Immunoglobulin Kappa Research Articles

Determination of biomarker candidates for the placenta accreta spectrum by plasma proteomic analysis

Placenta accreta spectrum (PAS) presents a significant obstetric challenge, associated with considerable maternal and fetal-neonatal morbidity and mortality. Nevertheless, it is imperative to acknowledge that a noteworthy subset of PAS cases remains undetected until the time of delivery, thereby contributing to an augmented incidence of morbidity among the affected individuals. The delayed identification of PAS not only hinders timely intervention but also exacerbates the associated health risks for both the maternal and fetal outcomes. This underscores the urgency to innovate strategies for early PAS diagnosis. In this study, we aimed to explore plasma proteins as potential diagnostic biomarkers for PAS. Integrated transcriptome and proteomic analyses were conducted to establish a novel diagnostic approach. A cohort of 15 pregnant women diagnosed with PAS and delivering at Inonu University Faculty of Medicine between 01/04/2021 and 01/01/2023, along with a matched control group of 15 pregnant women without PAS complications, were enrolled. Plasma protein identification utilized enzymatic digestion and liquid chromatography-tandem mass spectrometry techniques. Proteomic analysis identified 228 plasma proteins, of which 85 showed significant differences (P < 0.001) between PAS and control cases. We refined this to a set of 20 proteins for model construction, resulting in a highly accurate classification model (96.9% accuracy). Notable associations were observed for proteins encoded by P01859 (Immunoglobulin heavy constant gamma 2), P02538 (Keratin type II cytoskeletal 6A), P29622 [Kallistatin (also known as Serpin A4)], P17900 (Ganglioside GM2 activator Calmodulin-like protein 5), and P01619 (Immunoglobulin kappa variable 3–20), with fold changes indicating their relevance in distinguishing PAS from control groups. In conclusion, our study has identified novel plasma proteins that could serve as potential biomarkers for early diagnosis of PAS in pregnant women. Further research and validation in larger PAS cohorts are necessary to determine the clinical utility and reliability of these proteomic biomarkers for diagnosing PAS.

P134 Characterization for the similarity assessment between proposed biosimilar SB17 and ustekinumab reference product using state-of-the-art analytical method

Abstract Background SB17 is being developed as a proposed biosimilar to ustekinumab reference product (RP), a human monoclonal antibody (IgG1 kappa immunoglobulin) that binds to the common p40 subunit of cytokines interleukin (IL)-23 and IL-12. Binding to this subunit prevents interaction with their receptor, resulting in modulation the immune system responses which play a key role in the inflammatory disease. The objective of this study was to demonstrate structural, physicochemical, and biological similarity between ustekinumab RP and SB17 using various state-of-the-art analytical methods. Methods Comprehensive analytical characterization was conducted with side-by-side comparison of SB17 with the EU and US ustekinumab RP using various analytical methods. Comparisons included purity, product-related impurity, charge heterogeneity, primary structure, post-translational modification, higher order structure, quantity, Fab-related biological activities (potency and binding activity), and Fc-related biological activities. Results Based on the analytical similarity assessment, the structural, physicochemical, and biological characterization results demonstrated that SB17 is highly similar to the EU and US ustekinumab RP. In the structural aspects, it was confirmed that there is no clinically meaningful difference between post-translational modification profiles and higher order structures of SB17 compared to the ustekinumab RP. Product-related impurities in the form of aggregates were also confirmed to be similar. SB17 has a lower of acidic and basic peaks compared with ustekinumab RP, due to the difference in producing cell line. Ustekinumab RP is expressed in an Sp2/0 cell line, whereas SB17 is expressed in CHO cell line. However, the difference between SB17 and ustekinumab RP in the charge variants is not considered clinically meaningful since equivalent biological activity was observed. In case of mechanism of action (MoA)-related biological activities, SB17 is highly similar to the EU and US ustekinumab RP with respect to overall critical and non-critical quality attributes analyzed. Moreover, similarity or lack of activity in Fc-related biological activities was also confirmed. Based on these results, SB17 is expected to have highly similar safety and efficacy as compared to ustekinumab RP. Conclusion In summary, the overall analytical characterization and similarity assessment results show that SB17 is highly similar to the EU and US ustekinumab RP in terms of structural, physicochemical, biophysical, biological attributes.

Effects of early treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) on the bronchoalveolar lavage proteome and oxylipids during bovine respiratory syncytial virus (BRSV) infection.

Non-steroidal anti-inflammatory drugs (NSAID) are not recommended for use against pneumonia in humans, but are commonly utilised against bovine respiratory disease. This study aimed to determine if the use of NSAIDs in the early phase of bovine respiratory syncytial virus (BRSV)-infection limits pulmonary inflammation. Four to nine-week old calves were infected with BRSV by aerosol and were treated with either meloxicam intravenously on day (D)4 (n = 5, MEL), acetylsalicylat-DL-lysin intravenously on D4 and D5 (n = 5, ASA), or were left untreated as controls (n = 5, CTR). Clinical signs were monitored daily until necropsy on D7, BRSV-RNA was detected in nasal swabs and bronchoalveolar lavage (BAL) by RT-qPCR, inflammatory cells and proteins were identified in BAL by cytology and label-free quantitative mass spectrometry-based proteomics, respectively, and oxylipids were quantified in BAL and plasma by liquid chromatography tandem mass spectrometry with triple quadrupole mass detectors. The calves developed mild to moderate signs of respiratory disease and, with the exception of one MEL-treated and one ASA-treated calf, limited lung lesions. None of the treatments had a significant effect on virus replication, clinical signs or lung lesion extent. Relative to controls, both treatments initially induced a downregulation of proteins in BAL. Immunoglobulin (Ig)-related proteins, such as the Ig kappa and lambda locus and the joining chain of IgA and IgM, were downregulated in MEL-treated calves compared to controls. In addition, meloxicam induced an increased neutrophil influx in BAL in response to BRSV, possibly related to a reduction in plasma prostaglandin, and to a downregulation of The Liver X Receptor/ Retinoid X Receptor (LXR/RXR), the Farnesoid X Receptor (FXR)/RXR and the 24-Dehydrocholesterol Reductase (DHC24) signalling pathways in the lung. The risk of NSAIDs to increase neutrophil activity during stimulation with BRSV or other toll-like receptor 4 agonists needs to be investigated further. Since augmented neutrophil responses can be detrimental, the results of the present study do not support the use of NSAIDs to prevent the clinical expression of BRSV-infection.

