Immunoproteasome Subunit LMP7 Research Articles

Immunoproteasomal Processing of IsoLG-Adducted Proteins Is Essential for Hypertension.

Hypertension is characterized by CD8+ (cluster differentiation 8) T cell activation and infiltration into peripheral tissues. CD8+ T cell activation requires proteasomal processing of antigenic proteins. It has become clear that isoLG (isolevuglandin)-adduced peptides are antigenic in hypertension; however, IsoLGs inhibit the constitutive proteasome. We hypothesized that immunoproteasomal processing of isoLG-adducts is essential for CD8+ T cell activation and inflammation in hypertension. IsoLG adduct processing was studied in murine dendritic cells (DCs), endothelial cells (ECs), and B8 fibroblasts. The role of the proteasome and the immunoproteasome in Ang II (angiotensin II)-induced hypertension was studied in C57BL/6 mice treated with bortezomib or the immunoproteasome inhibitor PR-957 and by studying mice lacking 3 critical immunoproteasome subunits (triple knockout mouse). We also examined hypertension in mice lacking the critical immunoproteasome subunit LMP7 (large multifunctional peptidase 7) specifically in either DCs or ECs. We found that oxidant stress increases the presence of isoLG adducts within MHC-I (class I major histocompatibility complex), and immunoproteasome overexpression augments this. Pharmacological or genetic inhibition of the immunoproteasome attenuated hypertension and tissue inflammation. Conditional deletion of LMP7 in either DCs or ECs attenuated hypertension and vascular inflammation. Finally, we defined the role of the innate immune receptors STING (stimulator of interferon genes) and TLR7/8 (toll-like receptor 7/8) as drivers of LMP7 expression in ECs. These studies define a previously unknown role of the immunoproteasome in DCs and ECs in CD8+ T cell activation. The immunoproteasome in DCs and ECs is critical for isoLG-adduct presentation to CD8+ T cells, and in the endothelium, this guides homing and infiltration of T cells to specific tissues.

Abstract 054: Role Of The Immunoproteasome In Hypertension-associated Immune Activation

Background: Inflammation is essential for the development of hypertension (HTN), however the mechanisms underlying this process remain unclear. The immunoproteasome processes intracellular peptides for display within class I major histocompatibility complexes (MHC-I). LMP7 is the chymotrypsin subunit of the immunoproteasome. Isolevuglandins (isoLGs) are reactive oxygen species (ROS) that drive immune activation and contribute to the development of HTN. Methods: IsoLG adduct-MHC-I interaction was measured by fluorescence resonance energy transfer. Mice with LoxP sites flanking exons 1 and 2 of Psmb8 , the gene encoding the immunoproteasome subunit LMP7, were generated and crossed to mice expressing dendritic cell (DC) and endothelial cell (EC) specific cre-recombinase. Mice were treated with angiotensin II as a model of HTN and blood pressure was measured by radiotelemetry. Tissue inflammation was measured by flow cytometry. In vitro , DCs, ECs, and B8 fibroblasts were used to study IsoLG adduct processing. Mouse aortic ECs (MAECs) were studied in models of isoLG-mediated T cell proliferation, gene expression, and antigen presentation. Results: Overexpression of immunoproteasome subunits augments isoLG-adduct MHC-I presentation. Oxidative stress induces IsoLG adduct MHC-I presentation by ECs and DCs. EC-specific IsoLG adduct MHC-I presentation contributes to hypertensive T-cell proliferation. Conditional deletion of LMP7 in DCs attenuated both tissue inflammation and HTN (142.3 ± 13.3 mmHg in Psmb8 fl/fl mice compared to 127.1 ± 9.5 mmHg in Psmb8 fl/fl /CD11c-Cre mice, N = 5-6, p=0.0363). Similarly, EC-specific LMP7 loss-of function attenuated inflammation and HTN (142.9 ± 3.4 mm Hg in Psmb8 fl/fl mice compared to 119.2 ± 2.6 mm Hg in Psmb8 fl/fl /vecadherin-Cre mice, N = 5, p= 0.0094). IsoLGs increase LMP7 expression and STING activation in MAECs. Conclusions: These studies define a critical role of the immunoproteasome in the regulation of immune cell activation and tissue infiltration in HTN. Moreover, they define a mechanism of isoLG adduct MHC-I presentation as a driver of this process. Finally, these studies define a critical role of antigen presentation in T cell activation, proliferation, and migration in HTN.

