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.

PSMC3 proteasome subunit variants are associated with neurodevelopmental delay and type I interferon production.

A critical step in preserving protein homeostasis is the recognition, binding, unfolding, and translocation of protein substrates by six AAA-ATPase proteasome subunits (ATPase-associated with various cellular activities) termed PSMC1-6, which are required for degradation of proteins by 26S proteasomes. Here, we identified 15 de novo missense variants in the PSMC3 gene encoding the AAA-ATPase proteasome subunit PSMC3/Rpt5 in 23 unrelated heterozygous patients with an autosomal dominant form of neurodevelopmental delay and intellectual disability. Expression of PSMC3 variants in mouse neuronal cultures led to altered dendrite development, and deletion of the PSMC3 fly ortholog Rpt5 impaired reversal learning capabilities in fruit flies. Structural modeling as well as proteomic and transcriptomic analyses of T cells derived from patients with PSMC3 variants implicated the PSMC3 variants in proteasome dysfunction through disruption of substrate translocation, induction of proteotoxic stress, and alterations in proteins controlling developmental and innate immune programs. The proteostatic perturbations in T cells from patients with PSMC3 variants correlated with a dysregulation in type I interferon (IFN) signaling in these T cells, which could be blocked by inhibition of the intracellular stress sensor protein kinase R (PKR). These results suggest that proteotoxic stress activated PKR in patient-derived T cells, resulting in a type I IFN response. The potential relationship among proteosome dysfunction, type I IFN production, and neurodevelopment suggests new directions in our understanding of pathogenesis in some neurodevelopmental disorders.

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.

The Proteasome Activator PA200/PSME4: An Emerging New Player in Health and Disease.

Proteasomes comprise a family of proteasomal complexes essential for maintaining protein homeostasis. Accordingly, proteasomes represent promising therapeutic targets in multiple human diseases. Several proteasome inhibitors are approved for treating hematological cancers. However, their side effects impede their efficacy and broader therapeutic applications. Therefore, understanding the biology of the different proteasome complexes present in the cell is crucial for developing tailor-made inhibitors against specific proteasome complexes. Here, we will discuss the structure, biology, and function of the alternative Proteasome Activator 200 (PA200), also known as PSME4, and summarize the current evidence for its dysregulation in different human diseases. We hereby aim to stimulate research on this enigmatic proteasome regulator that has the potential to serve as a therapeutic target in cancer.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

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.

Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells.

Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome ß5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation.

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope.

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)-but not ERAP2-defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100209-217 -containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features.

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.

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.

Proteasome ß5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia.

Immunoproteasomes, harboring the active site subunits ß5i/LMP7, ß1i/LMP2, and ß2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in ß5i/LMP7-/- mice. Our data show that ß5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in ß5i/LMP7-/- mice. The impaired bacterial elimination in ß5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections.

Major Histocompatibility Complex (MHC) Class I Processing of the NY-ESO-1 Antigen Is Regulated by Rpn10 and Rpn13 Proteins and Immunoproteasomes following Non-lysine Ubiquitination.

The supply of MHC class I-restricted peptides is primarily ensured by the degradation of intracellular proteins via the ubiquitin-proteasome system. Depending on the target and the enzymes involved, ubiquitination is a process that may dramatically vary in terms of linkages, length, and attachment sites. Here we identified the unique lysine residue at position 124 of the NY-ESO-1 cancer/testis antigen as the acceptor site for the formation of canonical Lys-48-linkages. Interestingly, a lysine-less form of NY-ESO-1 was as efficient as its wild-type counterpart in supplying the HLA-A*0201-restricted NY-ESO-1157-165 antigenic peptide. In fact, we show that the regulation of NY-ESO-1 processing by the ubiquitin receptors Rpn10 and Rpn13 as a well as by the standard and immunoproteasome is governed by non-canonical ubiquitination on non-lysine sites. In summary, our data underscore the significance of atypical ubiquitination in the modulation of MHC class I antigen processing.