Noncanonical NF-κB Signaling Research Articles

Inhibition of S6K lowers age-related inflammation and increases lifespan through the endolysosomal system

Suppression of target of rapamycin complex 1 (TORC1) by rapamycin ameliorates aging in diverse species. S6 kinase (S6K) is an essential mediator, but the mechanisms involved are unclear. Here we show that activation of S6K specifically in Drosophila fat-body blocked extension of lifespan by rapamycin, induced accumulation of multilamellar lysosomes and blocked age-associated hyperactivation of the NF-κB-like immune deficiency (IMD) pathway, indicative of reduced inflammaging. Syntaxin 13 mediated the effects of TORC1–S6K signaling on lysosome morphology and inflammaging, suggesting they may be linked. Inflammaging depended on the IMD receptor regulatory isoform PGRP-LC, and repression of the IMD pathway from midlife extended lifespan. Age-related inflammaging was higher in females than in males and was not lowered in males by rapamycin treatment or lowered S6K. Rapamycin treatment also elevated Syntaxin 12/13 levels in mouse liver and prevented age-related increase in noncanonical NF-κB signaling, suggesting that the effect of TORC1 on inflammaging is conserved from flies to mammals.

NF-κB Inducing Kinase Attenuates Colorectal Cancer by Regulating Noncanonical NF-κB Mediated Colonic Epithelial Cell Regeneration

Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-kB signaling. Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis were initiated in whole-body NIK knockout mice (Nik-/-) and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium. Human transcriptomic data revealed dysregulated noncanonical NF-kB signaling. Invitro studies evaluating Nik-/- crypts and organoids derived from mature, nondividing CECs, and colonic stem cells exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed invivo, Nik-/- mice exhibited more severe colitis with dextran sulfate sodium administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC). Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue-dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.

Non-canonical NFKB signaling endows suppressive function through FOXP3-dependent regulatory T cell program

Regulatory T cells (Tregs) play a central role in modulating adaptive immune responses in humans and mice. The precise biological role of non-canonical nuclear factor ‘κ-light-chain-enhancer’ of activated B cells (NFKB) signaling in human Tregs has yet to be fully elucidated. To gain insight into this process, a Treg-like cell line (MT-2) was genetically modified using CRISPR/Cas9. Interestingly, NFKB2 knockout MT-2 cells exhibited downregulation of FOXP3, while NFKB1 knockout did not. Additionally, mRNA expression of FOXP3-dependent molecules was significantly reduced in NFKB2 knockout MT-2 cells. To better understand the functional role of the NFKB signaling, the NFKB1/NFKB2 loci of human primary Tregs were genetically edited using CRISPR/Cas9. Similar to MT-2 cells, NFKB2 knockout human Tregs displayed significantly reduced FOXP3 expression. Furthermore, NFKB2 knockout human Tregs showed downregulation of FOXP3-dependent molecules and a diminished suppressive function compared to wild-type and NFKB1 knockout Tregs. These findings indicate that non-canonical NFKB signaling maintains a Treg-like phenotype and suppressive function in human Tregs through the FOXP3-dependent regulatory T cell program.

Chlorogenic Acid Alleviated AFB1-Induced Hepatotoxicity by Regulating Mitochondrial Function, Activating Nrf2/HO-1, and Inhibiting Noncanonical NF-κB Signaling Pathway.

Aflatoxin B1 (AFB1), a kind of mycotoxin, imposes acute or chronic toxicity on humans and causes great public health concerns. Chlorogenic acid (CGA), a natural phenolic substance, shows a powerful antioxidant and anti-inflammatory effect. This study was conducted to investigate the effect and mechanism of CGA on alleviating cytotoxicity induced by AFB1 in L-02 cells. The results showed that CGA (160 μM) significantly recovered cell viability and cell membrane integrity in AFB1-treated (8 μM) cells. Furthermore, it was found that CGA reduced AFB1-induced oxidative injury by neutralizing reactive oxygen species (ROS) and activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. In addition, CGA showed anti-inflammatory effects as it suppressed the expression of inflammation-related genes (IL-6, IL-8, and TNF-α) and AFB1-induced noncanonical nuclear factor kappa-B (NF-κB) activation. Moreover, CGA mitigated AFB1-induced apoptosis by maintaining the mitochondrial membrane potential (MMP) and inhibiting mRNA expressions of Caspase-3, Caspase-8, Bax, and Bax/Bcl-2. These findings revealed a possible mechanism: CGA prevents AFB1-induced cytotoxicity by maintaining mitochondrial membrane potential, activating Nrf2/HO-1, and inhibiting the noncanonical NF-κB signaling pathway, which may provide a new direction for the application of CGA.