Deep and unbiased proteomics, pathway enrichment analysis, and protein-protein interaction of biomarker signatures in migraine.

Currently, there are no biomarkers for migraine. We aimed to identify proteomic biomarker signatures for diagnosing, subclassifying, and predicting treatment response in migraine. This is a cross-sectional and longitudinal study of untargeted serum and cerebrospinal fluid (CSF) proteomics in episodic migraine (EM; n = 26), chronic migraine (CM; n = 26), and healthy controls (HC; n = 26). We developed classification models for biomarker identification and natural clusters through unsupervised classification using agglomerative hierarchical clustering (AHC). Pathway analysis of differentially expressed proteins was performed. Of 405 CSF proteins, the top five proteins that discriminated between migraine patients and HC were angiotensinogen, cell adhesion molecule 3, immunoglobulin heavy variable (IGHV) V-III region JON, insulin-like growth factor binding protein 6 (IGFBP-6), and IGFBP-7. The top-performing classifier demonstrated 100% sensitivity and 75% specificity in differentiating the two groups. Of 229 serum proteins, the top five proteins in classifying patients with migraine were immunoglobulin heavy variable 3-74 (IGHV 3-74), proteoglycan 4, immunoglobulin kappa variable 3D-15, zinc finger protein (ZFP)-814, and mediator of RNA polymerase II transcription subunit 12. The best-performing classifier exhibited 94% sensitivity and 92% specificity. AHC separated EM, CM, and HC into distinct clusters with 90% success. Migraine patients exhibited increased ZFP-814 and calcium voltage-gated channel subunit alpha 1F (CACNA1F) levels, while IGHV 3-74 levels decreased in both cross-sectional and longitudinal serum analyses. ZFP-814 remained upregulated during the CM-to-EM reversion but was suppressed when CM persisted. CACNA1F was pronounced in CM persistence. Pathway analysis revealed immune, coagulation, glucose metabolism, erythrocyte oxygen and carbon dioxide exchange, and insulin-like growth factor regulation pathways. Our data-driven study provides evidence for identifying novel proteomic biomarker signatures to diagnose, subclassify, and predict treatment responses for migraine. The dysregulated biomolecules affect multiple pathways, leading to cortical spreading depression, trigeminal nociceptor sensitization, oxidative stress, blood-brain barrier disruption, immune response, and coagulation cascades. NCT03231241, ClincialTrials.gov.

Autoantibody profiling of patients with immune checkpoint inhibitor-associated myocarditis: a pilot study.

Immune checkpoint inhibitor (ICI)-associated myocarditis is a rare, but potentially fatal, immune-related adverse event. Hence, identifying biomarkers is critical for selecting and managing patients receiving ICI treatment. Serum autoantibodies (AAbs) in patients with ICI myocarditis may serve as potential biomarkers for predicting, diagnosing, and prognosing ICI myocarditis. We conducted a pilot study using a human proteome microarray with approximately 17,000 unique full-length human proteins to investigate AAbs associated with ICI myocarditis. AAb profiling was performed using sera collected from three patients with ICI myocarditis before the start of ICI treatment and immediately after myocarditis onset. All patients received anti-programmed death-1 antibody monotherapy. At baseline, 116, 296, and 154 autoantigens reacted positively to immunoglobulin G (IgG) in the serum samples from Cases 1, 2, and 3, respectively. Among these proteins, the recombination signal-binding protein for the immunoglobulin kappa J region (RBPJ) was recognized by all three samples, and 32 autoantigens were recognized by any two of the three samples. At the onset of ICI myocarditis, compared to baseline, 48, 114, and 5 autoantigens reacted more strongly with IgG in the serum samples from Cases 1, 2, and 3, respectively. Among these, antibodies against eukaryotic translation initiation factor 4E binding protein 3 (EIF4EBP3) were the most upregulated, with a 38-fold increase. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses highlighted that B-cell receptor signaling, leukocyte transendothelial migration, and thymus development were among the most affected pathways. Enrichment analyses using DisGeNET revealed that proteins reacting to AAbs detected in patients with ICI myocarditis are associated with several diseases, including dilated cardiomyopathy and muscle weakness. This pilot study provides the first integrated analysis of serum AAb profiling in patients with ICI myocarditis and identifies novel candidate markers associated with an increased risk of developing ICI myocarditis and its pathogenesis. However, our results require further independent validation in clinical trials involving a larger number of patients.

Clinical specificity of two assays for immunoglobulin kappa and lambda free light chains.

Free light chain (FLC) assays and the ratio of κ/λ are recommended for diagnosis, prognosis and monitoring of plasma cell dyscrasias (PCD). Limited data exists on FLC clinical specificity in patients diagnosed with other conditions. We assessed the κ, λ, and κ/λ FLC ratio using the FreeLite assay and the Sebia FLC ELISA assay in 176 patients with clinical presentations of fatigue, anemia, polyclonal hypergammaglobulinemia, joint disorders, kidney disease and non PCD-cancers with no monoclonal protein observed on serum protein electrophoresis or MASS-FIX immunoglobulin isotyping. Manufacturer defined reference intervals (RI) and glomerular filtration rate (GFR) specific RI (renal RI) were utilized. For the κ/λ ratio, 68.7 % (121/176) of specimens on the FreeLite and 87.5 % (154/176) of specimens on the Sebia assay were within RI. For κ, 68.2 % (120/176) and 72.2 % (127/176) of results were outside RI for FreeLite and Sebia respectively. For λ, 37.5 % (66/176) and 84.1 % (148/176) of FreeLite and Sebia results were outside RI. With FreeLite and Sebia, patients with kidney disease (n=25) had the highest κ/λ ratios. 44 patients (25.0 %) had GFR <60 mL/min/BSA. When renal RI were applied, 13.6 % had a FLCr outside the renal RI with FreeLite, and 4.5 % with Sebia. In a cohort of patients with signs and symptoms suggestive of PCDs, but ultimately diagnosed with other conditions, Sebia FLC had improved clinical specificity relative to FreeLite, if one was using an abnormal κ/λ ratio as a surrogate for monoclonality.

