Ubiquitin-conjugating Enzyme Research Articles

Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the UBA5 gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least 12 different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remain to be established. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype–phenotype relationships across the UBA5 allelic series. In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro platforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.

Allelic strengths of encephalopathy-associated UBA5 variants correlate between in vivo and in vitro assays.

Protein UFMylation downstream of the E1 enzyme UBA5 plays essential roles in development and endoplasmic reticulum stress. Variants in the UBA5 gene are associated with developmental and epileptic encephalopathy 44 (DEE44), an autosomal recessive disorder characterized by early-onset encephalopathy, movement abnormalities, global developmental delay, intellectual disability, and seizures. DEE44 is caused by at least 12 different missense variants described as loss of function (LoF), but the relationships between genotypes and molecular or clinical phenotypes remain to be established. We developed a humanized UBA5 fly model and biochemical activity assays in order to describe in vivo and in vitro genotype-phenotype relationships across the UBA5 allelic series. In vivo, we observed a broad spectrum of phenotypes in viability, developmental timing, lifespan, locomotor activity, and bang sensitivity. A range of functional effects was also observed in vitro across comprehensive biochemical assays for protein stability, ATP binding, UFM1 activation, and UFM1 transthiolation. Importantly, there is a strong correlation between in vivo and in vitro phenotypes, establishing a classification of LoF variants into mild, intermediate, and severe allelic strengths. By systemically evaluating UBA5 variants across in vivo and in vitro platforms, this study provides a foundation for more basic and translational UBA5 research, as well as a basis for evaluating current and future individuals afflicted with this rare disease.

SUMOylation is enriched in the nuclear matrix and required for chromosome segregation

As an important post-translational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging, and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or knockout of SUMO-conjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 knockout cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes.

Serious infections in patients with VEXAS syndrome: data from the French VEXAS registry

IntroductionVacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is an acquired autoinflammatory monogenic disease with a poor prognosis whose determinants are not well understood. We aimed to describe serious infectious...

Overexpression of UBE2E2 in Mouse Pancreatic β-Cells Leads to Glucose Intolerance via Reduction of β-Cell Mass.

Genome-wide association studies have identified several gene polymorphisms, including UBE2E2, associated with type 2 diabetes. Although UBE2E2 is one of the ubiquitin-conjugating enzymes involved in the process of ubiquitin modifications, the pathophysiological roles of UBE2E2 in metabolic dysfunction are not yet understood. Here, we showed upregulated UBE2E2 expression in the islets of a mouse model of diet-induced obesity. The diabetes risk allele of UBE2E2 (rs13094957) in noncoding regions was associated with upregulation of UBE2E2 mRNA in the human pancreas. Although glucose-stimulated insulin secretion was intact in the isolated islets, pancreatic β-cell-specific UBE2E2-transgenic (TG) mice exhibited reduced insulin secretion and decreased β-cell mass. In TG mice, suppressed proliferation of β-cells before the weaning period and while receiving a high-fat diet was accompanied by elevated gene expression levels of p21, resulting in decreased postnatal β-cell mass expansion and compensatory β-cell hyperplasia, respectively. In TG islets, proteomic analysis identified enhanced formation of various types of polyubiquitin chains, accompanied by increased expression of Nedd4 E3 ubiquitin protein ligase. Ubiquitination assays showed that UBE2E2 mediated the elongation of ubiquitin chains by Nedd4. The data suggest that UBE2E2-mediated ubiquitin modifications in β-cells play an important role in regulating glucose homeostasis and β-cell mass.

Role of the Mitochondrial E3 Ubiquitin Ligases as Possible Therapeutic Targets in Cancer Therapy.

Ubiquitination is a post-translational modification that targets specific proteins on their lysine residues. Depending on the type of ubiquitination, this modification ultimately regulates the stability or degradation of the targeted proteins. Ubiquitination is mediated by three different classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes and, most importantly, the E3 ubiquitin ligases. E3 ligases are responsible for the final step of the ubiquitin cascade, interacting directly with the target proteins. E3 ligases can also be involved in DNA repair, cell cycle regulation and response to stress; alteration in their levels can be involved in oncogenic transformation and cancer progression. Of all the six hundred E3 ligases of the human genome, only three of them are specific to the mitochondrion: MARCH5, RNF185 and MUL1. Their alterations (that reflect on the alteration of the mitochondria functions) can be related to cancer progression, as underlined by the increasing research performed in recent years on these three mitochondrial enzymes. This review will focus on the function and mechanisms of the mitochondrial E3 ubiquitin ligases, as well as their important targets, in cancer development and progression, also highlighting their potential use for cancer therapy.

Spatial regulation of DNA damage tolerance protein Rad5 interconnects genome stability maintenance and proteostasis networks.

