mRNA and protein levels are significantly upregulated in T. gondii-infected mouse brains, as shown by RT-qPCR and western blot analysis, compared with that in vehicle-treated brains, and their expression is localized in the microglia, astrocytes, and neurons of T. gondii-infected brains, as determined via immunofluorescence staining. Furthermore, the western blot mean gray value for the immunoproteasome subunits and the positive microglia and astrocyte immunohistochemical signals in the brains of T. gondii-infected mice were positively correlated, indicating that the observed relationships were highly significant. Therefore, it was concluded that the induction of the immunoproteasomes is a pathogenic mechanism underlying T. gondii infection-induced inflammation.

Gallic Acid Ameliorates Angiotensin II-Induced Atrial Fibrillation by Inhibiting Immunoproteasome- Mediated PTEN Degradation in Mice.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and is a major cause of stroke and heart failure. We and others have found that gallic acid (GA) plays a beneficial role in cardiac hypertrophic remodeling and hypertension. However, the effect of GA on angiotensin II (Ang II)-induced AF and atrial remodeling as well as the underlying mechanisms remain unknown. AF was induced in mice by Ang II infusion (2000 ng/kg/min) for 3 weeks. Blood pressure was measured using the tail-cuff method. Atrial volume was evaluated by echocardiography. Atrial remodeling was studied using hematoxylin and eosin, Masson’s trichrome, and immunohistochemical staining. Atrial oxidative stress was assessed by dihydroethidium staining. The gene expression of fibrotic and inflammatory markers and protein levels of signaling mediators were measured by quantitative real-time PCR and western blot analysis. In mice, GA administration significantly attenuated Ang II-induced elevation of blood pressure, AF incidence and duration, atrial dilation, fibrosis, inflammation, and oxidative stress compared with the vehicle control. Furthermore, GA downregulated Ang II-induced activity and expression of immunoproteasome subunits (β2i and β5i), which reduced PTEN degradation and led to the inactivation of AKT1 and downstream signaling mediators. Importantly, blocking PTEN activity by VO-Ohpic markedly reversed the GA-mediated protective effects on Ang II-induced AF and atrial remodeling. Therefore, our results provide novel evidence that GA exerts a cardioprotective role by inhibiting immunoproteasome activity, which attenuates PTEN degradation and activation of downstream signaling, and may represent a promising candidate for treating hypertensive AF.

Age-Dependent Effects of Immunoproteasome Deficiency on Mouse Adenovirus Type 1 Pathogenesis.

Acute respiratory infection with mouse adenovirus type 1 (MAV-1) induces activity of the immunoproteasome, an inducible form of the proteasome that shapes CD8 T cell responses by enhancing peptide presentation by major histocompatibility complex (MHC) class I. We used mice deficient in all three immunoproteasome subunits (triple-knockout [TKO] mice) to determine whether immunoproteasome activity is essential for control of MAV-1 replication or inflammatory responses to acute infection. Complete immunoproteasome deficiency in adult TKO mice had no effect on MAV-1 replication, virus-induced lung inflammation, or adaptive immunity compared to C57BL/6 (B6) controls. In contrast, immunoproteasome deficiency in neonatal TKO mice was associated with decreased survival and decreased lung gamma interferon (IFN-γ) expression compared to B6 controls, although without substantial effects on viral replication, histological evidence of inflammation, or expression of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-1β in lungs or other organs. T cell recruitment and IFN-γ production was similar in lungs of infected B6 and TKO mice. In lungs of uninfected B6 mice, we detected low levels of immunoproteasome subunit mRNA and protein that increased with age. Immunoproteasome subunit expression was lower in lungs of adult IFN-γ-deficient mice compared to B6 controls. Together, these results demonstrate developmental regulation of the immunoproteasome that is associated with age-dependent differences in MAV-1 pathogenesis.IMPORTANCE MAV-1 infection is a useful model to study the pathogenesis of an adenovirus in its natural host. Host factors that control MAV-1 replication and contribute to inflammation and disease are not fully understood. The immunoproteasome is an inducible component of the ubiquitin proteasome system that shapes the repertoire of peptides presented by MHC class I to CD8 T cells, influences other aspects of T cell survival and activation, and promotes production of proinflammatory cytokines. We found that immunoproteasome activity is dispensable in adult mice. However, immunoproteasome deficiency in neonatal mice increased mortality and impaired IFN-γ responses in the lungs. Baseline immunoproteasome subunit expression in lungs of uninfected mice increased with age. Our findings suggest the existence of developmental regulation of the immunoproteasome, like other aspects of host immune function, and indicate that immunoproteasome activity is a critical protective factor early in life.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism

GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection. Cryopreserved primary human hepatocytes (PHH) and differentiated HepaRG (dHepaRG) cells were infected with HBV and treated with GS-9620, conditioned media from human peripheral blood mononuclear cells treated with GS-9620 (GS-9620 conditioned media [GS-9620-CM]), or other innate immune stimuli. The antiviral and transcriptional response to these agents was determined. GS-9620 had no antiviral activity in HBV-infected PHH, consistent with low level TLR7 mRNA expression in human hepatocytes. In contrast, GS-9620-CM induced prolonged reduction of HBV DNA, RNA, and antigen levels in PHH and dHepaRG cells via a type I interferon (IFN)-dependent mechanism. GS-9620-CM did not reduce covalently closed circular DNA (cccDNA) levels in either cell type. Transcriptional profiling demonstrated that GS-9620-CM strongly induced various HBV restriction factors - although not APOBEC3A or the Smc5/6 complex - and indicated that established HBV infection does not modulate innate immune sensing or signaling in cryopreserved PHH. GS-9620-CM also induced expression of immunoproteasome subunits and enhanced presentation of an immunodominant viral peptide in HBV-infected PHH. Type I IFN induced by GS-9620 durably suppressed HBV in human hepatocytes without reducing cccDNA levels. Moreover, HBV antigen presentation was enhanced, suggesting additional components of the TLR7-induced immune response played a role in the antiviral response to GS-9620 in animal models of CHB. GS-9620 is a drug currently being tested in clinical trials for the treatment of chronic hepatitis B virus (HBV) infection. GS-9620 has previously been shown to suppress HBV in various animal models, but the underlying antiviral mechanisms were not completely understood. In this study, we determined that GS-9620 does not directly activate antiviral pathways in human liver cells, but can induce prolonged suppression of HBV via induction of an antiviral cytokine called interferon. However, interferon did not destroy the HBV genome, suggesting that other parts of the immune response (e.g. activation of immune cells that kill infected cells) also play an important role in the antiviral response to GS-9620.

Expression of immunoproteasome subunits in the porcine lung: Alterations during normal and inflammatory conditions

The elimination of infected cells by cytotoxic T lymphocytes (CTLs) occurs through interactions between T cell receptors (TCRs) and pathogen-derived antigenic peptide-major histocompatibility complex (MHC) class I complexes. The immunoproteasome (i-proteasome), which is a large proteolytic machine derived from the constitutive proteasome, is highly efficient at processing antigens for presentation on MHC class I molecules to activate CD8+ T lymphocytes; this in turn facilitates antiviral adaptive immune responses. To date, i-proteasome expression in the porcine lung has not been investigated. In this study, we systematically analyzed the expression of the i-proteasome in vivo in the porcine lung and in vitro in alveolar macrophages (AMs) under normal and inflammatory conditions such as with IFN-γ stimulation or PRRSV infection. AMs were shown to readily express low levels of i-proteasome subunits, which were confined to the cytoplasm and nucleus under normal conditions. While i-proteasome expression could also be detected in other lung parenchymal cells including alveolar type I and II cells and bronchial epithelial cells during inflammatory conditions. Results showed that i-proteasome expression is markedly increased in IFN-γ-stimulated AMs and PRRSV-infected lung tissue. In addition, PRRSV infection promoted i-proteasome expression in AMs during the early stage of infection, and this was independent of IFN-γ; expression was attenuated during the later stage of infection in vitro. These results suggested that i-proteasome subunit expression can be induced in the porcine lung, which facilitates the development of antiviral adaptive immune responses against intracellular infections. These results could facilitate the development of therapeutics that target intracellular pathogens.