Abstract A063: Epigenetic regulation of Neuregulin 1 promotes breast cancer progression associated to hyperglycemia

Abstract Hyperglycemia is a risk factor for breast cancer-related morbidity and mortality. Hyperglycemia induces Neuregulin 1 (Nrg1) overexpression in breast cancer, which subsequently promotes tumor progression. However, molecular mechanisms underlying hyperglycemia-induced Nrg1 overexpression remain poorly understood. Here, we show that hyperglycemia causes active histone modifications at the Nrg1 enhancer, forming enhanceosome complexes where recombination signal binding protein for immunoglobulin kappa J region (RBPJ), E1A binding protein p300 (P300), and SET domain containing 1A (SETD1A) are recruited to upregulate Nrg1 expression. Deletions in RBPJ-binding sites causes hyperglycemia-controlled Nrg1 levels to be downregulated, resulting in decreased tumor growth in vitro and in vivo. Mice with modest-temporary hyperglycemia, induced by low-dose short-exposure streptozotocin, display accelerated tumor growth and lapatinib resistance, whereas combining lapatinib with N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S42 phenylglycine t-butyl ester (DAPT) ameliorates tumor growth under these modest hyperglycemic conditions by inhibiting NOTCH and EGFR superfamilies. NOTCH activity is correlated with NRG1 levels, and high NRG1 levels predicts poor outcomes, particularly in HER2-positive breast cancer patients. Our findings highlight the hyperglycemia-linked epigenetic modulation of NRG1 as a potential therapeutic strategy for treating breast cancer patients with diabetes. Citation Format: Changhu Lee, Min Kim, Chanho Park, Woobeen Jo, Jiyoung Park. Epigenetic regulation of Neuregulin 1 promotes breast cancer progression associated to hyperglycemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A063.

Discovery of Unexpected Phospholipid-Binding Proteins in Antiphospholipid Syndrome Patients

INTRODUCTION: The antiphospholipid syndrome (APS), initially described nearly 4 decades ago, remains enigmatic with unclear pathophysiology. In addition to the lack of a mechanistic diagnostic test, APS exhibits considerable phenotypic heterogeneity, including the occurrence of “non-consensus” clinical manifestations (i.e. other than vascular thrombosis and pregnancy complications). Despite the evidence that phospholipid-binding factors are highly likely to be central to APS pathogenesis, the full complement of phospholipid-binding proteins has yet to be systemically assayed. Proteomic profiling by label-free quantitation (LFQ) may identify patterns of protein binding to phospholipid that are central to APS pathogenesis and thereby elucidate a basis for the heterogeneous clinical presentations of APS. METHODS: C18 silica beads were coated with phospholipid by incubation with a suspension of 30% phosphatidylserine/70% phosphatidylcholine. Plasmas from 4 groups: 1) APS patients (n=11), 2) APL positive patients who lacked clinical manifestations (“APL”) (n=11), 3) non-APL patients with thrombosis (“nonAPL”)(n=11), and 4) normal healthy controls (“normal”) (n=6) were incubated with the phospholipid-coated beads, and eluates were processed by gel electrophoresis followed by tryptic digestion. The resulting peptides were analyzed by LC-MS/MS on a Thermo Orbitrap Fusion Lumos. Protein identification, false discovery rate (FDR) estimation, and LFQ were performed using Proteome Discoverer 2.2 (Thermo). LFQ values were normalized by overall peptide abundance, and relative quantitation was obtained. Groupwise comparisons were performed using PD 2.2 in order to identify differentially enriched proteins. RESULTS: A total of 1,210 proteins were identified, with 875 proteins identified at high confidence (q&amp;lt;0.01). Based on gel electrophoresis, all samples had comparable amounts of total protein, and normalization of LFQ data on the basis of total proteins was therefore performed. Differentially expressed proteins between groups were identified using both fold-change and -log p value cutoffs (Figure 1a). Primary analysis focused on identifying proteins that were consistently preferentially bound from the APS plasmas compared to the normal controls. Novel findings included the following proteins related to coagulation, immune response, and to lipid binding that bound preferentially from APS plasmas compared to normal plasmas (APS:control ratios shown in parentheses): coagulation factor VII (10.4), heparin cofactor 2 (9.6), CD14 (6.1), apolipoprotein L1 (6.0), and apolipoprotein L2 (5.5). Additional proteins that preferentially bound from APS plasmas included OAF (13.7), BRSK1 (13.2), MINPP1 (12.5), fetuin B (10.5), SHBG (8.9), ADAMTS-like protein 4 (8.5), SERPIN A4 (7.6), alpha-2-HS glycoprotein (6.7) and inter-alpha-trypsin inhibitor heavy chains (H3, H4 and H1; 8.5, 6.6, 6.5 respectively). Consistent with prior knowledge of APS, and validating this LFQ approach immunoglobulins and complement proteins were also among the preferentially bound proteins and included: Ig heavy chain 1-3 (15.6), complement C9 (13.3), complement C4-B, (7.1), complement component C8 beta chain (5.6), Ig kappa variable 1D-8 (5.6), and Ig lambda-like polypeptide 1 (3.9). Interestingly, a number of identified proteins showed intermediate abundance (between APS and control) in the APL positive patients without clinical manifestations (“APL”) and APL negative patients with thrombosis (“nonAPL”) (see figure 1B-D for examples), suggesting a potential dose response effect. CONCLUSIONS: 1) These results are a first demonstration of concept for the ability of LFQ proteomics to address the complexity of APS through analysis of phospholipid-bound proteins. 2) A number of phospholipid-binding proteins, many of which have not been previously implicated in APS pathogenesis, are preferentially present in APS patient plasma 3) Some plasmas from APL and non-APL patients also show increased binding of some of these proteins compared to the normal controls, suggesting that LFQ may identify markers for subgroups of patients within these 2 categories.