The Rad5/HLTF protein has a central role in the tolerance to DNA damage by mediating an error-free mode of bypassing unrepaired DNA lesions, and is therefore critical for the maintenance of genome stability. We show in this work that, following cellular stress, Rad5 is regulated by relocalization into two types of nuclear foci that coexist within the same cell, which we termed 'S' and 'I'. Rad5 S-foci form in response to genotoxic stress and are associated with Rad5's function in maintaining genome stability, whereas I-foci form in the presence of proteotoxic stress and are related to Rad5's own proteostasis. Rad5 accumulates into S-foci at DNA damage tolerance sites by liquid-liquid phase separation, while I-foci constitute sites of chaperone-mediated sequestration of Rad5 at the intranuclear quality control compartment (INQ). Relocalization of Rad5 into each type of foci involves different pathways and recruitment mechanisms, but in both cases is driven by the evolutionarily conserved E2 ubiquitin-conjugating enzyme Rad6. This coordinated differential relocalization of Rad5 interconnects DNA damage response and proteostasis networks, highlighting the importance of studying these homeostasis mechanisms in tandem. Spatial regulation of Rad5 under cellular stress conditions thus provides a useful biological model to study cellular homeostasis as a whole.

Ubiquitin-conjugating enzyme E2 for regulating autophagy in diabetic cardiomyopathy: A mini-review.

The prevalence of diabetic cardiomyopathy (DCM) increases year by year with the increase in the prevalence of diabetes mellitus (DM), which is one of the most serious cardiovascular complications of DM and a major cause of death in diabetic patients. Although the pathological molecular features of DCM have not been fully elucidated, increasing evidence suggests that impaired autophagy in cardiomyocytes plays a nonnegligible role in the development of DCM. It has been shown that SUMOylation [SUMO = small ubiquitin-like modifier], a post-translational modification of proteins, and its associated ubiquitin-proteasome system mediates protein quality control in the heart and plays an important role in the proteotoxic environment of the heart. Specifically, the expression of ubiquitin-conjugating enzyme E2 (Ubc9), the only SUMO-E2 enzyme, exerts a positive regulatory effect on autophagy in cardiomyocytes with potential cardioprotective effects. This review focuses on the role that autophagy plays in DCM and the potential for Ubc9-regulated autophagy pathways to ameliorate DCM, highlighting the potential of Ubc9 as an interventional target in DCM and providing new insights into the pathogenesis of the disease.

A ubiquitin-proteasome system-related signature to predict prognosis, immune infiltration, and therapy efficacy for breast cancer.

The ubiquitin-proteasome system (UPS) is an essential regulatory system for maintaining homeostasis, and its dysfunction may cause various diseases. The activity of proteasome and ubiquitin-conjugating enzymes has been found to be greatly increased in breast cancer (BC), indicating that the heterogeneity of UPS may be related to the progression of BC. Gene data was obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases and performed in multiple algorithms to construct a UPS-related signature for BC. Patients in the UPS low-risk group had greater overall and recurrence-free survival probability than those in the UPS high-risk group. This signature was closely associated with functional enrichment. Some high metabolism-related pathways were more active in the UPS high-risk group. The UPS low-risk group had more abundant anti-tumor immune cells, while in the UPS high-risk group, immunosuppressive cells were dominant. More importantly, we found that the UPS low-risk group was more sensitive to immunotherapy, while the UPS high-risk group responded better to radiotherapy. Drug sensitivity analysis identified more effective chemotherapy drugs in different UPS-related risk groups. This UPS-related signature may serve as a novel biomarker and independent prognostic factor for BC. It can effectively predict prognosis, immune infiltration, and therapy efficacy, providing new strategies for individualized treatment.

UBE2J1 is the E2 ubiquitin-conjugating enzyme regulating androgen receptor degradation and antiandrogen resistance

Prostate cancer (PCa) is primarily driven by aberrant Androgen Receptor (AR) signaling. Although there has been substantial advancement in antiandrogen therapies, resistance to these treatments remains a significant obstacle, often marked by continuous or enhanced AR signaling in resistant tumors. While the dysregulation of the ubiquitination-based protein degradation process is instrumental in the accumulation of oncogenic proteins, including AR, the molecular mechanism of ubiquitination-driven AR degradation remains largely undefined. We identified UBE2J1 as the critical E2 ubiquitin-conjugating enzyme responsible for guiding AR ubiquitination and eventual degradation. The absence of UBE2J1, found in 5–15% of PCa patients, results in disrupted AR ubiquitination and degradation. This disruption leads to an accumulation of AR proteins, promoting resistance to antiandrogen treatments. By employing a ubiquitination-based AR degrader to adeptly restore AR ubiquitination, we reestablished AR degradation and inhibited the proliferation of antiandrogen-resistant PCa tumors. These findings underscore the fundamental role of UBE2J1 in AR degradation and illuminate an uncharted mechanism through which PCa maintains heightened AR protein levels, fostering resistance to antiandrogen therapies.

