Subunits LMP7 Research Articles

Targeting Immunoproteasome in Polarized Macrophages Ameliorates Experimental Emphysema Via Activating NRF1/2-P62 Axis and Suppressing IRF4 Transcription.

Chronic obstructive pulmonary disease (COPD) stands as the prevailing chronic airway ailment, characterized by chronic bronchitis and emphysema. Current medications fall short in treatment of these diseases, underscoring the urgent need for effective therapy. Prior research indicated immunoproteasome inhibition alleviated various inflammatory diseases by modulating immune cell functions. However, its therapeutic potential in COPD remains largely unexplored. Here, an elevated expression of immunoproteasome subunits LMP2 and LMP7 in the macrophages isolated from mouse with LPS/Elastase-induced emphysema and polarized macrophages in vitro is observed. Subsequently, intranasal administration of the immunoproteasome-specific inhibitor ONX-0914 significantly mitigated COPD-associated airway inflammation and improved lung function in mice by suppressing macrophage polarization. Additionally, ONX-0914 capsulated in PLGA nanoparticles exhibited more pronounced therapeutic effect on COPD than naked ONX-0914 by targeting immunoproteasome in polarized macrophages. Mechanistically, ONX-0914 activated autophagy and endoplasmic reticulum (ER) stress are not attribute to the ONX-0914 mediated suppression of macrophage polarization. Intriguingly, ONX-0914 inhibited M1 polarization through the nuclear factor erythroid 2-related factor-1 (NRF1) and NRF2-P62 axis, while the suppression of M2 polarization is regulated by inhibiting the transcription of interferon regulatory factor 4 (IRF4). In summary, the findings suggest that targeting immunoproteasome in macrophages holds promise as a therapeutic strategy for COPD.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

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.

Immunohistochemical Identification and Assessment of the Location of Immunoproteasome Subunits LMP2 and LMP7 in Acquired Cholesteatoma.

Cholesteatoma, accompanied by chronic inflammatory response, is characterized by invasive growth and osteolytic activity. As specific proteasome isoforms, the immunoproteasomes serve as an important modulator of inflammatory responses. The aim of the present study was to determine the biological activity of cholesteatoma through the analysis of the expression and localization of immunoproteasome subunits of low molecule weight protein (LMP) 2 and LMP7. Cholesteatoma specimens were obtained from 15 adults who underwent ear surgery due to acquired attic cholesteatoma. Normal skin specimens were taken from retro-auricular skin incisions from the same patients. The specimens were stained with anti-LMP7 antibody, using immunohistochemistry techniques based on the binding of biotinylated secondary antibody with the enzyme-labeled streptavidin and the Envision FLEX system. In all specimens of cholesteatoma, the immunohistochemical reaction with the antibody against the LMP2 was positive, in both the cytoplasm of the cholesteatoma matrix and the perimatrix. A negative reaction with anti-LMP2 was observed in the cytoplasm and nuclei of control skin cells. A positive nuclear and cytoplasmic immunohistochemical reaction with anti-LMP7 has been demonstrated in numerous cells, in both the matrix and perimatrix of cholesteatoma. We present evidence of the presence of expressions of LMP2 and LMP7 within cholesteatoma tissue. Our results might bring new information concerning immunoproteasome-dependent pathophysiologic mechanisms in cholesteatoma.

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.

Adaptation of the Tumor Antigen Presentation Machinery to Ionizing Radiation.

Ionizing radiation (IR) can reprogram proteasome structure and function in cells and tissues. In this article, we show that IR can promote immunoproteasome synthesis with important implications for Ag processing and presentation and tumor immunity. Irradiation of a murine fibrosarcoma (FSA) induced dose-dependent de novo biosynthesis of the immunoproteasome subunits LMP7, LMP2, and Mecl-1, in concert with other changes in the Ag-presentation machinery (APM) essential for CD8+ T cell-mediated immunity, including enhanced expression of MHC class I (MHC-I), β2-microglobulin, transporters associated with Ag processing molecules, and their key transcriptional activator NOD-like receptor family CARD domain containing 5. In contrast, in another less immunogenic, murine fibrosarcoma (NFSA), LMP7 transcripts and expression of components of the immunoproteasome and the APM were muted after IR, which affected MHC-I expression and CD8+ T lymphocyte infiltration into NFSA tumors invivo. Introduction of LMP7 into NFSA largely corrected these deficiencies, enhancing MHC-I expression and invivo tumor immunogenicity. The immune adaptation in response to IR mirrored many aspects of the response to IFN-γ in coordinating the transcriptional MHC-I program, albeit with notable differences. Further investigations showed divergent upstream pathways in that, unlike IFN-γ, IR failed to activate STAT-1 in either FSA or NFSA cells while heavily relying on NF-κB activation. The IR-induced shift toward immunoproteasome production within a tumor indicates that proteasomal reprogramming is part of an integrated and dynamic tumor-host response that is specific to the stressor and the tumor and therefore is of clinical relevance for radiation oncology.