Genomic Determinants of Therapy Response in ETV6::RUNX1 Leukemia

ETV6::RUNX1 leukemia is the second most common childhood B-cell acute lymphoblastic leukemia subtype. Although it has a low overall relapse risk, a significant proportion of relapses occur within this subtype due to its relatively high incidence. No consistent genomic biomarkers have been identified that predict therapy response beyond early therapy response as measured by minimal residual disease at the end of induction. In this study, our aim was to use multiomic data from 175 ETV6::RUNX1 leukemias treated according to the NOPHO ALL2000 and ALL2008 protocols to identify genomic features that predict therapy response at presentation. Cases were classified into three responder groups based on MRD at end-of-induction (EOI) therapy: fast responders with negative MRD, intermediate responders with positive but &amp;lt;0.1% MRD, and slow responders with &amp;gt;0.1% MRD. Response data was also collected at mid-induction (day 15) and end-of-consolidation (EOC), when available. Diagnostic and remission DNA were analyzed using array-based copy number data (n=142) or whole genome sequencing (WGS) (n=33), and diagnostic bulk RNA sequencing (n=51) was complemented with single cell multiomics (RNA, surface protein and VDJ sequencing) data from both healthy (n=8) and leukemic bone marrow cells upon chemotherapy (n=8). Overall, the frequency of somatic genetic changes was not significantly associated with treatment response suggesting that genetic diversification through elevated random mutagenesis events may not underlie the drug resistant phenotype. However, analysis of mutational signatures revealed that fast-responding leukemic blasts frequently exhibited the APOBEC mutational signature and had higher cell cycle activity (Figure 1A). Accordingly, we show that expression of A3B is highly specific to cycling cells also in healthy bone marrow hematopoietic progenitor cells, pro- and pre-B cells. We also examined RAG-mediated genetic instability that manifests in focal structural variations and has highest activity in ETV6::RUNX1leukemia (Figure 1B). We confirmed several recurrent RAG off-target sites, including significant enrichment of the known risk locus TBX1LR1 in slow responding cases. Strikingly, slow-responding leukemias had also frequent rearrangements in the immunoglobulin kappa genes and the kappa-deleting elements, despite their pro-B-like transcriptome state upon diagnosis. Based on the single cell multiomics profiles, we found that upon treatment the blast phenotype shifts towards an immature-B-like transcriptome, with concomitant expression of Ig light chains with productive sequence, thereby permitting activation of BCR signaling that provides a survival advantage. At CNV level, we found that the chromosomal alterations related to the translocation event associated with treatment response. Specifically, slow responders had more often the amplification of der(21)t(12;21) chromosome, impacting expression of USP5, TIGAR and SLC2A3, associated in earlier studies to chemoresistance and tumorigenesis. In comparison, fast responders were often missing a region of chromosome 12p with lower expression of several genes including oncogenic KRAS. In summary, combining multiomic and response data, we characterized genetic alterations and mutational processes that affect the treatment response in ETV6::RUNX1leukemia. Our findings highlight the genetic and transcriptomic heterogeneity of this common pediatric leukemia subtype and its implications for treatment sensitivity, paving the way for better classification of the disease at presentation.

Targeting Loss of the Tumor Suppressor TENT5C in Multiple Myeloma

Introduction: Multiple myeloma (MM) is a genetically complex and heterogeneous neoplasm in which multiple genomic events lead to tumor development and progression. Loss of TENT5C through mutation or deletion on chromosome 1p occurs in more than 20% of newly diagnosed MM patients and is associated with a poor progression free and overall survival. Although, many studies have identified TENT5C as a tumor suppressor gene in MM, the impact of gene loss on MM physiopathology remain to be elucidated. Here we sought to identify drugs which may specifically target TENT5C-null MM cells using targeted and high-throughput drug screens (HTS). Methods: We generated CRISPR/Cas9 knockout (ko) of TENT5C in wild type (wt) U266 and KMS-11 MM cell lines and over-expressed (oe) TENT5C in MM cell lines with biallelic inactivation (H929 -/- and LP-1 -/-). Modified cell lines were compared to their original counterparts for gene expression, proliferation, and cell cycle differences. Targeted and HTS were performed to identify synthetic lethal pathways related to loss of TENT5C in MM that will result in specific cell death. Results: In TENT5C ko cells the RNA poly(A) polymerase activity was decreased at two TENT5C top targeted substrates, immunoglobulin kappa light chain and signal sequence receptor. Consistent with TENT5C tumor suppressor function, cell proliferation analysis showed that TENT5C ko cells had a higher growth rate than TENT5C wt cells. Cell cycle analysis, after nocodazole synchronization, showed TENT5C-null cells had an increased G2/M population compared to TENT5C-positive cells (U266 TENT5C ko 19.86% vs. TENT5C wt 2.6%, p&amp;lt;0.001; H929 TENT5C -/- 8.22% vs. TENT5C oe 1.18%, p&amp;lt;0.001), indicating an increase in cell cycle progression in TENT5C-null cells. Given our prior data indicating an increase in splicing abnormalities and non-homologous end joining (NHEJ) activation in TENT5C -/- patient samples, we performed growth inhibition curves using appropriate drugs. The spliceosome inhibitors pladienolide B and isoginkgetin affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=0.58 vs. TENT5C wt GI 50=1.43 nM, U266 TENT5C ko GI 50=7.03 vs. TENT5C wt GI 50=13.72 µM, p&amp;lt;0.001, respectively) as did NHEJ inhibitors STL127705 and SCR7 (U266 TENT5C ko GI 50=22.5 vs. TENT5C wt GI 50=62.55 µM, U266 TENT5C ko GI 50=41.93 vs. TENT5C wt GI 50=117.38 µM, p&amp;lt;0.01). These results were confirmed on the other modified cells to ensure TENT5C specificity. A HTS using the LOPAC1280 library also identified several hits specifically affecting TENT5C null cells. The hits were categorized according to their targets to identify pathway enrichment. Inhibitors targeting the cell cycle (roscovitine, apigenin, SP60012) and angiogenesis (amsacrine) were selected for validation and further investigation. The differential effect observed in the HTS data was confirmed with full dose kill-curves on the same cell line (U266) and validated in another TENT5C ko cell line (KMS-11) to ensure target specificity. For example, amsacrine, an angiogenesis inhibitor, exhibited differential activity onT ENT5C ko and TENT5C wt cells (U266 TENT5C ko GI 50=5.63 µM vs. U266 TENT5C wt GI 50=9.19, p&amp;lt;0.001; KMS-11 TENT5C ko GI 50=9.26 µM vs. KMS-11 TENT5C wt GI 50=14.38, p&amp;lt;0.001). Similarly, roscovitine, a CDK inhibitor, affected TENT5C ko cells at lower concentrations than TENT5C wt cells (U266 TENT5C ko GI 50=23.33 vs. U266 TENT5C wt GI 50=34.91 µM; KMS-11 TENT5C ko GI 50=13.47 vs. KMS-11 TENT5C wt GI 50=29.33 µM, p&amp;lt;0.001). Additionally, cell cycle analysis showed that roscovitine induced a G 0/G 1 arrest in a dose-response manner in U266 and KMS-11 cells. Interestingly, TENT5C ko cells were more sensitive to the CDK inhibitor, and the arrested KMS-11 G 0/G 1 populations increased from 3.35 in TENT5C wt to 6.46 % in TENT5C ko and from 15.51 in TENT5C wt to 21.15 % in TENT5C ko (p&amp;lt;0.05), after treatment with 12.5 and 25 µM of roscovitine, respectively. The same trend was also validated in U266 TENT5C ko cells to ensure target specificity. Conclusion: Our results indicate a tumor suppressor role of TENT5C in MM, with antiproliferative properties and a susceptibility to spliceosome and NHEJ inhibitors. The HTP screen identified other targets that are currently undergoing validation leading to the identification of the molecular mechanisms underlying this function.