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged >50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (>80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

UBA1 Mutations Identify a Rare but Distinct Subtype of Myelodysplastic Syndromes

Background Mutations in UBA1 are associated with VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, an adult-onset inflammatory disorder (Beck DB et al. NEJM 2020). Approximately 40% of VEXAS patients are also diagnosed with myelodysplastic syndromes (MDS). We previously profiled UBA1 p.M41 mutations in a select subset of the International Working Group for MDS (IWG-PM) cohort (Bernard E et al. NEJM Evid 2022) including 375 male patients lacking disease-defining mutations or established disease classification by WHO guidelines, and identified 28 patients (7%) with UBA1 p.M41T/V/L mutations (Cohort A, Sirenko M et al. Blood 2022). However, the prevalence and characteristics of UBA1 mutations in MDS have not been systematically evaluated in a representative MDS population. Methods We used targeted next-generation sequencing of the full UBA1 locus to profile 2027 diagnostic and treatment-naive MDS samples (Cohort B, 61% male) ascertained through the IWG-PM (Figure 1). Clinical associations of UBA1 mutations were evaluated and clinical history was reviewed for inflammatory conditions when available. Results In Cohort B, 35 UBA1 mutations in 34 (1.7%) patients were identified, of which 20 (0.97%) had likely pathogenic variants and 14 had variants of unknown significance (VUS). Likely pathogenic variants included 13 p.M41T/V/L mutation (variant allele fraction [VAF] range 0.013-0.94), and 7 non-p.M41 mutations where 6/7 had VAF>0.35: 2 with p.S56F (VAF 0.87 and 0.93), 2 p.A478S (VAF 0.72 and 0.80), 2 p.S621C (VAF 0.80 and 0.82) and 1 p.Y55H (VAF 0.025). Three VUS were in female patients. 204 cases were profiled by both assays and of those, all 8 ddPCR positive cases were identified by NGS with concordant VAF (R2=0.999 p<0.0001). Integration of Cohort A and B (n=2,198) yielded 40 patients (all male, median age 72; range 44-89 years) with pathogenic UBA1 variants. The WHO 2016 classification (available for n=38 of 40) was MDS-SLD/MLD (25), MDS-EB1 (4), MDS-U (3), CMML (2), MDS-RS-MLD (1), aCML (1), MDS/MPN-RS-T (1), MDS/MPN-U (1). Patients had a median of 1 myeloid gene mutation in addition to UBA1 (range: 0-4) with most frequent co-occurring events in TET2 (n=12), DNMT3A (n = 10), ASXL1 (n=3), SF3B1 (n=3) and loss of the Y chromosome (n=5). In 8 patients with pathogenic UBA1 > 2% VAF and co-occurring DNMT3A mutations, DNMT3A and UBA1 VAF were correlated (slope = 0.88, r = 0.87, p = 0.0005), suggesting that co-mutation may lead to clonal expansion. Conversely, TET2 co-mutations were either subclonal (n=4) or clonal (n=8) to UBA1. Among patients with pathogenic UBA1 variants, the majority had IPSS-M Very-Low/Low risk (73% 27/37). In contrast, patients with VUS were more likely to have Moderate or High risk (64% 7/11). Partial clinical history was available for 33 cases with pathogenic UBA1 variants. 50% (9/18) had inflammatory-rheumatic disease (IRD) including psoriatic arthritis, relapsing polychondritis, Sweet syndrome, bronchiolitis obliterans with organizing pneumonia, ear chondritis, and rosacea. 3 patients had more than one IRD. 8 were treated for IRD with steroids (n=6) or methotrexate (n=2). 4 patients had MGUS. Other manifestations included vacuoles (4/8), thromboembolic disease (5/16), non-infection fever (5/14), weight loss (4/14), ocular symptoms (5/15), arthralgia (3/16), chondritis (5/15), and other inflammation (6/14). IRD diagnosis usually preceded MDS (average time 0.28 years; range 16.5 years prior to 1.4 years after). Three patients transformed to acute myeloid leukemia (AML). One patient had a low VAF UBA1 p.M41V (0.0002 by ddPCR) and TET2, SF3B1, FLT3, and ASXL2 co-mutations at baseline. The second had UBA1 p.S56F (VAF 0.868) and a Chr 7q deletion. The third had VUS UBA1 p.R869L (VAF 0.222) and IRF1, NFE2 and RRAS co-mutations. Among the other UBA1-mutant patients that died or were censored after 1 year (n=37), none transformed to AML. Conclusion Within the large, representative, diagnostic and well-characterized IWG-PM MDS cohort, we find likely pathogenic UBA1 mutations in 1% of patients, with enrichment in male patients with few or no mutations in myeloid driver genes (7%). UBA1-mutant patients were predominantly IPSS-M low risk with a median of 1 additional mutation, usually in DNMT3A or TET2. UBA1 mutations may define a distinct subset of MDS and its recognition in future guidelines will improve the management of patients with MDS/VEXAS overlap.