ONX0914 inhibition of immunoproteasome subunit LMP7 ameliorates diabetic cardiomyopathy via restraining endothelial-mesenchymal transition.

Diabetic cardiomyopathy (DCM) is a chronic metabolic disease with few effective therapeutic options. Immunoproteasome is an inducible proteasome that plays an important role in the regulation of many cardiovascular diseases, while its role in DCM remains under discussion. The present study aims to demonstrate whether inhibiting immunoproteasome subunit low molecular weight polypeptide 7 (LMP7) could alleviate DCM. Here, we established a type I diabetes mellitus mouse model by streptozotocin (STZ) in 8-week-old male wild-type C57BL/6J mice. We found that immunoproteasome subunit LMP7 was overexpressed in the heart of diabetic mice, while inhibiting LMP7 with pharmacological inhibitor ONX0914 significantly alleviated myocardial fibrosis and improved cardiac function. Besides, compared with diabetic mice, ONX0914 treatment reduced protein levels of mesenchymal markers (Vimentin, α-smooth muscle actin, and SM22α) and increased endothelial markers (VE-cadherin and CD31). In TGFβ1 stimulated HUVECs, we also observed that ONX0914 could inhibit endothelial-mesenchymal transition (EndMT). Mechanistically, we prove that ONX0914 could regulate autophagy activity both in vivo and vitro. Meanwhile, the protective effect of ONX0914 on TGFβ1 stimulated HUVECs could be abolished by 3-methyladenine (3MA) or hydroxychloroquine (CQ). All in all, our data highlight that inhibition of LMP7 with ONX0914 could ameliorate EndMT in diabetic mouse hearts at least in part via autophagy activation. Thus, LMP7 may be a potential therapeutic target for the DCM.

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.

Suppression of prostate cancer and amelioration of the immunosuppressive tumor microenvironment through selective immunoproteasome inhibition

ABSTRACT New treatment options to battle hormone-refractory prostate carcinoma (PC) are a pressing medical need. Chronic inflammation has been implicated in PC etiology. The pro-inflammatory cytokines IL-6, IL-23 and IL-17 are key mediators to promote growth of PC. Here, we evaluate the potential of immunoproteasome inhibition for anti-inflammatory and direct anti-tumorigenic therapy of PC. The anti-tumor effect of immunoproteasome inhibitor ONX 0914 was tested in mouse and human PC cells and the in vivo therapeutic efficacy of immunoproteasome inhibition was analyzed in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice in preventive and therapeutic settings and in castration-resistant (CR)PC after castration. Inhibition of the immunoproteasome subunit LMP7 induced apoptotic cell death in PC cell lines. In TRAMP mice, ONX 0914-treatment resulted in significant inhibition of PC growth with a decreased frequency of malignant prostatic lesions and inhibition of metastasis formation. The number of immunosuppressive myeloid cells in PC was greatly reduced in response to ONX 0914. Thus, immunoproteasome inhibition shows remarkable efficacy against PC progression in vivo and impedes tumor recurrence in CRPC-TRAMP mice by blocking the immunosuppressive inflammatory response in the tumor microenvironment. In conclusion, we show that the immunoproteasome is a promising drug target for the treatment of PC.

Translational PK/PD Modeling of Tumor Growth Inhibition and Target Inhibition to Support Dose Range Selection of the LMP7 Inhibitor M3258 in Relapsed/Refractory Multiple Myeloma.