Immunoglobulin Light Chains Control Permissiveness to Malignant B-Cell Transformation By RAS-Pathway Lesions

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) cause constitutive ERK-activation which can lead to divergent outcomes. ERK-activation not only drives early stages of pre-B cell differentiation (Yasuda et al., 2008) but also induces negative selection and cell death to eliminate aberrantly activated B-cells at later stages of development (Limnander et al., 2011). Germline mutations in this pathway cause lymphoproliferative disorder and autoimmunity but not leukemia. Furthermore, activating lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development, but they are rarely found in B-cell receptor (BCR)-expressing mature B-cell derived tumors ( Figure 1). These findings are consistent with previous observations that transgenic mouse models for RAS-oncogene were able to transform BCR-negative B-cell precursors to develop B-cell acute lymphoblastic leukemia (Chan et al., 2020). In contrast, transgenic expression of these oncogenes fails to transform BCR-positive mature B-cells (Mullins et al., 2013; Chung et al., 2014). Signals from the BCR, consisting of immunoglobulin (Ig) heavy chains (μ HC) and conventional light chains (kappa LC or lambda LC), and its precursor the pre-BCR, consisting of μ HC and surrogate light chains (SLC; λ5 and VpreB), regulate the development and function of B-cells. Here, we investigated whether structural elements of the (pre-) BCR determine the outcome of oncogenic RAS activation and how oncogenic RAS in B-ALL avoids cell death to promote leukemogenesis. Results: We first studied the impact of oncogenic RAS-activation in BCR-positive mantle cell lymphoma cells. Expression of NRAS G12D in BCR-positive mantle cell lymphoma (JeKo-1) cells transiently reduced the surface expression of μ HC and eventually resulted in depletion of cells from culture in growth competition assays. We then examined whether CRISPR/Cas9-based genetic ablation of BCR components will enable permissiveness to oncogenic RAS signaling. Despite loss of μ HC function, oncogenic RAS caused depletion of JeKo-1 cells from culture. In contrast, this effect was partially reversed by genetic ablation of the constant region of immunoglobulin kappa LC ( IGKC). We next examined whether reconstitution of surrogate light chain (SLC) components of the pre-BCR will confer permissiveness to oncogenic RAS signaling in JeKo-1 cells. We engineered JeKo-1 cells to express Dox-inducible expression of λ5 (Igll1) and VpreB. In the presence of kappa LC, overexpression of SLC alone did not reverse the deleterious impact of oncogenic RAS expression. Notably, reconstitution of SLC components following genetic deletion of IGKC amplified the effect of IGKC-deletion alone and largely rescued cells from oncogenic RAS-induced survival disadvantage. Taken together, our results showed that immunoglobulin light chains control the consequences of oncogenic RAS activation in B-cells - while SLC of the pre-BCR plays a productive role in enabling oncogenic RAS-signaling, conventional light chain of the BCR plays a counterproductive role. Supporting this scenario, we found that SLC enabled oncogenic RAS to massively induce the proto-oncogene Bcl6 at the expense of the tumor suppressor Prdm1 to avoid cell death, promoting RAS-driven transformation of B-cell precursors. Upon genetic ablation of λ5, upregulation of Bcl6 was abrogated while Prdm1 was de-repressed. Consequently, RAS-driven malignant transformation of B-cell precursors was subverted. Reflecting the critical role of SLC in enabling RAS-BCL6 signaling, mature B-cells, which express BCR and lack SLC, failed to induce Bcl6 upon oncogenic RAS-activation, leading to upregulation of Prdm1. This observation may explain why persistent activation of the RAS-pathway in mature B-cells can lead to negative selection and cell death (Limnander et al., 2011). Conclusions: Our results demonstrated that immunoglobulin light chains determine permissiveness to RAS-driven malignant transformation of B-cells. Expression of SLC enables RAS-BCL6 activation, allowing B-cell precursors to avoid PRDM1-mediated tumor suppression to promote leukemogenesis. In contrast, conventional LC opposes oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations in BCR-expressing mature B-cell lymphomas.