Sensitivity to Targeted UBA1 Inhibition in a Myeloid Cell Line Model of Vexas Syndrome

Somatic UBA1 mutations in hematopoietic cells are a hallmark of VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, which is a late-onset, male-predominant myeloid autoinflammatory disease associated with bone marrow failure and high mortality. UBA1 resides on the X chromosome and encodes the E1 enzyme isoforms UBA1a and UBA1b, which are essential for nuclear and cytosolic protein ubiquitination, respectively. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous missense mutations affecting methionine-41 (M41), leading to loss of UBA1b, gain of a truncated protein isoform (UBA1c),and globally reduced polyubiquitination. The relationship between impaired polyubiquitination, autoinflammation, and dyspoiesis in VEXAS syndrome is poorly understood, highlighting the need for experimental models that reproduce the genetic, biochemical, morphological, and inflammatory features associated with somatic UBA1 M41 missense mutations. To investigate the effects of VEXAS syndrome-associated UBA1 mutations, we used CRISPR-Cas9-based homology directed repair (HDR) in the male myeloid cell line 32D to generate isogenic single cell clones expressing Uba1 WT or Uba1 M41L from the endogenous mouse Uba1 locus. Sanger sequencing analysis of genomic and complementary DNA indicated the presence of a single Uba1 allele in Uba1 WT and Uba1 M41L single cell clones. Consistent with prior analyses of VEXAS syndrome patient samples, UBA1 and ubiquitin immunoblots showed that hemizygous Uba1 M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform and reduced polyubiquitination. Uba1 M41L cells also contained abnormal cytoplasmic vacuoles, which were largely absent from Uba1 WT cells. Electron microscopy revealed that vacuoles in Uba1 M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Similar to cytokine profiles in VEXAS syndrome patient samples, concentrations of IL1β and CXCL10 were significantly increased in supernatants from Uba1 M41L cells compared to Uba1 WT cells; Uba1 M41L cell supernatants also contained higher concentrations of CCL5, CCL17, CXCL9, and CXCL12. Thus, hemizygous Uba1 M41L expression in a myeloid cell line recapitulates key biochemical, morphological, and inflammatory features of VEXAS syndrome. The UBA1c protein isoform generated via UBA1 M41 missense mutations has reduced capacity to form the UBA1-ubiquitin thioester bond necessary for ubiquitin transfer to E2 enzymes. We hypothesized that decreased UBA1 activity would render Uba1 M41L cells vulnerable to the UBA1 inhibitor TAK243. In cell proliferation assays, Uba1 M41L cells were more sensitive to TAK243 compared to Uba1 WT cells (IC 50 mean±s.d., 1.4±0.6nM vs. 63±28nM, respectively). TAK243 promoted apoptosis in Uba1 M41L cells, as indicated by increased annexin-V/propidium iodide staining, H2AX, H2AX pS139, and PARP1 cleavage. TAK243 also led to preferential loss of Uba1 M41L cells in competition assays with Uba1 WT cells. To determine if on-target UBA1 inhibition was responsible for the increased TAK243 sensitivity of Uba1 M41L cells, we used CRISPR-HDR to generate a TAK243 drug-binding mutant, Uba1 A580S. Knock-in of Uba1 A580S rendered Uba1 M41L cells resistant to TAK243; the TAK243 IC 50 was significantly higher in Uba1 M41L;A580S cells compared to Uba1 M41L cells (mean±s.d., 73±16nM vs. 3.8±1.2nM, respectively). Together, these data demonstrate that hemizygous expression of Uba1 M41L in 32D cells confers TAK243 sensitivity via on-target inhibition of UBA1. Notably, increased TAK243 sensitivity and reduced polyubiquitination in Uba1 M41L cells were rescued by overexpression of UBA1b, but not UBA1b/C632A (catalytically inactive) or luciferase, indicating that loss of UBA1b enzymatic function contributes to the phenotype conferred by Uba1 M41L. In summary, we present a myeloid cell line with hemizygous expression of Uba1 M41L that exhibits the biochemical, morphological, and inflammatory features of VEXAS syndrome. We find that loss of UBA1b is a cause of reduced polyubiquitination and renders Uba1 M41L cells vulnerable to targeted UBA1 inhibition by TAK243. Our Uba1 M41L knock-in cell line is a faithful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Serious Infections in Patients with Vexas Syndrome: A Study from the French Vexas Group

Background VEXAS (Vacuoles, E1 Enzyme, X-Linked, Autoinflammatory, Somatic) syndrome is an autoinflammatory monogenic disease caused by inactivating somatic mutations in the UBA1 gene and characterized by heterogenous systemic auto-inflammation and progressive hematologic manifestations. Its management is not consensual but often include biologic DMARDs or azacitidine in case of association with myelodysplastic syndrome. Prognosis appears to be poor, with substantial morbidity and mortality mainly caused by infection. The aim of this study was to describe the spectrum of serious infectious complications and their potential risk factors in VEXAS patients. Methods We conducted a retrospective multicenter study including patients with genetically proven VEXAS syndrome from the French VEXAS registry. Episode of serious infections (defined as an infection leading to hospitalization and/or intravenous infectious treatments and/or death) were described, and their risk factors analyzed with multivariable Cox proportional hazard models.These patients were compared to a cohort of 50 VEXAS patients without serious infection after at least one year of follow-up since diagnosis. Results Seventy-four patients (99% male, median [IQR] age at VEXAS onset of 68 [63-75] years) with 133 serious infections were included. Infections occurred despite anti-infective prophylaxis in 46% of cases. The main immunosuppressive drugs received at the time of infection were JAK inhibitors (29%), biologics (21%) and azacitidine (11%), while 16% of infections occurred without treatment (no immunosuppressant or corticosteroids ≤ 10 mg/d).The most common sites of infection were lung (59%), skin (10%) and urinary tract (9%). Microbiological confirmation was obtained for 76%: 52% bacterial, 30% viral, 15% fungal and 3% mycobacterial. Among pulmonary infections, the main infectious agents were SARS-CoV-2 (28%), Legionella pneumophila (21%) and Pneumocystis jivoreci (19%) (Figure 1). Invasive fungal infections accounted for 11% of all infections. Nearly 20% of pulmonary infections occurred in the absence of treatment with a high prevalence of L. pneumophila (42%) and P. jivoreci (17%) infections. In multivariate analysis, age >75 years (HR [95%CI] 1.91 [1.05-3.47]), p.Met41Val mutation (2.44 [1.05-5.63]) and arthralgia (2.03 [1.16-3.56]) were associated with the risk of first serious episode of infections. Among treatments, JAK inhibitors were the most associated with serious infections (4.51 [2.19-9.26] compared to biologics and azacitidine. After a median follow-up of 4.4 [2.5-7.7] years, 27 (36%) patients died including 15 (56%) due to serious infection. Conclusion VEXAS syndrome is associated with a high incidence of serious infections especially in patients carrying the p.Met41Val mutation. The high frequency of atypical infections such as legionellosis and invasive fungal infections in patient without immunosuppressive treatment might suggest an intrinsic immunodeficiency of the disease. JAK inhibitors, used as first-line treatment, are particularly at risk of serious infections occurring early after initiation.