M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, β5i, PSMB8) proteolytic subunit of the immunoproteasome, a component of the cellular protein degradation machinery, highly expressed in malignant hematopoietic cells including multiple myeloma. Here we describe the fit-for-purpose pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy modeling of M3258 based on preclinical data from several species. The inhibition of LMP7 activity (PD) and tumor growth (efficacy) were tested in human multiple myeloma xenografts in mice. PK and efficacy data were correlated yielding a free M3258 concentration of 45 nM for half-maximal tumor growth inhibition (KC50). As M3258 only weakly inhibits LMP7 in mouse cells, both in vitro and in vivo bridging studies were performed in rats, monkeys, and dogs for translational modeling. These data indicated that the PD response in human xenograft models was closely reflected in dog PBMCs. A PK/PD model was established, predicting a free IC50 value of 9 nM for M3258 in dogs in vivo, in close agreement with in vitro measurements. In parallel, the human PK parameters of M3258 were predicted by various approaches including in vitro extrapolation and allometric scaling. Using PK/PD/efficacy simulations, the efficacious dose range and corresponding PD response in human were predicted. Taken together, these efforts supported the design of a phase Ia study of M3258 in multiple myeloma patients (NCT04075721). At the lowest tested dose level, the predicted exposure matched well with the observed exposure while the duration of LMP7 inhibition was underpredicted by the model. SIGNIFICANCE STATEMENT: M3258 is a novel inhibitor of the immunoproteasome subunit LMP7. The human PK and human efficacious dose range of M3258 were predicted using in vitro-in vivo extrapolation and allometric scaling methods together with a fit-for-purpose PK/PD and efficacy model based on data from several species. A comparison with data from the Phase Ia clinical study showed that the human PK was accurately predicted, while the extent and duration of PD response were more pronounced than estimated.

Airway epithelial immunoproteasome subunit LMP7 protects against rhinovirus infection

Immunoproteasomes (IP) serve as an important modulator of immune responses to pathogens and other pathological factors. LMP7/β5i, one of the IP subunits, plays a critical role in autoimmune diseases by downregulating inflammation. Rhinovirus (RV) infection is a major risk factor in the exacerbations of respiratory inflammatory diseases, but whether LMP7 regulates RV-mediated inflammation in the lung particularly in the airway epithelium, the first line of defense against RV infection, remains unclear. In this study, we determined whether airway epithelial LMP7 promotes the resolution of RV-mediated lung inflammation. Inducible airway epithelial-specific LMP7-deficient (conditional knockout, CKO) mice were generated to reveal the in vivo anti-inflammatory and antiviral functions of LMP7. By using LMP7-deficient primary human airway epithelial cells generated by CRISPR-Cas9, we confirmed that airway epithelial LMP7 decreased pro-inflammatory cytokines and viral load during RV infection. Additionally, airway epithelial LMP7 enhanced the expression of a negative immune regulator A20/TNFAIP3 during viral infection that may contribute to the anti-inflammatory function of LMP7. We also discovered that induction of LMP7 by a low dose of polyinosinic:polycytidylic acid (PI:C) reduced RV-mediated inflammation in our CKO mice infected with RV. Our findings suggest that airway epithelial LMP7 has anti-inflammatory and antiviral functions that is critical to the resolution of RV-mediated lung inflammation. Induction of airway epithelial LMP7 may open a novel avenue for therapeutic intervention against RV infection.

Structure-Based Optimization and Discovery of M3258, a Specific Inhibitor of the Immunoproteasome Subunit LMP7 (β5i).

Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (β1, β2, and β5). LMP7 (β5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.

M3258 Is a Selective Inhibitor of the Immunoproteasome Subunit LMP7 (β5i) Delivering Efficacy in Multiple Myeloma Models.

