Immunoproteasome function maintains oncogenic gene expression in KMT2A-complex driven leukemia.

Pharmacologic targeting of chromatin-associated protein complexes has shown significant responses in KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) but resistance frequently develops to single agents. This points to a need for therapeutic combinations that target multiple mechanisms. To enhance our understanding of functional dependencies in KMT2A-r AML, we have used a proteomic approach to identify the catalytic immunoproteasome subunit PSMB8 as a specific vulnerability. Genetic and pharmacologic inactivation of PSMB8 results in impaired proliferation of murine and human leukemic cells while normal hematopoietic cells remain unaffected. Disruption of immunoproteasome function drives an increase in transcription factor BASP1 which in turn represses KMT2A-fusion protein target genes. Pharmacologic targeting of PSMB8 improves efficacy of Menin-inhibitors, synergistically reduces leukemia in human xenografts and shows preserved activity against Menin-inhibitor resistance mutations. This identifies and validates a cell-intrinsic mechanism whereby selective disruption of proteostasis results in altered transcription factor abundance and repression of oncogene-specific transcriptional networks. These data demonstrate that the immunoproteasome is a relevant therapeutic target in AML and that targeting the immunoproteasome in combination with Menin-inhibition could be a novel approach for treatment of KMT2A-r AML.

Immunogenic cell death triggered by impaired deubiquitination in multiple myeloma relies on dysregulated type I interferon signaling.

Introduction: Proteasome inhibition is first line therapy in multiple myeloma (MM). The immunological potential of cell death triggered by defects of the ubiquitin-proteasome system (UPS) and subsequent perturbations of protein homeostasis is, however, less well defined. Methods: In this paper, we applied the protein homeostasis disruptors bortezomib (BTZ), ONX0914, RA190 and PR619 to various MM cell lines and primary patient samples to investigate their ability to induce immunogenic cell death (ICD). Results: Our data show that while BTZ treatment triggers sterile type I interferon (IFN) responses, exposure of the cells to ONX0914 or RA190 was mostly immunologically silent. Interestingly, inhibition of protein de-ubiquitination by PR619 was associated with the acquisition of a strong type I IFN gene signature which relied on key components of the unfolded protein and integrated stress responses including inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR) and general control nonderepressible 2 (GCN2). The immunological relevance of blocking de-ubiquitination in MM was further reflected by the ability of PR619-induced apoptotic cells to facilitate dendritic cell (DC) maturation via type I IFN-dependent mechanisms. Conclusion: Altogether, our findings identify de-ubiquitination inhibition as a promising strategy for inducing ICD of MM to expand current available treatments.

Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach.

MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.

Hematopoietic stem cell transplantation in a patient with proteasome-associated autoinflammatory syndrome (PRAAS).

BACKGROUND: Proteasome-associated autoinflammatory syndromes (PRAASs) form a family of recently described rare autosomal recessive disorders of disturbed proteasome assembly and proteolytic activity caused by mutations in genes coding for proteasome subunits. The treatment options for these proteasome disorders consist of lifelong immunosuppressive drugs or Janus kinase inhibitors, which may have partial efficacy and noticeable side effects. Because proteasomes are ubiquitously expressed, it is unknown whether hematopoietic stem cell transplantation (HSCT) may be a sufficient treatment option. OBJECTIVE: Our aim was to report the case of a young boy with a treatment-resistant cutaneous vasculitis that was initially suspected to be associated with a gene variant in SH2D1A. METHODS: Whole-exome sequencing was performed to identify the genetic defect. Molecular and functional analyses were performed to assess the impact of variants on proteasomal function. The immune characterization led to the decision to perform HSCT on our patient and conduct follow-up over the 7-year period after the transplant. Because loss of myeloid chimerism after the first HSCT was associated with relapse of autoinflammation, a second HSCT was performed. RESULTS: After the successful second HSCT, the patient developed mild symptoms of lipodystrophy, which raised the suspicion of a PRAAS. Genetic analysis revealed 2 novel heterozygous variants in PSMB4 (encoding proteasomal subunit ß7). Retrospective analysis of patient cells stored before the first HSCT and patient cells obtained after the second HSCT demonstrated that HSCT successfully rescued proteasome function, restored protein homeostasis, and resolved the interferon-stimulated gene signature. Furthermore, successful HSCT alleviated the autoinflammatory manifestations in our patient. CONCLUSION: Patients with treatment-resistant PRAAS can be cured by HSCT.

Immunoproteasome Function in Normal and Malignant Hematopoiesis.

The ubiquitin-proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.