Prevalence and Outcome of Vexas Syndrome in Unrelated Hematopoietic Stem Cell Transplantation

Introduction Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic (VEXAS) syndrome is a newly identified monogenic disorder defined by somatic UBA1 mutations in hematopoietic progenitor cells, resulting in bone marrow failure (BMF) and systemic inflammation. This syndrome is frequently associated with myelodysplastic syndrome (MDS) or plasma cell dyscrasia. Although high-dose corticosteroids alleviate symptoms and cytopenia, allogeneic hematopoietic stem cell transplantation (HSCT) has been suggested as a potentially curative treatment. This study evaluated the safety and efficacy of unrelated HSCT for treating VEXAS syndrome and its prevalence in patients with hematologic diseases. Methods We conducted retrospective screening for UBA1 mutations in patients with BMF or plasma cell neoplasms who underwent their first HSCT from unrelated donors in Japan between 1995 and 2020. Pre-HSCT blood DNA samples and clinical data were collected from the Japanese Data Center for Hematopoietic Cell Transplantation. Multiplex real-time PCR was used to detect the most common pathogenic UBA1 mutations, including p.M41L (c.121A>C), p.M41V (c.121A>G), and p.M41T (c.122T>C), with detection limits of 0.13%, 0.5%, and 1%, respectively. The presence of these mutations was confirmed using Sanger sequencing and droplet digital PCR. In addition, we examined UBA1 mutations in 2 patients previously diagnosed with MDS but suspected of having VEXAS syndrome based on specific clinical features. Results Among 4340 patients with either BMF or plasma cell neoplasms who underwent unrelated HSCT, 1771 had available pre-HSCT blood DNA samples. The diagnoses of the 1771 patients included MDS (n = 1264), acquired aplastic anemia (n = 375), inherited BMF syndromes (n = 89), plasma cell neoplasms (n = 23), and other acquired BMF syndromes (n = 20). The median patient age at HSCT was 46 (range, 0-75; interquartile range, 20-59) years old, and the male-to-female ratio was 1.6:1. Pathogenic UBA1 mutations were detected in 2 of the 1771 patients (0.11%), both of whom were male MDS patients older than the median age (cases 1 and 2); thus, the prevalence of VEXAS syndrome within this patient subgroup was 0.36%. Both patients received chemotherapy before unrelated donor bone marrow transplantation (BMT), including induction chemotherapy and azacitidine, and achieved complete and partial responses, respectively. In addition, UBA1 mutations were detected in the 2 MDS patients at Kanazawa University Hospital (cases 3 and 4). Case 3 also underwent unrelated BMT after experiencing cytopenia relapse and inflammatory symptoms during corticosteroid reduction. The clinical characteristics and outcomes of the 4 patients with VEXAS syndrome are summarized in Table 1. Variant allele frequencies (VAFs) of UBA1 mutations ranged from 2% to 71%. The 3 patients who underwent unrelated BMT received fludarabine and busulfan-based conditioning regimens and graft-versus-graft disease (GVHD) prophylaxis with tacrolimus + methotrexate or cyclosporine + methotrexate. All 3 patients achieved neutrophil and platelet engraftment. One patient required systemic corticosteroids for acute skin GVHD. One patient died of idiopathic interstitial pneumonia 5 months after BMT. The other 2 patients were alive without relapse, chronic GVHD, or other complications at 18 and 28 months after BMT. Discussion Our results support the curative potential of allogeneic HSCT for VEXAS syndrome without excessive toxicity. The occurrence of transplant-related mortality due to idiopathic interstitial pneumonia may be related to the reported poor outcome of the p.M41V mutation following non-transplant therapy. The low prevalence of VEXAS syndrome in the current cohort may be due to the late age of onset and the rare progression to advanced MDS. The low VAFs of UBA1 mutations in the 2 patients who responded to chemotherapy before HSCT may indicate the efficacy of chemotherapy in selectively eradicating UBA1 mutant clones. Our rapid, sensitive, and cost-effective screening method for VEXAS syndrome can help optimize treatment strategies for this rare disease in future studies.