Large multifunctional peptidase 7 (LMP7/β5i/PSMB8) is a proteolytic subunit of the immunoproteasome, which is predominantly expressed in normal and malignant hematolymphoid cells, including multiple myeloma, and contributes to the degradation of ubiquitinated proteins. Described herein for the first time is the preclinical profile of M3258; an orally bioavailable, potent, reversible and highly selective LMP7 inhibitor. M3258 demonstrated strong antitumor efficacy in multiple myeloma xenograft models, including a novel model of the human bone niche of multiple myeloma. M3258 treatment led to a significant and prolonged suppression of tumor LMP7 activity and ubiquitinated protein turnover and the induction of apoptosis in multiple myeloma cells both in vitro and in vivo Furthermore, M3258 showed superior antitumor efficacy in selected multiple myeloma and mantle cell lymphoma xenograft models compared with the approved nonselective proteasome inhibitors bortezomib and ixazomib. The differentiated preclinical profile of M3258 supported the initiation of a phase I study in patients with multiple myeloma (NCT04075721).

Comparative Assessment of the WNT/β-Catenin Pathway, CacyBP/SIP, and the Immunoproteasome Subunit LMP7 in Various Histological Types of Renal Cell Carcinoma

ObjectiveThe Wnt/ß-catenin pathway plays an important role in pathogenesis of variety cancers. Most studies on changes in WNT/β-catenin pathway in renal cell carcinoma (RCC) apply only to clear cell RCC, while there are no comparative assessments of this signaling pathway in various histological types of renal tumors in the available literature. Additionally, considering the close relationship between WNT/β-catenin signaling, CacyBP/SIP and proteasomal activity, it seemed worth comparing WNT/β-catenin pathway, CacyBP/SIP and LMP7 immunoproteasome subunit in human samples of clear cell, papillary, and chromophobe RCC.MethodsTests were performed on sections of three types of kidney tumors together with surrounding unchanged tissue fragments collected from 50 patients. Samples were divided into three groups depending on the histological type of cancer: clear cell, papillary and chromophobe RCC. Immunohistochemistry and PCR methods were used to identify WNT10A, Fzd5, β-catenin, GSK-3ß, CacyBP/SIP, LMP7, and gene expression.ResultsImmunoreactivity and expression of WNT10A, Fzd5, β-catenin, GSK-3ß, CacyBP/SIP, LMP7 in clear cell RCC was markedly increased compared to non-cancerous kidney tissue. In papillary RCC, immunoreactivity and expression of WNT/β-catenin pathway, CacyBP/SIP, LMP7 was also increased compared to non-malignant kidneys, but it was less pronounced than in clear cell RCC. The least substantial increase in immunoreactivity and expression of WNT/β-catenin pathway, CacyBP/SIP, LMP7 was found in chromophobe RCC, compared to other RCC histological subtypes studied.ConclusionsStudy results suggest an important role of WNT/β-catenin pathway, CacyBP/SIP and LMP7 in RCC carcinogenesis, and may indicate new aspects of pathomechanisms leading to differences in the biology of clear cell, papillary and chromophobe RCC.

Short-Term ONX-0914 Administration: Performance and Muscle Phenotype in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease. Although the lack of dystrophin protein is the primary defect responsible for the development of DMD, secondary disease complications such as persistent inflammation contribute greatly to the pathogenesis and the time-dependent progression of muscle destruction. The immunoproteasome is a potential therapeutic target for conditions or diseases mechanistically linked to inflammation. In this study, we explored the possible effects of ONX-0914 administration, an inhibitor specific for the immunoproteasome subunit LMP7 (ß5i), on motor performance, muscular pathology and protein degradation in 7-week old MDX mice, an age when the dystrophic muscles show extensive degeneration and regeneration. ONX-0914 (10 mg/kg) was injected subcutaneously on Day 2, 4, and 6. The mice were evaluated for physical performance (walking speed and strength) on Day 1 and 8. We show that this short-term treatment of ONX-0914 in MDX mice did not alter strength nor walking speed. The physical performance findings were consistent with no change in muscle inflammatory infiltration, percentage of central nuclei and proteasome content. Taken together, muscle structure and function in the young adult MDX mouse model are not altered with ONX-0914 treatment, indicating the administration of ONX-0914 during this critical time period does not exhibit any detrimental effects and may be an effective treatment of secondary complications of muscular dystrophy after further investigations.