Immunoproteasome impairment via ß5i/LMP7-deletion leads to sustained pancreatic injury from experimental pancreatitis.

Uncovering potential new targets involved in pancreatitis may permit the development of new therapies and improvement of patient's outcome. Acute pancreatitis is a primarily sterile disease characterized by a severe systemic inflammatory response associated with extensive necrosis and a mortality rate of up to 24%. Considering that one of the reported disease mechanisms comprises the endoplasmic reticulum (ER) stress response and that the immunoproteasome is a key regulator to prevent proteotoxic stress in an inflammatory context, we investigated its role in acute pancreatitis. In this study, we demonstrate that immunoproteasome deficiency by deletion of the ß5i/LMP7-subunit leads to persistent pancreatic damage. Interestingly, immunoproteasome-deficient mice unveil increased activity of pancreatic enzymes in the acute disease phase as well as higher secretion of Interleukin-6 and transcript expression of the Interleukin IL-1ß, IFN-ß cytokines and the CXCL-10 chemokine. Cell death was increased in immunoproteasome-deficient mice, which appears to be due to the increased accumulation of ubiquitin-protein conjugates and prolonged unfolded protein response. Accordingly, our findings suggest that the immunoproteasome plays a protective role in acute pancreatitis via its role in the clearance of damaged proteins and the balance of ER stress responses in pancreatic acini and in macrophages cytokine production.

The Ubiquitin-Proteasome System in Immune Cells.

The ubiquitin-proteasome system (UPS) is the major intracellular and non-lysosomal protein degradation system. Thanks to its unique capacity of eliminating old, damaged, misfolded, and/or regulatory proteins in a highly specific manner, the UPS is virtually involved in almost all aspects of eukaryotic life. The critical importance of the UPS is particularly visible in immune cells which undergo a rapid and profound functional remodelling upon pathogen recognition. Innate and/or adaptive immune activation is indeed characterized by a number of substantial changes impacting various cellular processes including protein homeostasis, signal transduction, cell proliferation, and antigen processing which are all tightly regulated by the UPS. In this review, we summarize and discuss recent progress in our understanding of the molecular mechanisms by which the UPS contributes to the generation of an adequate immune response. In this regard, we also discuss the consequences of UPS dysfunction and its role in the pathogenesis of recently described immune disorders including cancer and auto-inflammatory diseases.

Report of the first patient with a homozygous OTUD7A variant responsible for epileptic encephalopathy and related proteasome dysfunction.

Heterozygous microdeletions of chromosome 15q13.3 (MIM: 612001) show incomplete penetrance and are associated with a highly variable phenotype that may include intellectual disability, epilepsy, facial dysmorphism and digit anomalies. Rare patients carrying homozygous deletions show more severe phenotypes including epileptic encephalopathy, hypotonia and poor growth. For years, CHRNA7 (MIM: 118511), was considered the candidate gene that could account for this syndrome. However, recent studies in mouse models have shown that OTUD7A/CEZANNE2 (MIM: 612024), which encodes for an ovarian tumor (OTU) deubiquitinase, should be considered the critical gene responsible for brain dysfunction. In this study, a patient presenting with severe global developmental delay, language impairment and epileptic encephalopathy was referred to our genetics center. Trio exome sequencing (tES) analysis identified a homozygous OTUD7A missense variant (NM_130901.2:c.697C>T), predicted to alter an ultraconserved amino acid, p.(Leu233Phe), lying within the OTU catalytic domain. Its subsequent segregation analysis revealed that the parents, presenting with learning disability, and brother were heterozygous carriers. Biochemical assays demonstrated that proteasome complex formation and function were significantly reduced in patient-derived fibroblasts and in OTUD7A knockout HAP1 cell line. We provide evidence that biallelic pathogenic OTUD7A variation is linked to early-onset epileptic encephalopathy and proteasome dysfunction.

Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas.

The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. The fraction of protein passing through the ER represents a large proportion of the total protein in the cell. Protein folding, glycosylation, sorting and transport are essential tasks of the ER and a compromised ER folding network has been recognized to be a key component in the disease pathogenicity of common neurodegenerative, metabolic and malignant diseases. On the other hand, the ER protein folding machinery also holds significant potential for therapeutic interventions. Many causes can lead to ER stress. A disturbed calcium homeostasis, the generation of reactive oxygen species (ROS) and a persistent overload of misfolded proteins within the ER can drive the course of adisease. In this review the role of ER-stress in diseases of the liver and pancreas will be examined using pancreatitis and Wilson´s disease as examples. Potential therapeutic targets in ER-stress pathways will also be discussed.