Deep Learning Based Blood Abnormalities Detection As a Tool for Vexas Syndrome Screening

Introduction The VEXAS syndrome (vacuoles, E1 enzyme, X-linked, auto-inflammatory, somatic) described in 2020 caused by mutations of the UBA1 gene, displayed a large pleomorphic array of clinical and biological features. Nevertheless, these criteria do not allow to discriminate VEXAS from other myeloid malignancies on complete blood count + differential, notably due to the absence of peripheral blood characterization of the disease. This study aimed at singling out dysplastic features indicative of VEXAS among peripheral blood (PB) polymorphonuclears (PMN) from VEXAS patients compared to healthy patients and myelodysplastic (MDS) patients. As such task is tedious and subject to operator bias, a multicentric dataset has been used to design a deep learning algorithm for automatic detection of these features, finally tested on an external validation cohort. Patient, material and methods Patients written consents were obtained and five academic centers (number from 1 to 5) were involved in this 3 steps study. Firstly, 3 academic centers (number 1 to 3) enrolled 20 patients distributed as follow: 9 patients with UBA1 mut and 6 patients UBA1 wt with no other genetic mutation as well as 5 samples from MDS patients. A total of 25 PB smears and especially PMN images were gathered and screened for various abnormalities. Two independent morphologists blindly quantified PMN predefined characteristics. This dataset of 2,824 multilabelled PMN was evaluated and tested by a two-sample Wilcoxon Rank Sum test for statistical significance between UBA1 mut and the others. Secondly, in order to automate the detection of these significant features on PMN images, a convolutional neural network (CNN) was trained using a multicentric image dataset gathered from 4 academic centers (number 2 to 5). Patients were selected based on clinical and biological symptoms suggesting VEXAS syndrome, namely fever, skin lesions, chondrites, vasculitis and/or anemia. After evaluation of the UBA1 mutation status, this multicentric cohort was separated into confirmed VEXAS (n = 19) and UBA1 WT (n=20). A total of 6,615 annotated PMN images were collected from DI systems (Cellavision ®, Lund, Sweden) with 1 to 12 PB smears per patient, yielding a 48 UBA1 mut and 33 UBA1 wt smears. All patients were males, the median age was 74 year-old (IQR 67-77) and that of control patients 72 year- old (61-79). Finally, the CNN was evaluated on an external cohort from another academic hospital (center 1), including inflammatory patients, and patients screened for VEXAS syndrome for a total cohort of 15 patients and 1,887 images. Results Four specific abnomalities were observed in PMNs from VEXAS patients as compared with healthy or myelodysplastic controls. The automatic recognition of anomalies was then cast in a multilabel classification task, where each PMN image could be assigned to one or more labels during interference. In order to take into account this specificity, a CNN was trained, composed of different layers of convolutions and pooling (Figure1). The model was set-up to output an independent binary prediction for the presence or absence of each of the four anomalies of interest. Automatic detection of these 4 anomalies by the proposed model yielded area under the curve (AUC) of 0.827; 0.837; 0.927 and 0.947 (Figure 2). Regarding the general performance on the external validation cohort, the CNN achieved a Hamming loss of 0.141, and macro and micro F1 scores of 0.588 and 0.668 respectively. Discussion This study suggests that computer-assisted analysis of PB smears, focusing on suspected VEXAS cases, could provide valuable insights to determine which patients should undergo molecular testing. A deep-learning approach leveraging previously identified peripheral blood indicators and automatic analyzers is thus presented, which can help hematologists orient their suspicion before initiating further analysis.

The UBE2J2/UBE2K-MARCH5 Ubiquitination Machinery Regulates Apoptosis in Response to Venetoclax in Acute Myeloid Leukemia

Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins on the mitochondrial membrane. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, however, some patients display upfront resistance, and most patients will ultimately relapse. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. To systematically identify key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the AML cell line MV4-11. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA ( PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL ( BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells. Furthermore, depletion of the E2 ubiquitin-conjugating enzymes, UBE2J2 and UBE2K, ranked highly among the venetoclax sensitizers. We validated that deletion of either UBE2J2 or UBE2K increased induction of apoptosis in multiple AML cell lines and patient-derived xenograft (PDX) cells upon venetoclax treatment and was deleterious as single gene perturbation in some models. Exploiting the Broad Institute Cancer DepMap dataset, which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on the E3 ligase MARCH5. We previously identified the repression of MARCH5 as a strong inducer of apoptosis in AML. Since E2s coordinate with ubiquitin E3 ligases to execute ubiquitination, this DepMap result suggested that UBE2J2 and UBE2K serve as MARCH5 E2 partners. To test this hypothesis, we utilized NanoBiT technology, a structural complementation reporter system, to detect protein interactions between MARCH5 and the E2 candidates. LgBIT and SmBIT, two split subunits of luciferase, were fused with MARCH5 and the E2 proteins, respectively. The luminescent signal was activated upon the co-expression of LgBIT-MARCH5 with either of the SmBIT-tagged E2 proteins but not an empty vector, suggesting that MARCH5 and UBE2J2/UBE2K constitute ubiquitination machinery that regulates apoptosis in AML. We next tested the possible redundancy between the two E2s and showed that the double knockout of UBE2J2/ UBE2K further enhanced the venetoclax sensitivity. MARCH5 depletion results in increased NOXA expression, an important node in dictating venetoclax response. Several reports indicated that NOXA is also a critical downstream mediator of MARCH5. In contrast, we previously showed that MARCH5 can regulate apoptosis independently of NOXA in AML. To reassess the role of NOXA and other pro-apoptotic proteins in MARCH5-mediated apoptosis, we conducted an unbiased CRISPR rescue screen in a MARCH5-dTAG degradation system derived from PDX17-14, a complex karyotype, MLL-AF10 PDX model. BAX was the top rescuing target, emphasizing that apoptosis induction is the main mechanism accounting for the growth inhibition of MARCH5-depleted cells. However, depletion of other pro-apoptotic BCL2 members, including NOXA and BIM, did not rescue MARCH5 depletion in this screen consistent with our previously published data. Here, we confirmed that NOXA KO does not rescue MARCH5 depletion in additional AML models. Similarly, KO of UBE2J2 or UBE2K can repress AML cell growth and increase venetoclax sensitivity even in the context of NOXA KO. Our study highlights that UBE2J2 and UBE2K are two important functional partners of MARCH5 in regulating apoptosis in AML cells and can serve as additional targets for enhancing venetoclax efficacy. Unbiased screening and low-throughput target validation further emphasize that the MARCH5 ubiquitination machinery regulates apoptosis in AML cells largely in a NOXA-independent manner. Additional studies are needed to dissect the MARCH5/UBE2J2/UBE2K complex-mediated apoptosis regulation in AML.

Genotype-Phenotype Mapping Via Single-Cell Multi-Omics Identifies Therapeutic Vulnerabilities in Vexas Syndrome

VEXAS (Vacuoles E1 enzyme X-linked Autoinflammatory Somatic) syndrome originates from hematopoietic stem cells (HSCs) carrying a somatic UBA1 p.M41 mutation. UBA1 p.M41 mutation leads to systemic inflammation and bone marrow failure, with poor survival. Other than allogenic stem cell transplantation, no curative therapy is available for VEXAS. This arises from the challenge of defining the phenotypic effects of the UBA1 p.M41 mutation in primary human samples, confounded due to the mixture of wildtype and mutated cells. Thus, it is essential to resolve the admixture of wildtype and UBA1 p.M41 mutated cells to understand the effect of the UBA1 p.M41 mutation in the disease-initiating HSCs. By doing so, we can identify potential mutant-specific therapeutic vulnerabilities to specifically eliminate UBA1 p.M41 HSCs. To map genotype-phenotype relationship in human VEXAS samples, we deployed and developed single-cell multi-omics including Genotyping of Transcriptomes (GoT) and Genotyping of Targeted loci with Chromatin Accessibility (GoT-ChA), allowing the capture of UBA1 genotype, cell identity, transcriptome, chromatin accessibility and signaling pathways at single cell resolution ( Fig.1a). We applied GoT to CD34+ enriched bone marrow mononuclear cells (BMMNCs) from VEXAS primary human samples ( n = 31,406 cells across 6 patients). Integrating UBA1 genotyping with gene expression data showed an enrichment of the mutation in myeloid-committed cells and depletion in B and T cells. Interestingly, this mutation is also enriched in natural killer (NK) cells and plasmacytoid dendritic cells (both originating from lymphoid progenitors), which have not been previously reported. This suggests that VEXAS HSPCs display a skewed differentiation towards myeloid, NK, and plasmacytoid dendritic cells, which may contribute to inflammation in VEXAS. High-resolution mapping of UBA1 p.M41 vs. UBA1 wt HSCs revealed myeloid priming of mutated HSCs. UBA1 p.M41 HSCs showed an increased inflammation signature (Interferon alpha and gamma response pathways) along with increased unfolded protein response (UPR), glycolysis and decreased autophagy pathways. We validated UPR pathway activity by showing the expected increase in XBP1 splicing in UBA1 p.M41 HSPCs using targeted capture of the unconventional splice site. Interestingly, transcriptome data also showed a global translational downregulation accompanied by specific ATF4 target gene enrichment ( Fig.1b). These results suggest a preferential induction of the PERK arm of the UPR. Surface protein expression analysis supported the transcriptomic data, showing upregulation of CD38, CD13 and HLA-DR, denoting myeloid bias and increased inflammatory response in UBA1 p.M41 HSCs. Next, we performed GoT-ChA on CD34 + cells from VEXAS patients (n = 27,894 cells across 3 patients) to study mutant-specific chromatin accessibility. Complementary to the transcriptome data, UBA1 p.M41 HSCs showed increased motif accessibility for TFs associated with myelopoiesis, indicative of myeloid priming. We also observed enrichment for ATF4 and CEPBD TF motifs - downstream of PERK pathway of UPR ( Fig.1b). To validate these findings, we developed GoT-ChA integrated with intracellular protein capture and demonstrated an increased expression of phosphorylated eIF2 and ATF4 proteins in the UBA1 p.M41 HSCs. Finally, to functionally validate the specific role of the PERK pathway on the fitness of UBA1 p.M41 HSCs, we performed assays using mobilized CD34 + cells in-vitro. UBA1 Inhibition in normal CD34 + cells led to ATF4 up-regulation, which correlated with cell survival. The addition of PERK inhibition reduced ATF4 expression and resulted in markedly reduced CD34+ viability. In contrast, inhibiting IRE1 pathway along with UBA1 inhibition did not affect HSC viability. Altogether, we demonstrated that human VEXAS HSCs show a cell-intrinsic inflammatory phenotype and are myeloid biased. Moreover, mutated HSCs tolerate proteome toxicity through activation of the PERK pathway as a part of UPR, providing a fitness advantage to the UBA1 p.M41 mutated HSCs. Targeting the PERK pathway may thus represent a new strategy for overcoming VEXAS syndrome via precision targeting of UBA1 p.M41 mutated HSCs.