Interplay between proteasome function and inflammatory responses in e-cig vapor condensate-challenged lung epithelial cells.

Exposure to cigarettes and other nicotine-based products results in persistent inflammation in the lung. In recent years, electronic cigarettes (e-cigs) have become extremely popular among adults and youth alike. E-cigarette vapor-induced oxidative stress promotes protein breakdown, DNA damage and cell death, culminating in a variety of respiratory diseases. The proteasome, a multi-catalytic protease, superintends protein degradation within the cell. When cells are stimulated with inflammatory cytokines such as IFN-γ and TNF-α, the constitutive catalytic proteasome subunits are replaced by the inducible subunits-low-molecular mass polypeptide (LMP)2 (β1i), multi-catalytic endopeptidase complex-like (MECL)1 (β2i), and LMP7 (β5i), which are required for the production of certain MHC class I-restricted T-cell epitopes. In this study, we used human alveolar epithelial cells (A549) and exposed them to filtered air or (1%) tobacco-flavored (TF) electronic cigarette vapor condensate(ECVC) ± nicotine (6mg/ml) (TF-ECVC ± N) for 24h. We observed an increase in the levels of IFN-γ, TNF-α, and inducible proteasome subunits (LMP7/PSMB8, LMP2/PSMB9, MECL1/PSMB10), and a reduced expression of constitutive proteasome subunits (β1/PSMB6 and β2/PSMB7) in challenged A549 cells. Interestingly, knockdown of the inducible proteasome subunit LMP7 reversed ECVC-induced expression of NADPH oxidase and immunoproteasome subunits in A549 cells. In addition, pre-exposure to an LMP7 inhibitor (ONX-0914) abrogated the mRNA expression of several NOX subunits and rescued the excessive production/release of inflammatory cytokines/chemokines (IL-6, IL-8, CCL2, and CCL5) in ECVC-challenged cells. Our findings suggest an important role of LMP7 in regulating the expression of inflammatory mediators during ECVC exposure. Overall, our results provide evidence for proteasome-dependent ROS-mediated inflammation in ECVC-challenged cells.

Abstract 2997: Tumor hypoxia blocks the induction of antigen processing machinery

Abstract Antigen presentation is critical for adaptive immune response to cancer but is often subverted to evade immune response. While hypoxia is a pervasive feature of solid tumors, it’s role in tumor antigen processing remains understudied. To assess the effects of reduced oxygen on antigen processing machinery, we used interferon (IFN)-γ to induce the expression of the immunoproteasome (IP), a proteasome isoform critical for the tumor antigen processing, in A549 human lung cancer cells cultured under 20% O2, or 2% O2. Treatment with IFN-γ induced protein expression of IP subunits LMP2, LMP7, and MECL-1 in a dose-dependent manner under 20% O2 as assessed by western blotting. In contrast, IFN-γ failed to induce protein expression of these subunits under hypoxic conditions. We observed similar blockades of other antigen processing pathway components in response to IFN-γ under hypoxic conditions including TAP, tapasin, and calreticulin, as well as secondary mediators of IFN-γ-stimulated genes including several members of the interferon regulatory factor (IRF) family. Surprisingly, under identical conditions, induction of IP subunit mRNAs appear intact under hypoxia as assessed by RT-PCR, with similar levels of LMP2, LMP7, and MECL-1 upregulated by IFN-γ regardless of oxygen level, suggesting hypoxic IP blockade occurs post-transcriptionally or post-translationally. In line with this notion, primary mediators of IFN-γ signaling, including phosphorylated (Tyr701) STAT1 levels were unaffected by oxygen levels. To investigate the mechanism behind this phenomenon, we created hypoxia-inducible factor (HIF)-1a and/or HIF-2a knockout A549 cell lines or used cobalt chloride (CoCl2), a hydroxylase inhibitor, to directly interrogate the hypoxia signaling pathway. Blockade of IP subunit induction in response to IFN-γ was not observed after CoCl2 treatment, nor did HIF-1a and/or HIF-2a genetic deletion rescue IP subunit blockade in A549 cells cultured under hypoxia, indicating that hypoxia-induced IP blockade is independent of the HIF pathway. To test if this phenomenon is regulated by epigenic means, we treated cells with 5-Azacytidine (5-aza), a cystine analog nucleoside, and observed a re-establishment of IP subunit protein expression under hypoxia in response to IFN-γ. Given that 5-aza preferentially integrates RNA over DNA, and that IP subunit mRNA levels appear unaffected by hypoxia, suggests that hypoxia-induced IP blockade may be due to hypoxia-specific cytosine modifications within mRNA transcripts. Current studies include efforts to map mRNA methylation patterns and interrogate mRNA methylation transferases under hypoxic conditions. These studies introduce a novel link between hypoxia and reduced cancer cell antigen processing machinery and may help identify druggable epigenetic targets that can be used to enhance the immunogenicity of hypoxic tumors. Citation Format: Alexis Rebecca Ramos, Matthew Smith, Heena Panchal, Adam Mailloux. Tumor hypoxia blocks the induction of antigen processing machinery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2997.