Transcriptome Sequencing of the Oral Mucosa Reveals Gene Expression Differences in Patients With Parry-Romberg Syndrome

Objective: Parry-Romberg syndrome (PRS) is an acquired disease characterized by progressive unilateral atrophy of the facial skin, subcutaneous tissue, muscle, and bone. There are various hypotheses to try to explain the occurrence of the disease, but the specific etiology and pathogenesis remain unclear. This study aimed to explore the potential molecular pathogenesis of the disease by using next-generation RNA-sequencing technology. Methods: The authors collected oral mucosal tissue from the affected side and the healthy side from 3 patients with PRS. Tissue samples were subjected to RNA extraction, whole transcriptome sequencing, and bioinformatics analysis. Differentially expressed genes were obtained from both groups of samples and then analyzed for functional enrichment. Results: A total of 186 differentially expressed genes were screened from the 2 groups of samples. Compared with the healthy side, several immune-related genes, including immunoglobulin kappa variable (IGKV)2D-28, IGKV1D-33, IGKV1-33, and NLRP10, were significantly upregulated in the affected tissue. In addition, the differential genes were significantly enriched in metabolic pathways including pancreatic secretion, protein and fat digestion, and absorption. Conclusions: The authors described the gene expression differences between the affected and healthy tissues of patients with PRS for the first time. Immune responses may play a role in the pathogenesis of PRS.

Proteome Analysis of Bevacizumab Intervention in Experimental Central Retinal Vein Occlusion.

Bevacizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the management of macular edema in central retinal vein occlusion (CRVO). Studying retinal protein changes in bevacizumab intervention may provide insights into mechanisms of action. In nine Danish Landrace pigs, experimental CRVO was induced in both eyes with argon laser. The right eyes received an intravitreal injection of 0.05 mL bevacizumab (n = 9), while the left control eyes received 0.05 mL saline water (NaCl). Retinal samples were collected 15 days after induced CRVO. Label-free quantification nano-liquid chromatography-tandem mass spectrometry identified 59 proteins that were regulated following bevacizumab treatment. Following bevacizumab intervention, altered levels of bevacizumab components, including the Ig gamma-1 chain C region and the Ig kappa chain C region, were observed. Changes in other significantly regulated proteins ranged between 0.58-1.73, including for the NADH-ubiquinone oxidoreductase chain (fold change = 1.73), protein-transport protein Sec24B (fold change = 1.71), glycerol kinase (fold change = 1.61), guanine-nucleotide-binding protein G(T) subunit-gamma-T1 (fold change = 0.67), and prefoldin subunit 6 (fold change = 0.58). A high retinal concentration of bevacizumab was achieved within 15 days. Changes in the additional proteins were limited, suggesting a narrow mechanism of action.

The Effectiveness of Blinatumomab in Clearing Next-Generation Sequencing Measurable Residual Disease in Pediatric Patients with B-Cell Acute Lymphoblastic Leukemia

Introduction Blinatumomab is a bispecific T-cell engager antibody designed to redirect CD3+ T cells to bind to CD19+ target cells in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). Measurable residual disease (MRD) is a significant prognostic factor for relapse and overall survival in B-ALL. Blinatumomab consolidation therapy has been shown to improve MRD remission (&amp;lt;10 -4), as detected by multiparameter flow cytometry (MFC) or reverse transcriptase-polymerase chain reaction (PCR), and survival outcomes in children with high-risk first relapsed B-ALL. The next-generation sequencing (NGS)-MRD is a highly sensitive technique that can detect very low levels of MRD (&amp;lt;10 -6) in patients who were considered “MRD negative” by less sensitive MFC or PCR technologies. Early achievement of NGS-MRD negativity (&amp;lt;10 -6) can help in identifying patients who have a very low risk of relapse. It has been suggested that blinatumomab treatment could help to attain deeper responses than that measured with a MRD sensitivity level of 10 -4. However, data on blinatumomab clearing MRD with high sensitivity of 10 -6 remain scarce. Hence, we evaluated the effectiveness of blinatumomab in eradicating extremely low level of MRD (&amp;lt;10 -6), as detected by NGS, in children with B-ALL. Methods Pediatric B-ALL patients who were MFC-MRD negative (&amp;lt;10 -4) but NGS-MRD positive (sensitivity of 10 -6) after chemotherapy followed by blinatumomab consolidation therapy between October 2021 and February 2023, were studied retrospectively. At diagnosis, NGS analysis was performed by high-throughput deep level sequencing using the Seq-MRD ® (ImmuQuad Biotech, Hangzhou, China) to detect clonal rearrangements. Genomic DNA was extracted from bone marrow samples using QIAamp DNA Mini kit (Qiagen, Hilden, Germany), amplified by quantitative PCR and sequenced using the Illumina® Novaseq PE150 (Illumina ® Novaseq, San Diego, USA) platform. The sequences of immunoglobulin heavy chain (IGH) variable (V), diversity (D), and joining (J) (IGH -VDJ), IGH-DJ, immunoglobulin kappa light chain (IGK), immunoglobulin kappa deleting element (IGKDE) and immunoglobulin light chain (IGL) were assessed for clonality with dominant index sequence(s). Results Overall, 19 patients were enrolled. The baseline patient characteristics are shown in Table 1. Median age at diagnosis was 4.4 years (range 0.3-10.6), with males accounting for 84% of patients. Eight patients (42%) had at least 1 unfavorable genetic abnormality (i.e., KMT2A rearrangement, E2A-PBX1). Seventeen (89%) patients had ≥2 Ig clonal rearrangement at diagnosis. In total, NGS detected 71 clonal rearrangements, including 27 IGH clonal rearrangements in 15 (79%) patients, 9 IGH-DJ in 6 (32%), 14 IGK in 8 (42%), 6 IGKDE in 9 (32%), and 12 IGL in 8 (42%) patients. All patients achieved hematological complete response (CR) with MFC-MRD &amp;lt;10 -4 after the induction or consolidation chemotherapy. However, all these patients were identified to be MRD positive by NGS, including NGS-MRD &amp;gt;10 -6 and positive but not quantifiable (NGS-MRD &amp;lt;10 -6). Though the patients were MFC-MRD negative, 19 IGH clones, 4 IGH-DJ clones, 8 IGK clones, 7 IGKDE clones and 3 IGL clones were still persisted. After blinatumomab, MRD negativity rate by MFC was 95% (18/19) and the NGS-MRD negativity rate was 68% at 10 -6 (13/19). The rate of MRD clearance after blinatumomab per NGS was 69% and 67% in CR1 and CR2 patients, respectively. The clearance rates of IGH, IGK, IGL, IGH-DJ, and IGKDE were 75%, 67%, 33%, 100%, and 83%, respectively (Table 2). Three NGS-MRD negative patients with available NGS-MRD at pre-, mid-, and post-treatment achieved MRD clearance rapidly after blinatumomab treatment that was maintained durably through day 28. Three of 6 NGS-MRD positive patients relapsed after blinatumomab with 3 being IGH positive and 2 being IGL positive. Conclusion To the best of our knowledge, for the first time it has been demonstrated that blinatumomab could further eradicate MRD in patients achieving MFC-MRD &amp;lt;10 -4. Advanced MRD detection using NGS and blinatumomab made deeper molecular remission in B-ALL patients achievable. Further large-scale studies are warranted to verify the prognostic value of NGS in detecting lower levels of MRD in ALL patients treated in the current immunotherapy era.