UBE2N Regulates Oncoprotein Networks in Myeloid Malignancies

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) originate from clonal mutant hematopoietic stem and progenitor cells, referred to as leukemic stem and progenitor cells (LSPCs). Ubiquitination is an essential process for various cellular maintenance functions, including protein degradation and scaffolding, which is often perturbed in a variety of cancers. Ubiquitination of protein substrates involves three related enzymes: the E1 activating enzyme, the E2 conjugating enzyme, and the E3 ligase. In humans, there are up to 40 E2 conjugating enzymes, among which Ubiquitin conjugating enzyme E2 N (UBE2N) stands out as one of the most crucial and overexpressed E2 enzymes in AML cells. Our recent study reported that UBE2N is a druggable target in MDS and AML (Barreyro et al., Science Translational Medicine, 2022), and we found that its catalytic active site cysteine 87 is required for various AML mouse models, including MLL-AF9, FLT3-ITD, AML1-ETO9a, and MN1 (unpublished). These observations indicate that the catalytic function of UBE2N is critical for AML cells but dispensable for normal hematopoiesis. To examine the active state of UBE2N, we investigated the “charged” version of UBE2N, which contains a ubiquitin bound to cysteine 87, in normal hematopoietic cells and in a AML cell line panels. Our findings confirmed that the activated form of UBE2N is highly expressed in human AML cells compared to normal hematopoietic cells. UBE2N exclusively synthesizes Lysine-63 (K63)-linked polyubiquitin chains on target proteins in conjunction with selected E3 ligases, leading to the stabilization and/or activation of protein substrates. To identify the relevant targets of UBE2N in AML, we conducted a ubiquitin-enrichment followed by a proteomic analysis in isogenic UBE2N-deficient AML cells compared to WT AML cells. We identified ~300 proteins that are ubiquitinated by UBE2N. Importantly, UBE2N regulates a network of leukemia-associated oncoproteins, including SYNCRIP, HUWE1, NPM1, TYK2, IKBKG, VAV1, BAX, IGF2BP2, IRF4, IRAK4, RBMX, STAT3, and BTK. UBE2N can interact with various E3 ligases in a cell- and context-dependent manner. To gain insight into the major E3 ligase that cooperates with activated UBE2N in AML, we performed a proximity-based labeling assay followed by mass spectrometry. This interactome screen identified Tripartite motif containing 21 (TRIM21) as the top E3 ligase that partners with UBE2N in AML cells. TRIM21 is overexpressed in AML patients compared to healthy controls. Moreover, deletion of TRIM21 in AML cells impaired cell viability and proliferation in vitro and prolonged survival in a xenograft model in vivo, which phenocopies the loss of UBE2N. Importantly, restoration of TRIM21 rescued the functional defect of UBE2N-deficient AML cells, indicating that the UBE2N-TRIM21 axis is operational in AML. To prioritize the UBE2N-dependent oncoprotein network, we focused on UBE2N-TRIM21 regulated signaling in AML. For this, a gene expression analysis was performed in UBE2N-deficient AML cells that were restored with TRIM21. The top UBE2N-TRIM21 oncoprotein network that emerged was related to interferon-STAT signaling. Additionally, both UBE2N and TRIM21 were found to regulate STAT3 ubiquitination. Deletion or inhibition of UBE2N catalytic activity significantly reduced total and activated STAT3 in AML cells. Finally, the expression of constitutively active STAT3 partially restored the functional defect of AML cells treated with a UBE2N inhibitor, indicating that STAT3 function, a known driver of LSPCs, is regulated via the UBE2N-TRIM21 axis in AML. In summary, our data reveal that UBE2N regulates oncoprotein networks in AML and that inhibiting UBE2N catalytic function, such as with small molecule inhibitors, effectively suppresses LSPCs.

An adaptive stress response that confers cellular resilience to decreased ubiquitination

Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.