Dysregulation of Gene Expression of Key Signaling Mediators in PBMCs from People with Type 2 Diabetes Mellitus.

Diabetes is currently the fifth leading cause of death by disease in the USA. The underlying mechanisms for type 2 Diabetes Mellitus (DM2) and the enhanced susceptibility of such patients to inflammatory disorders and infections remain to be fully defined. We have recently shown that peripheral blood mononuclear cells (PBMCs) from non-diabetic people upregulate expression of inflammatory genes in response to proteasome modulators, such as bacterial lipopolysaccharide (LPS) and soybean lectin (LEC); in contrast, resveratrol (RES) downregulates this response. We hypothesized that LPS and LEC will also elicit a similar upregulation of gene expression of key signaling mediators in (PBMCs) from people with type 2 diabetes (PwD2, with chronic inflammation) ex vivo. Unexpectedly, using next generation sequencing (NGS), we show for the first time, that PBMCs from PwD2 failed to elicit a robust LPS- and LEC-induced gene expression of proteasome subunit LMP7 (PSMB8) and mediators of T cell signaling that were observed in non-diabetic controls. These repressed genes included: PSMB8, PSMB9, interferon-γ, interferon-λ, signal-transducer-and-activator-of-transcription-1 (STAT1), human leukocyte antigen (HLA DQB1, HLA DQA1) molecules, interleukin 12A, tumor necrosis factor-α, transporter associated with antigen processing 1 (TAP1), and several others, which showed a markedly weak upregulation with toxins in PBMCs from PwD2, as compared to those from non-diabetics. Resveratrol (proteasome inhibitor) further downregulated the gene expression of these inflammatory mediators in PBMCs from PwD2. These results might explain why PwD2 may be susceptible to infectious disease. LPS and toxins may be leading to inflammation, insulin resistance, and thus, metabolic changes in the host cells.

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.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 enables prevention of transplant arteriosclerosis.

The loss of vascular wall cells in allotransplanted arteries is the initial event leading to transplant arteriosclerosis (TA) and ensuing loss of allograft function. Pharmacological agents able to prevent TA are currently lacking. We previously showed that selective inhibition of the immunoproteasome prevented the chronic rejection of renal allografts. However, the role and mechanisms of selective inhibition of a single immunoproteasome subunit to prevent immune-mediated vascular allograft rejection and TA is not clear. The effect and potential mechanism of combined or individual inhibition of peptidolytically active immunoproteasome LMP7 (β5i) and LMP2 (β1i) subunits on immune rejection-mediated TA was investigated using the epoxyketone inhibitor ONX 0914, and the recently developed LMP7-selective inhibitor KZR-329 and LMP2-selective inhibitor KZR-504 in a rat aorta transplantation model. We find that co-inhibition of LMP7 and LMP2 in allogeneic recipients significantly suppressed T-cell activation and function by expressing inhibitory surface markers and then activating inhibitory signals. Moreover, co-inhibition of LMP7 and LMP2 substantially reduced the number of immunoglobulin G-secreting cells and plasma cells and production of alloantibodies through activating the unfolded protein response and incapacitating the survival niche of plasma cells in the bone marrow. Consequentially, the accumulation of inflammatory cytokines, complement, and antibodies is reduced and the apoptosis of vascular wall cells decreased in aortic allografts via LMP7 and LMP2 co-inhibition with ONX 0914 treatment or combined KZR-329 and KZR-504 treatment. However, neither individual inhibition of LMP7 by KZR-329 nor individual inhibition of LMP2 by KZR-504 showed suppression of immune rejection and TA. We define a critical role of LMP7 and LMP2 in TA and strongly propose co-inhibition of both immunoproteasome subunits as promising therapeutic approach to suppress TA and allograft rejection.