The immunoproteasome is induced by cytokines and regulates apoptosis in human islets.

In addition to degrading misfolded and damaged proteins, the proteasome regulates the fate of cells in response to stress. The role of the proteasome in pro-inflammatory cytokine-induced human beta-cell apoptosis is unknown. Using INS-1, INS-1E and human islets exposed to combinations of IFNγ, IL-1β and TNFα with or without addition of small molecules, we assessed the role of the immunoproteasome in pancreatic beta-cell demise. Here, we show that cytokines induce the expression and activity of the immuno-proteasome in INS-1E cells and human islets. Cytokine-induced expression of immuno-proteasome subunits, but not activity, depended upon histone deacetylase 3 activation. Inhibition of JAK1/STAT1 signaling did not affect proteasomal activity. Inhibition of the immuno-proteasome subunit PSMB8 aggravated cytokine-induced human beta-cell apoptosis while reducing intracellular levels of oxidized proteins in INS-1 cells. While cytokines increased total cellular NFκB subunit P50 and P52 levels and reduced the cytosolic NFκB subunit P65 and IκB levels, these effects were unaffected by PSMB8 inhibition. We conclude that beta cells upregulate immuno-proteasome expression and activity in response to IFNγ, likely as a protective response to confine inflammatory signaling.

Subversion of immunoproteasome subunit expression in dengue virus serotype 2-infected HepG2 cells.

Infection with all serotypes of dengue virus (DV) results in augmented antigen presentation by MHC class I molecules. However, the upregulation of immunoproteasome subunits only results from infection with two serotypes. This study aims to elucidate changes in the expression of immunoproteasome subunits resulting from infection with DV, particularly DV serotype 2 (DV2). HepG2 cells were grown in various culture milieu. Total cellular RNA and proteins were extracted and quantified. Results demonstrated sequestration of immunoproteasome subunits LMP2 and LMP7 in DV2-infected cells. This study provides insights into the mechanisms underlying immune evasion by DV.

Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA

ABSTRACTOverexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2′-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.

Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits

Nonstructural protein 1 of tick-borne encephalitis virus activates the expression of immunoproteasome subunits

Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome

The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2'-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.

Protein oxidative damage in the hippocampus in a mouse model of acute hyperammonemia