Clonal Bone Marrow Plasma Cells Are Common in Patients with Vexas Syndrome

Introduction Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder. Its prevalence increases with age, reaching 5.3% at 70 years and older. MGUS progression rate to multiple myeloma (MM) or lymphoplasmacytic malignancies is about 1% per year. Patients with autoimmune diseases are at increased risk for developing MGUS and MM. VEXAS ( vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic syndrome), an autoinflammatory disorder caused by somatic mutations in ubiquitin-activating enzyme, UBA1 gene, has been associated with plasma cell disorders, specifically MGUS, but the rate of progression is unknown. Cyclin D1, important in cell cycle progression, is regulated by ubiquitin-proteasome mediated degradation, and deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. We aim to assess marrow characteristics of plasma cell disorders (MGUS and MM) in VEXAS, and the impact of a defective UBA1 gene product on protein expression of cyclin D1. Methods We assessed 28 patients with VEXAS for the presence of plasma cell disorders, and studied the clinical and laboratory characteristics of those with clonal plasma cells (PC) in the marrow. Bone marrow biopsies and aspirates were evaluated by morphology, immunohistochemistry (IHC) dual staining CD138/cyclin D1, dual staining MUM-1/CD20), in situ hybridization (Kappa and Lambda light chains) and flow cytometry (FC) using antibodies for CD45, CD19, CD20, CD38, CD138, CD56, CD27, CD81, and cytoplasmic Kappa and Lambda immunoglobulins. Serum free kappa and lambda light chains, IgG, IgM, and IgA, and level of M paraprotein by serum protein electrophoresis with immunofixation were obtained. Fluorescent in situ hybridization (FISH) analysis for MM was performed in most patients with clonal PCs. Results Patients' characteristics are shown in Table 1. Eight out of 28 (28.6%) patients with VEXAS syndrome, all males, had clonal plasma cells in their bone marrows: 3 MGUS, and 5 MM (3 smoldering, SMM; 2 active MM). Median age at diagnosis of plasma cell disorder was 69.5 years (range 60-75). All patients had pathogenic UBA1 somatic mutations (5 M41T, 1 each with M41V, M41L, and splice). Clinically, monoclonal protein size was small, with no specific selection of the type of M protein. Strikingly, most (7/8) clonal plasma cells showed cyclin D1 expression by IHC (dual immunohistochemical stain cyclinD1/CD138 shows double-positive plasma cells; figure not shown), displaying nuclear immunoreactivity in 6 cases, and cytoplasmic immunoreactivity in 1 case. FC showed positive CD20 expression of clonal plasma cells in 4 cases, dim to negative CD19 expression in 4 cases, variable CD56 (positive in 3 cases, partial positive in 1 case, negative in 2 cases), dim to negative CD27 in 3 cases, dim to negative CD81 in 3 cases. FISH for MM revealed: t(11;14) in 4 cases, gain of 11q (CCND1) in 2 cases (in one case, CCND1 gain was present in addition to t(11;14); in the second case, CCND1 gain was associated with gains of 1q and 17p(TP53). The cyclin D1-negative MM case was negative for t(11;14) but had t(4;14). All cases with t(11;14) were positive for cyclin D1 by IHC (nuclear staining). The one MM case with negative nuclear staining for cyclin D1 but positive cytoplasmic staining, was negative for t(11;14) but had gain of 11q (CCND1).One patient evolved from SMM to active MM after 4 years. No evolution to MM was seen in patients with MGUS with median follow-up of 30 months. Conclusions Patients with VEXAS have a high prevalence of clonal plasma cell disorders. Clonal plasma cells in VEXAS patients are associated with t(11;14), and with nuclear cyclin D1 expression by IHC. Cytoplasmic cyclin D1 staining in the absence or weak nuclear staining was observed with CCND1 gain and absence of t(11;14). Cytoplasmic cyclin D1 expression may be related to lack of cyclin D1 protein degradation by a defective UBA1 gene product. Our results suggest that defective UBA1 and autoinflammation pose an increased risk for developing MGUS or multiple myeloma.

Establishing the best combination of the kappa free light chain index and oligoclonal bands for an accurate diagnosis of multiple sclerosis.