Abstract DDT02-01: First-time disclosure of M3258: A selective inhibitor of the immunoproteasome subunit LMP7 with potential for improved therapeutic utility in multiple myeloma compared to pan-proteasome inhibitors

Abstract Large multifunctional peptidase 7 (LMP7, β5i, PSMB8) is a chymotrypsin-like proteolytic subunit of the immunoproteasome, which degrades ubiquitinated proteins and generates peptides for presentation on MHC class I. In contrast to the constitutive proteasome, which is broadly expressed, the immunoproteasome is specifically present in normal and malignant hematopoietic cells and can be induced in non-hematopoietic cells by inflammatory stimuli such as IFNγ. Pan-proteasome inhibitors like Bortezomib, which is approved in multiple myeloma and mantle cell lymphoma, indiscriminately inhibit the proteolytic activities of multiple subunits of the constitutive proteasome and immunoproteasome, including LMP7. Widespread proteasome inhibition is believed to underpin the adverse safety profiles of these agents and limit their therapeutic potential. Selective LMP7 targeting could achieve improved antitumor activity as a result of enhanced target inhibition, meanwhile circumventing the dose-limiting severe toxicities associated with pan-proteasome inhibitors. Based on the aforementioned hypothesis, a drug discovery program was initiated to identify selective inhibitors of LMP7. This led to the discovery of M3258, a covalent-reversible, potent, selective and orally-bioavailable inhibitor of LMP7. M3258 demonstrated strong in vivo antitumor activity, up to complete regression, in multiple myeloma xenograft models at daily oral doses as low as 1 mg/kg. This was associated with significant and prolonged suppression of tumor LMP7 activity. Furthermore, M3258 was efficacious in several multiple myeloma models that were refractory to Bortezomib. Subacute GLP toxicology studies with M3258, applied orally on a once-daily schedule, identified the lymphatic and hematopoietic systems in rat and dog and intestinal system in dog alone as main target organs. Importantly, M3258 was without effect on the peripheral and central nervous systems and cardiac and respiratory organs. Overall, M3258 demonstrated a superior preclinical therapeutic window and more restricted spectrum of toxicities compared to pan-proteasome inhibitors. Supported by robust preclinical data, clinical phase I assessment of M3258 in multiple myeloma patients is planned to begin in 2019. Citation Format: Michael Sanderson, Michael Busch, Christina Esdar, Manja Friese-Hamim, Mireille Krier, Jianguo Ma, Djordje Musil, Felix Rohdich, Willem Sloot, Gina Walter, Ugo Zanelli, Oliver Schadt, Markus Klein. First-time disclosure of M3258: A selective inhibitor of the immunoproteasome subunit LMP7 with potential for improved therapeutic utility in multiple myeloma compared to pan-proteasome inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr DDT02-01.

Comparative evaluation of CacyBP/SIP protein, β-catenin, and immunoproteasome subunit LMP7 in the heart of rats with hypertension of different etiology.

Despite extensive research into the pathogenesis of hypertension and disease-related end organ damage, the mechanisms leading to cardiac complications of hypertensive patients are still not fully elucidated. The aim of the presented research was immunodetection and evaluation of CacyBP/SIP, β-catenin, and proteasomes in the hearts of rats with hypertension of different etiology. Our results show an innovative and important network of interactions between proteins potentially involved in the development and progression of heart problems in various types of hypertension. This report might contribute to deeper understanding of the role of the CacyBP/SIP protein, β-catenin, and proteasomes in heart function. Our results might also bring new information concerning the intracellular processes and signal pathways involved in the regulation of cardiomyocytes functioning in hypertension state. In addition to cognitive significance, the results of presented studies may contribute to further successes in preventing and treatment of cardiac complications associated with hypertension.