Nrf1 can be processed and activated in a proteasome-independent manner.

In response to proteasome inhibition, the transcription factor Nrf1 facilitates de novo synthesis of proteasomes by inducing proteasome subunit (PSM) genes [1,2]. Previously, we showed that activation of the p120 form of Nrf1, a membrane-bound protein in the endoplasmic reticulum (ER) with the bulk of its polypeptide in the lumen, involves its retrotranslocation into the cytosol in a manner that depends on the AAA-ATPase p97/VCP [3]. This is followed by proteolytic processing and mobilization of the transcriptionally active p110 form of Nrf1 to the nucleus. A subsequent study suggested that site-specific proteolytic processing of Nrf1 by the proteasome yields an active 75 kDa fragment [4]. We show here that under conditions where all three active sites of the proteasome are completely blocked, p120 Nrf1 can still be proteolytically cleaved to the p110 form, which is translocated to the nucleus to activate transcription of PSM genes. Thus, our results indicate that a proteasome-independent pathway can promote the release of active p110 Nrf1 from the ER membrane.

TCF11/Nrf1-Mediated Induction of Proteasome Expression Prevents Cytotoxicity by Rotenone.

AIMS: Precise regulation of cellular protein degradation is essential for maintaining protein and redox homeostasis. The ubiquitin proteasome system (UPS) represents one of the major degradation machineries, and UPS disturbances are strongly associated with neurodegeneration. We have previously shown that the transcription factor TCF11/Nrf1 induces antioxidant response element-mediated upregulation of UPS components in response to proteotoxic stress. Knockout of TCF11/Nrf1 is embryonically lethal, and therefore, the present investigation describes the role of oxidative stress in regulating TCF11/Nrf1-dependent proteasome expression in a model system relevant to Parkinson's disease. RESULTS: Using the human dopaminergic neuroblastoma cell line SH-SY5Y and mouse nigrostriatal organotypic slice cultures, gene and protein expression analysis and functional assays revealed oxidative stress is induced by the proteasome inhibitor epoxomicin or the mitochondrial complex I inhibitor rotenone and promotes the upregulation of proteasome expression and function mediated by TCF11/Nrf1 activation. In addition, we show that these stress conditions induce the unfolded protein response. TCF11/Nrf1, thus, has a cytoprotective function in response to oxidative and proteotoxic stress. Innovation and Conclusion: We here demonstrate that adaption of the proteasome system in response to oxidative stress is dependent on TCF11/Nrf1 in this model system. We conclude that TCF11/Nrf1, therefore, plays a vital role in maintaining redox and protein homeostasis. This work provides a vital insight into the molecular mechanisms of neurodegeneration due to oxidative stress by rotenone, and further studies investigating the role of TCF11/Nrf1 in the human condition would be of considerable interest. Antioxid. Redox Signal. 25, 870-885.

Exposure to Melan-A/MART-126-35 tumor epitope specific CD8(+)T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS).

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing.

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.

Autosomal recessive mutations in proteasome subunit ß 8 (PSMB8), which encodes the inducible proteasome subunit ß5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes ß7), PSMB9 (encodes ß1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.

The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

MicroRNA-101 Suppresses Tumor Cell Proliferation by Acting as an Endogenous Proteasome Inhibitor via Targeting the Proteasome Assembly Factor POMP.

Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation.

Oxidation matters: the ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation.

During innate immune responses the delicate balance of protein synthesis, quality control and degradation is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins. The regulated degradation of ubiquitin-tagged proteins by the ubiquitin proteasome system (UPS) represents one major pathway for the maintenance of cellular proteostasis and regulatory processes under these conditions. In addition, MHC class I antigen presentation is strictly dependent on an appropriate peptide supply by the UPS to efficiently prime CD8(+) T cells and to initiate an adaptive immune response. We here discuss recent efforts in defining the link between innate immune mechanisms like cytokine and ROS production, the induction of an efficient adaptive immune response and the specific involvement of the UPS therein. Cytokines and/or infections induce translation and the production of free radicals, which in turn confer oxidative damage to nascent as well as folded proteins. In parallel, the same signaling cascades are able to accelerate the protein turnover by the concomitantly induced ubiquitin conjugation and degradation of such damaged polypeptides by immunoproteasomes. The ability of immunoproteasomes to efficiently degrade such oxidant-damaged ubiquitylated proteins protects cells from accumulating toxic ubiquitin-rich aggregates. At the same time, this innate immune mechanism facilitates a sufficient peptide supply for MHC class I antigen presentation and connects it to initiation of adaptive immunity.