Hepatic encephalopathy (HE) is a common neuropsychiatric complication of both chronic and acute liver failure that is characterized by psychomotor, intellectual and cognitive impairment [1]. The mechanism of neuronal dysfunction in HE has not yet been conclusively investigated and possibly implies numerous pathological processes. It is widely accepted that among the factors contributing to HE, hyperammonemia, due to ammonia dysmetabolism and inflammation as a response to infection, play a major role in the pathogenesis. A growing body of evidence, e.g. RNA oxidation and protein nitrosylation, further indicate that oxidative stress occurs in neural cells as a result of ammonia intoxication and may lead to subsequent tissue damage [2,3]. Particular interest of this study was to elucidate the effect of an acute ammonia load on protein homeostasis in the hippocampus, the brain area of learning, memory formation and consolidation. To this end we analysed the accumulation of oxidized proteins, protein ubiquitination, proteasome activity and expression of immunoproteasome subunits after a single intraperitoneal injection of NH4Ac (10 mmol/kg body weight) in 6 weeks old C57B/6 mice. Following ammonia administration the animals fall into coma with a survival rate of 10%. After approximately one hour of coma, the motor ability and reflexes were restored. To analyse whether ammonia intoxication has a delayed effect on CNS tissue, animals were sacrificed 24 hours post injection, perfused with PBS and hippocampi were dissected. Western blot detection of protein carbonylation by OxiBlot technique revealed a significant accumulation of oxidized proteins in the hippocampus of animals with ammonia-precipitated encephalopathy compared to the saline-treated control group. Involvement of oxidative stress in disturbed protein homeostasis was further supported by detection of increased expression of the antioxidant enzymes hemeoxygenase 1 (HO-1) and thioredoxin 1 (Trx1). Similar to the animal model used in this study, up-regulation of HO-1 has been previously described in CNS tissue of patients with HE [4]. Moreover we found that protein oxidation was associated with elevated levels of ubiquitinated proteins and activation of proteasome (chemotrypsin-like) activity, representing the major cellular machinery for degradation of misfolded or damaged proteins. These data indicate that the ubiquitin-proteasome system may play an important role in the cellular response to ammonia-induced protein damage. To analyse whether hyperammonemia has a direct effect on neural cells, in an in vitro approach we exposed mixed glial cultures and the BV2 microglial cell line to 5 mM NH4Cl. Here, comparable to our findings in animals with ammonia-induced encephalopathy, 24 hours treatment increased the total amount of oxidized and ubiquitinated proteins, proteasome activity and expression of antioxidant enzymes. Notably, ammonia-induced protein damage was associated with a diminished functional role of microglia in terms of myelin debris clearance as it was observed using a myelin phagocytosis assay. Treatment with the antioxidant compound N-acetylcysteine rescued the effect of ammonia on impaired myelin engulfment and accumulation of ubiquitinated proteins. Taken together, our data show that ammonia intoxication induces protein oxidation in hippocampal tissue. This result corresponds to findings of other groups indicating impaired long-term potentiation in the hippocampus of animals with hyperammonemia and impaired cognitive function. We observed that an alteration of the protein homeostasis may persist longer after initial onset of neurological deterioration and thus, may contribute to a delayed effect of ammonia toxicity on neuronal activity. Upregulation of anti-oxidant enzymes in both mouse experimental hyperammonemia and patients with HE indicates that similar oxidative processes may underlie the CNS tissue damage. Induction of proteasome activity observed in this study may have a crucial role in the cellular adaptation in response to disturbed protein homeostasis. Moreover, our data indicate that hyperammonemia alone is able to affect microglia (the brain’s innate immune cells) and may have a negative impact on homeostatic and/or inflammatory function of microglia during the course of hepatic encephalopathy.

Antileukemic activity and mechanism of drug resistance to the marine Salinispora tropica proteasome inhibitor salinosporamide A (Marizomib).

Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome β-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50 = 5.1 nM), whereas their bortezomib-resistant sublines were 9- and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit β5-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. β-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conserved mechanism of resistance to proteasome inhibitors.

Anti-leukemic activity and mechanisms underlying resistance to the novel immunoproteasome inhibitor PR-924

PR-924 is a novel prototypic immunoproteasome inhibitor bearing markedly enhanced specificity for the β5i immunoproteasome subunit, compared to the classical proteasome inhibitor bortezomib. Here, we assessed the growth inhibitory potential of PR-924 in three human hematologic malignancy cell lines (CCRF-CEM, THP1, and 8226) and their bortezomib-resistant sublines. Parental cells displayed equal sensitivity to PR-924 (IC50: 1.5–2.8μM), whereas their bortezomib-resistant tumor lines displayed a 10–12 fold cross-resistance to PR-924. However, PR-924 cross-resistance factors for bortezomib-resistant sublines were markedly lower compared to the resistance factors to bortezomib. Proteasome inhibition experiments confirmed that PR-924 specifically inhibited β5i activity, even far below concentrations that exerted anti-proliferative activity. We further determined whether PR-924 activity might be compromised by acquisition of drug resistance phenomena. Indeed, CEM cells rendered stepwise resistant to 20μM PR-924 (CEM/PR20) displayed 13-fold PR-924-resistance and 10-fold cross-resistance to bortezomib. CEM/PR20 cells were devoid of mutations in the PSMB8 gene (encoding β5i), but acquired Met45Ile mutation in the PSMB5 gene (encoding constitutive β5), consistent with β5 mutations observed in bortezomib-resistant cells. Furthermore, compared to parental CEM cells, CEM/PR20 cells exhibited 2.5-fold upregulation of constitutive proteasome subunit expression, whereas immunoproteasome subunit expression was 2-fold decreased. In conclusion, PR-924 displayed potent anti-leukemic activity including toward bortezomib-resistant leukemia cells. Despite the specificity of PR-924 to the β5i immunoproteasome subunit, its anti-leukemic effect required concentrations that blocked both β5 and β5i subunits. This is underscored by the emergence of mutations in PSMB5 rather than in PSMB8.