Induction of the Immunoproteasome Subunit Lmp7 Links Proteostasis and Immunity in α-Synuclein Aggregation Disorders

Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders.

Expression of Immunoproteasome Subunit LMP7 in Breast Cancer and Its Association with Immune-Related Markers.

PurposeIn the presence of interferon, proteasome subunits are replaced by their inducible counterparts to form an immunoproteasome (IP) plays a key role in generation of antigenic peptides presented by MHC class I molecules, leading to elicitation of a T cell‒mediated immune response. Although the roles of IP in other cancers, and inflammatory diseases have been extensively studied, its significance in breast cancer is unclear.Materials and MethodsWe investigated the expression of LMP7, an IP subunit, and its relationship with immune system components in two breast cancer cohorts.ResultsIn 668 consecutive breast cancer cohort, 40% of tumors showed high level of LMP7 expression, and tumors with high expression of LMP7 had more tumor-infiltrating lymphocytes (TILs) in each subtype of breast cancer. In another cohort of 681 triple-negative breast cancer patients cohort, the expression of LMP7 in tumor cells was significantly correlated with the amount of TILs and the expression of interferon-associated molecules (MxA [p < 0.001] and PKR [p < 0.001]), endoplasmic reticulum stress-associated molecules (PERK [p=0.012], p-eIF2a [p=0.001], and XBP1 [p < 0.001]), and damage-associated molecular patterns (HMGN1 [p < 0.001] and HMGB1 [p < 0.001]). Patients with higher LMP7 expression had better disease-free survival outcomes than those with no or low expression in the positive lymph node metastasis group (p=0.041).ConclusionClose association between the TIL levels and LMP7 expression in breast cancer indicates that better antigen presentation through greater LMP7 expression might be associated with more TILs.

Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation

Chronic antibody-mediated rejection is the major cause of fading allograft function and loss after renal transplantation. Currently, pharmacological agents for the suppression of chronic antibody-mediated rejection are lacking. Non-selective proteasome inhibitors suppress antibody-mediated allograft rejection. However, extensive adverse side effects of these inhibitors severely limit their application. In contrast, immunoproteasome inhibition is effective in preclinical models of autoimmune diseases and was applied over weeks without obvious adverse side effects. ONX 0914, an immunoproteasome subunit LMP7 (β5i)-selective inhibitor, impeded the chronic rejection of kidneys transplanted from Fischer to allogeneic Lewis rats. ONX 0914 inhibited immunoproteasome induction both inimmune organs and renal allografts. Selective immunoproteasome inhibition reduced the numbers of B and plasma cells, and suppressed donor-specific alloantibody production. The infiltration of T cells, B cells and macrophages as well as interferon-γ, interleukin-17, IgG and complement deposition were reduced in renal allografts of ONX 0914-treated recipients. Chronic nephropathy was ameliorated and renal allograft function preserved, enabling long-term survival of recipients. Thus, our studies define a critical role of the immunoproteasome in chronic kidney allograft rejection and suggest immunoproteasome inhibition as a promising therapeutic approach to suppress chronic antibody-mediated rejection.

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the Tcell repertoire.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin-like activities suggesting distinct functions in thymic Tcell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP118-125 -specific Tcells although the class I mediated presentation of GP118-125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7-deficient CD8+ Tcells into RAG1-deficient mice we show that LMP7-deficient mice lacked GP118-125 -specific Tcell precursors and that LMP7 was required in radioresistant cells - most likely thymic epithelial cells - to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP118-125 -specific Tcell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic Tcell repertoire.