The immunoglobulin kappa free light chain (KFLC) index has been proposed as a potentially suitable alternative to oligoclonal IgG bands (OCGB) for diagnosing multiple sclerosis (MS), offering automation and reduced processing time. However, there is no consensus on the preferred approach or how to combine both techniques. This prospective cohort study aimed to determine the best utilization of OCGB and KFLC index in patients with a clinically isolated syndrome (CIS) followed for at least two years. OCGB and KFLC were assessed using isoelectric focusing and immunoblotting and turbidimetry, respectively. Sensitivity, specificity, and accuracy for diagnosing MS were calculated for each method. The study included 371 patients, with 260 (70.1 %) being women, and a median age of 34.9 (27.8 - 43.9) years. Using a cut-off value of 6.1, the KFLC index demonstrated a sensitivity and specificity of 86.3% and 93.9%, respectively. The sensitivity of OCGB (95.3%) was higher (p < 0.001 vs. KFLC index) and the specificity (100%) was comparable to that of the KFLC index (p = 0.5). The concordance between the methods was not uniform across all patients, with 97.8% agreement in patients with KFLC index ≥ 6.1 and 56.0 % in patients with KFLC index < 6.1. In patients with a KFLC index < 6.1, OCGB still identified 75.0 % of MS patients due to its higher sensitivity. An algorithm using the KFLC index as a screening tool and OCGB as an alternative for patients with a negative KFLC index result achieved an accuracy of 96.3 %. Combining the KFLC index and OCGB can provide an easily reproducible and accurate method for diagnosing MS, with OCGB primarily reserved for patients with a KFLC index < 6.1.

NOTCH2 sensitizes the chondrocyte to the inflammatory response of tumor necrosis factor α

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain invitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.

Is tear proteome profile a predictor of developing uveitis in ANA-positive patients with oligoarticular juvenile idiopathic arthritis?

Although less than one-third of anti-nuclear antibody (ANA) positive patients with oJIA develop uveitis, ANA positivity is still the most well-known marker for assessing the risk of uveitis in oligoarticular JIA (oJIA). Therefore, novel biomarkers are needed to better assess the risk of developing uveitis. For this purpose, we performed a comparative tear proteome analysis of uveitis patients to reveal the identity of differentially regulated proteins. Tear samples were collected using the Schirmer strips in 7 oJIA and 7 oJIA patients with uveitis (oJIA-U). All oJIA-U patients had developed bilateral anterior uveitis and were inactive and topical treatment-free. The nHPLC LC-MS/MS system was used for protein identification and label-free proteome comparisons. The PANTHER and STRING analyses were carried out using UniProt accession numbers of the identified proteins. Patient characteristics, e.g., age, gender, disease duration, and treatments were similar. For protein identification, three different databases were searched. Twenty-two, 147, and 258 database searches, respectively. Of these, 15 were common to all three proteome databases. Of these 15 proteins, 10 proteins were upregulated, and 2 were downregulated, based on the twofold regulation criteria. The upregulated proteins were, namely, cystatin-S, secretoglobin family 1D member, opiorphin prepropeptide, mammaglobin-B, lysozyme C, mesothelin, immunoglobulin kappa constant, extracellular glycoprotein lacritin, beta-2-microglobulin, and immunoglobulin J chain. The downregulated proteins were dermcidin and prolactin-inducible protein. Among the differentially regulated proteins, cystatin-S was the most regulated protein with an 18-fold upregulation ratio in tear samples from uveitis patients. Here, the identities and regulation ratios of several proteins were revealed when tear samples from uveitis patients were compared to patients without uveitis. These proteins are putative biomarkers for assessing uveitis risk and require further attention.

Predictive biomarkers for latent Mycobacterium tuberculosis infection

Tuberculosis is a leading cause of infectious death worldwide, with almost a fourth of the world's population latently infected with its causative agent, Mycobacterium tuberculosis. Current diagnostic methods are insufficient to differentiate between healthy and latently infected populations. Here, we used a machine learning approach to analyze publicly available proteomic data from saliva and serum in Ethiopia's healthy, latent TB (LTBI) and active TB (ATBI) people. Our analysis discovered a profile of six proteins, Mast Cell Expressed Membrane Protein-1, Hemopexin, Lamin A/C, Small Proline Rich Protein 2F, Immunoglobulin Kappa Variable 4-1, and Voltage Dependent Anion Channel 2 that can precisely differentiate between the healthy and latently infected populations. This data suggests that a combination of six host proteins can serve as accurate biomarkers to diagnose latent infection. This is important for populations living in high-risk areas as it may help in the surveillance and prevention of severe disease.

Macrophage PTEN controls STING-induced inflammation and necroptosis through NICD/NRF2 signaling in APAP-induced liver injury

BackgroundPhosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling has been known to play a critical role in maintaining cellular and tissue homeostasis, which also has an essential role in the inflammatory response. However, it remains unidentified whether and how the macrophage PTEN may govern the innate immune signaling stimulator of interferon genes (STING) mediated inflammation and hepatocyte necroptosis in APAP-induced liver injury (AILI).MethodsMyeloid-specific PTEN knockout (PTENM−KO) and floxed PTEN (PTENFL/FL) mice were treated with APAP (400 mg/kg) or PBS. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated Notch1 knockout (KO) or CRISPR/Cas9-mediated STING activation vector followed by LPS (100 ng/ml) stimulation.ResultsHere, we report that myeloid-specific PTEN knockout (PTENM−KO) mice were resistant to oxidative stress-induced hepatocellular injury with reduced macrophage/neutrophil accumulation and proinflammatory mediators in AILI. PTENM−KO increased the interaction of nuclear Notch intracellular domain (NICD) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) in the macrophage nucleus, reducing reactive oxygen species (ROS) generation. Mechanistically, it is worth noting that macrophage NICD and NRF2 co-localize within the nucleus under inflammatory conditions. Additionally, Notch1 promotes the interaction of immunoglobulin kappa J region (RBPjκ) with NRF2. Disruption of the Notch1 signal in PTEN deletion macrophages, reduced RBPjκ and NRF2 binding, and activated STING signaling. Moreover, PTENM−KO macrophages with STING activated led to ROS generation and TNF-α release, resulting in hepatocyte necroptosis upon co-culture with primary hepatocytes.ConclusionsOur findings demonstrate that the macrophage PTEN-NICD/NRF2-STING axis is critical to regulating oxidative stress-induced liver inflammation and necroptosis in AILI and implies the therapeutic potential for managing sterile liver inflammation.-_SzJnLNvYmjVKnhJWJTYKVideo Graphical