The Novel Immunoproteasome Inhibitor PR-924: Anti-Leukemic Activity and Mechanisms Of Resistance

Proteasome inhibition with bortezomib (BTZ) is an effective treatment for hematological malignancies and an emerging treatment strategy for acute leukemia. However, the side-effects of BTZ and emergence of BTZ-resistance call for alternative therapeutic approaches. The immunoproteasome may serve as a candidate since its expression is higher than constitutive proteasome expression in cells of hematological malignancies, including acute lymphocytic leukemia (ALL). To this end, the new immunoproteasome inhibitor PR-924 may elicit anti-leukemic activity, since it bears greater specificity for the β5i subunit of the immunoproteasome compared to other proteasome inhibitors including BTZ and carfilzomib. The aim of the current study was to examine the proteasome inhibition capacity and cell growth inhibitory impact of PR-924 in acute leukemia cells and sublines with acquired resistance to BTZ. We further determined whether PR-924 itself would be prone to resistance development and if so, to explore the underlying molecular basis. To assess the anti-leukemic activity of PR-924, its cytotoxicity was determined in two human cell lines of hematological origin: the T-ALL cell line CCRF-CEM and the acute myeloid leukemia cell line THP1, and their 150-fold BTZ-resistant sublines CEM/BTZ200, and THP1/BTZ200, respectively. Parental CEM and THP1 cells displayed similar sensitivity to PR-924 (IC50 CEM: 1.8 µM ± 0.4, THP1: 1.5 µM ± 0.2), whereas their BTZ-resistant lines displayed a moderate 10-12 fold cross-resistance to PR-924 (IC50 CEM/BTZ200: 21.1 µM ± 0.5, THP1/BTZ200: 15.6 µM ± 1.6). Flow cytometric analysis revealed that PR-924-induced cell death was mediated by induction of apoptosis. Moreover, PR-924 exposure resulted in a (up to 50%) reduction of cell surface expression of HLA Class I.To determine whether PR-924 activity relies on specific inhibition of β5i, proteasome activity inhibition experiments were performed over a range of 10 nM – 10 µM PR-924. Notably, in parental cells, β5i activity was already inhibited by 20% at 10 nM PR-924 and > 90% inhibition was achieved at 100 nM PR-924. At higher PR-924 concentrations of 1-10 µM, inhibition of both β5 and β1i activities was observed, thus indicating that PR-924 blocks the β5i activity far below concentrations that exert anti-proliferative activity (1.5 µM), consistent with earlier studies of PR-924 (Parlati et al. Blood, 2009).Next, acquired resistance to PR-924 was provoked in CEM and THP1 cells by step-wise increasing PR-924 concentrations in cell culture, starting at the IC50 concentrations. Following this process, CEM cells resistant to 20 µM PR-924 (CEM/PR20) and THP1 cells resistant to 12 mM PR-924 (THP1/PR12) exhibited IC50 values of 22.1 µM PR-924 (resistance factor 13) and 14.3 µM PR-924 (resistance factor 10), respectively. In addition, these PR-924 resistant cells displayed 10-fold cross-resistance to BTZ. To explore mechanisms of PR-924 resistance, we first sequenced exon 2/3 of the PSMB8 gene (encoding β5i), a similar functioning coding region that was previously reported to harbor PSMB5 mutations in BTZ-resistant leukemia cells (Franke et al. Leukemia, 2012). However, no mutations were found in PSMB8 exon 2/3. Remarkably, we did identify mutations in exon 2 of the PSMB5 gene (encoding the S1 pocket of β5) in both CEM/PR20 (Met45Ile) and of THP1/PR12 (Ala49Thr); these amino acid substitutions are similar to those observed in BTZ-resistant sublines. Finally, we characterized proteasome subunit expression by ProCISE analysis in the PR-924 resistant cell lines. This method is a subunit-specific active-site ELISA assay, which utilizes a purified proteasome standard curve to calculate ng of subunit per µg total protein. Expression of constitutive proteasome subunits in CEM/PR20 and THP1/PR8 cells were upregulated up to 2.5-fold, compared to their parental counterparts, whereas immunoproteasome subunit expression was moderately decreased (up to 2-fold) in CEM/PR20 and increased in THP1/PR8 (up to 1.6-fold).In conclusion, PR-924 displayed significant anti-leukemic activity. Although there was cross-resistance to BTZ, PR-924 retained activity in BTZ-resistant leukemia cells. Despite PR-924 specificity to the β5i subunit of the proteasome, its anti-leukemic effect requires concentrations that block both β5 and β5i subunits. This notion is underscored by emergence of acquired mutations in PSMB5 rather than in PSMB8. Disclosures:Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Anderl:Onyx: Employment.