No prolongation of skin allograft survival by immunoproteasome inhibition in mice

The immunoproteasome, a distinct class of proteasomes, which is inducible under inflammatory conditions and constitutively expressed in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Moreover, inhibition of the immunoproteasome subunit LMP7 ameliorates clinical symptoms of autoimmune diseases in vivo and was shown to suppress the development of T helper cell (Th) 1 and Th17 cells and to promote regulatory T-cell (Treg) generation independently of its function in antigen processing. Since Th1 and Th17 cells are detrimental and Treg cells are critical for transplant acceptance, we investigated the influence of the LMP7-selective inhibitor ONX 0914 in a mixed lymphocyte reaction (MLR) in vitro as well as on allograft rejection in a MHC-disparate (C57BL/6 to BALB/c) and a multiple minor histocompatibility antigen (miHA)-disparate (B10.Br to C3H) model of skin transplantation in vivo. Although we observed reduced allo-specific IL-17 production of T cells in vitro, we found that selective inhibition of LMP7 had neither an influence on allograft survival in an MHC-mismatch model nor in a multiple minor mismatch skin transplantation model. We conclude that inhibition of the immunoproteasome is not effective in prolonging skin allograft survival in skin allotransplantation.

Inhibition and deficiency of the immunoproteasome subunit LMP7 suppress the development and progression of colorectal carcinoma in mice.

New treatment options and drug targets for colorectal carcinoma are a pressing medical need. Inflammation and pro-inflammatory cytokines produced by Th1 and Th17 cells like IL-6, TNF, IL-17 and IL-23 promote the development and growth of colorectal cancer (CRC). The immunoproteasome is a proteasome subtype highly expressed in immune cells but also in the intestine. Since the immunoproteasome promotes Th1 and Th17 differentiation and pro-inflammatory cytokine production, we investigated here whether deficiency or inhibition of the immunoproteasome subunit LMP7 would interfere with CRC development and exacerbation in preventive and therapeutic mouse models. Treatment with the LMP7 inhibitor ONX 0914 blocked tumor initiation and progression in either chemically-induced (AOM/DSS) or transgenic mouse models (ApcMin/+) of colon carcinogenesis. ONX 0914 treatment strongly reduced tumor numbers and CRC-associated loss of body weight while the survival rates were significantly enhanced. Moreover, genetic LMP7 deficiency markedly reduced the tumor burden in AOM/DSS induced wild type and ApcMin/+ mice. In conclusion, we show that the immunoproteasome is involved in CRC development and progression and we identify LMP7 as a new potential drug target for the treatment of CRC.

Prevention of colitis-associated cancer by selective targeting of immunoproteasome subunit LMP7.

Chronic inflammation is a well-known risk factor in development of intestinal tumorigenesis, although the exact mechanisms underlying development of colitis-associated cancer (CAC) still remain obscure. The activity and function of immunoproteasome has been extensively analyzed in the context of inflammation and infectious diseases. Here, we show that the proteasomal immunosubunit LMP7 plays an essential role in development of CAC. Mice devoid of LMP7 were resistant to chronic inflammation and formation of neoplasia, and developed virtually no tumors after AOM/DSS treatment. Our data reveal that LMP7 deficiency resulted in reduced expression of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3 as well as adhesion molecule VCAM-1. As a consequence, an impaired recruitment and activity of tumor-infiltrating leukocytes resulting in decreased secretion of cytokines IL-6 and TNF-α was observed. Further, the deletion or pharmacological inhibition of LMP7 and consequent blockade of NF-κB abrogated the production of IL-17A, which possesses a strong carcinogenic activity in the gut. Moreover, in vivo administration of the selective LMP7 inhibitor ONX-0914 led to a marked reduction of tumor numbers in wild-type (WT) mice. Collectively, we identified the immunoproteasome as a crucial mediator of inflammation-driven neoplasia highlighting a novel potential therapeutic approach to limit colonic tumorigenesis.