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.

Correction: Ablation and Inhibition of the Immunoproteasome Catalytic Subunit LMP7 Attenuate Experimental Abdominal Aortic Aneurysm Formation in Mice.

Correction: Ablation and Inhibition of the Immunoproteasome Catalytic Subunit LMP7 Attenuate Experimental Abdominal Aortic Aneurysm Formation in Mice.

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.

Inhibition of immunoproteasome attenuates NLRP3 inflammasome formation in tumor necrosis factor α-stimulated intestinal epithelial cell

Excessive release of inflammatory cytokines has been considered as a major cause of chronic inflammation, resulting in intestinal barrier disruption that leads to inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα) is one of the well-known inflammatory cytokines that activates formation of NLRP3 inflammasome, thus resulting in excessive secretion of inflammatory cytokines causing IBD. Although immunoproteasome inhibitors have been reported to inhibit inflammatory cytokine release, immunoproteasome inhibition has not yet been addressed for attenuation of NLRP3 inflammasome activity in intestinal epithelial cell. Here, we observed that NLRP3 inflammasome assembly was attenuated by peptide epoxyketone YU102, a LMP2 subunit immunoproteasome inhibitor, in intestinal epithelial cell. YU102 also inhibited maturation of active caspase-1 and secretion of IL-1β, which are subsequent inflammatory cascade after the formation of NLRP3 inflammasome. Progression of epithelial-mesenchymal transition and increase of cellular permeability, which were induced by TNFα, were also suppressed through inhibition of immunoproteasome. Furthermore, we found that YU102 does not inhibit degradation of IкBα and its following NF-кB activation that leads to transcription of NLRP3. These findings suggest that inhibition of immunoproteasome with YU102 offers a potential therapeutic premise for prevention of TNFα-induced chronic inflammation through attenuation of NLRP3 inflammasome assembly.

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).

The Immunoproteasome Subunits LMP2, LMP7 and MECL-1 Are Crucial Along the Induction of Cerebral Toxoplasmosis.

Cell survival and function critically relies on the fine-tuned balance of protein synthesis and degradation. In the steady state, the standard proteasome is sufficient to maintain this proteostasis. However, upon inflammation, the sharp increase in protein production requires additional mechanisms to limit protein-associated cellular stress. Under inflammatory conditions and the release of interferons, the immunoproteasome (IP) is induced to support protein processing and recycling. In antigen-presenting cells constitutively expressing IPs, inflammation-related mechanisms contribute to the formation of MHC class I/II-peptide complexes, which are required for the induction of T cell responses. The control of Toxoplasma gondii infection relies on Interferon-γ (IFNγ)-related T cell responses. Whether and how the IP affects the course of anti-parasitic T cell responses along the infection as well as inflammation of the central nervous system is still unknown. To answer this question we used triple knockout (TKO) mice lacking the 3 catalytic subunits of the immunoproteasome (β1i/LMP2, β2i/MECL-1 and β5i/LMP7). Here we show that the numbers of dendritic cells, monocytes and CD8+ T cells were reduced in Toxoplasma gondii-infected TKO mice. Furthermore, impaired IFNγ, TNF and iNOS production was accompanied by dysregulated chemokine expression and altered immune cell recruitment to the brain. T cell differentiation was altered, apoptosis rates of microglia and monocytes were elevated and STAT3 downstream signaling was diminished. Consequently, anti-parasitic immune responses were impaired in TKO mice leading to elevated T. gondii burden and prolonged neuroinflammation. In summary we provide evidence for a critical role of the IP subunits β1i/LMP2, β2i/MECL-1 and β5i/LMP7 for the control of cerebral Toxoplasma gondii infection and subsequent neuroinflammation.