Adenovirus E1A interacts directly with, and regulates the level of expression of, the immunoproteasome component MECL1

Proteasomes represent the major non-lysosomal mechanism responsible for the degradation of proteins. Following interferon γ treatment 3 proteasome subunits are replaced producing immunoproteasomes. Adenovirus E1A interacts with components of the 20S and 26S proteasome and can affect presentation of peptides. In light of these observations we investigated the relationship of AdE1A to the immunoproteasome. AdE1A interacts with the immunoproteasome subunit, MECL1. In contrast, AdE1A binds poorly to the proteasome β2 subunit which is replaced by MECL1 in the conversion of proteasomes to immunoproteasomes. Binding sites on E1A for MECL1 correspond to the N-terminal region and conserved region 3. Furthermore, AdE1A causes down-regulation of MECL1 expression, as well as LMP2 and LMP7, induced by interferon γ treatment during Ad infections or following transient transfection. Consistent with previous reports AdE1A reduced IFNγ-stimulated STAT1 phosphorylation which appeared to be responsible for its ability to reduce expression of immunoproteasome subunits.

Synaptic Proteins Linked to HIV-1 Infection and Immunoproteasome Induction: Proteomic Analysis of Human Synaptosomes

Infection of the central nervous system with human immunodeficiency virus type 1 (HIV-1) can produce morphological changes in the neocortical synaptodendritic arbor that are correlated with neurocognitive impairment. To determine whether HIV-1 infection influences the protein composition of human synapses, a proteomic study of isolated nerve endings was undertaken. Synaptosomes from frontal neocortex were isolated using isopyknic centrifugation from 19 human brain specimens. Purity and enrichment were assessed by measuring pre- and postsynaptic protein markers. Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to screen for proteins differentially expressed in HIV/AIDS. The concentrations of 31 candidate protein spots were potentially abnormal in HIV-infected decedents with HIV encephalitis and/or increased expression of immunoproteasome subunits. Immunoblots showed that the concentration of some of them was related to HIV-1 infection of the brain and immunoproteasome (IPS) induction. Synapsin 1b and stathmin were inversely related to brain HIV-1 load; 14-3-3ζ and 14-4-4ε proteins were higher in subjects with HIV-1 loads. Perturbed synaptosome proteins were linked with IPS subunit composition, and 14-3-3ζ was histologically colocalized with IPS subunits in stained neocortical neurons. Proteomics illustrates that certain human proteins within the synaptic compartment are involved with changes in the synaptodendritic arbor and neurocognitive impairment in HIV-1-infected people.