RelB mediated by GSK3β/IκBα as a potential therapeutic target in pilocarpine seizure model rats and drug-resistant epilepsy patients

BackgroundDrug-resistant epilepsy (DRE) affects more than 20 million people worldwide. DRE patients do not respond to anti-seizure medications. Shifting from anti-seizure to anti-epileptic and disease-modifying therapy will be an important aim for future research. The canonical nuclear factor kappa B (NF-κB) signaling pathway plays a pivotal role in epilepsy and neuroinflammation. However, the expression and regulation of RelB, which can be activated via both canonical and non-canonical NF-κB signaling, are obscure in epilepsy. To clarify the expression and localization of RelB in the DRE phenotype and to determine the proteins related to RelB regulation, we conducted the following studies. MethodsQuantitative PCR was performed to detect the transcription of RELB in pilocarpine-induced epileptic rats. Western blotting was used to determine the abundance of RelB and proteins related to RelB regulation in brain tissue from both epilepsy model rats and DRE patients and in liposaccharide-induced HT22 cells. Immunofluorescence staining and immunohistochemistry were used to locate RelB in brain sections from DRE patients. ELISA was used to determine inflammatory cytokines secreted by HT22 cells into the culture medium. ResultsRELB transcription and expression were increased in the hippocampus and cortex of epileptic rats during the acute and latent phases and in epileptic foci of patients with temporal lobe epilepsy. Additionally, RelB levels were mainly increased in epileptic rat neurons and accumulated in the nuclei of hippocampal neurons. Further research demonstrated increased GSK3β phosphorylation at the Ser9 inhibitory site and decreased IκBα levels, which contributed to RelB accumulation in hippocampal neurons after seizure and in liposaccharide-stimulated HT22 cells. ConclusionsThis study is the first to demonstrate that RelB is widely distributed and increased in epileptogenic foci neurons in epileptic phenotypes. These results indicate that RelB may play roles in DRE and is mediated by GSK3β and IκBα, providing a new target for anti-epileptic and disease-modifying therapy.

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

Cyanine Dye Conjugation Enhances Crizotinib Localization to Intracranial Tumors, Attenuating NF-κB-Inducing Kinase Activity and Glioma Progression.

Glioblastoma (GBM) is a highly aggressive form of brain cancer with a poor prognosis and limited treatment options. The ALK and c-MET inhibitor Crizotinib has demonstrated preclinical therapeutic potential for newly diagnosed GBM, although its efficacy is limited by poor penetration of the blood brain barrier. Here, we identify Crizotinib as a novel inhibitor of nuclear factor-κB (NF-κB)-inducing kinase, which is a key regulator of GBM growth and proliferation. We further show that the conjugation of Crizotinib to a heptamethine cyanine dye, or a near-infrared dye (IR-Crizotinib), attenuated glioma cell proliferation and survival in vitro to a greater extent than unconjugated Crizotinib. Moreover, we observed increased IR-Crizotinib localization to orthotopic mouse xenograft GBM tumors, which resulted in impaired tumor growth in vivo. Overall, IR-Crizotinib exhibited improved intracranial chemotherapeutic delivery and tumor localization with concurrent inhibition of NIK and noncanonical NF-κB signaling, thereby reducing glioma growth in vitro, as well as in vivo, and increasing survival in a preclinical rodent model.

IGF2BP1 Stabilizes and Inhibits Degradation of Pro-Oncogenic Transcripts in ETV6-RUNX1 Positive B-Cell Acute Lymphoblastic Leukemia

Insulin like growth factor 2 mRNA binding protein 1(IGF2BP1) belongs to a family of oncofetal proteins expressed in embryonic life followed by re-emergence during malignant transformation. It is an RNA binding protein known to be dysregulated and overexpressed in multiple malignancies. We have demonstrated its specific overexpression in ETV6-RUNX1 translocated B-cell Acute Lymphoblastic Leukemia (B-ALL). RNA immunoprecipitation using anti-IGF2BP1 antibody, followed by sequencing (RIP-Seq) in Reh cell line (ETV6-RUNX1 positive) identified multiple oncogenic transcripts belonging to the non-canonical NF-κB and PI3K signaling pathways. Overexpression of genes from these pathways (IL6ST, GADD45A, NFAT5, MDM2, CDK6, FGFR1) was also observed in our B-ALL patient samples specifically in the ETV6-RUNX1 positive patients (Total n=111; ETV6-RUNX1 positive: 39) in comparison to MACS sorted, peripheral blood CD19 positive B-cells from healthy volunteers as well as in comparison to non ETV6-RUNX1 translocated B-Acute lymphoblastic leukemia patients. This was also corroborated by public B-ALL gene expression data from the TARGET database in the cBioportal. However, knockout of IGF2BP1 in Reh cell line using CRISPR-Cas9 technology led to significant downregulation of only some of these targets. This implied that the expression of some of the mRNA targets binding to IGF2BP1 was not influenced by its knockout. To better elucidate the role played by IGF2BP1 in the stability of its binding mRNA targets, we decided to study the rate of degradation of target transcripts. mRNA stability was assessed to quantify the rate of degradation of target mRNA after inhibition of transcription using actinomycin. Since actinomycin causes global transcriptional block, we first quantified the effect of actinomycin on the expression of multiple (n=7) housekeeping genes against each other, and selected 3 ( HGPRT, B2M and PPIA) which showed least change after actinomycin treatment. These 3 housekeeping genes were used for further quantification of target transcripts. Dual specificity phosphatase 1( DUSP1) a transcript known to be rapidly degraded, was used to validate the transcriptional inhibition by actinomycin. We quantified the rate of degradation of target transcripts in Reh cells using BTYNB, a small molecule inhibitor of IGF2BP1. BTYNB binds to IGF2BP1 and prevents binding to target transcripts which leads to their destabilization and reduced half-life. We used a combination of BTYNB and actinomycin to study rate of degradation after IGF2BP1 inhibition. Effect of IGF2BP1 on the stability of three target mRNAs involved in non-canonical NF-κB signalling pathway was quantified by comparing their rate of degradation in BTYNB treated cells against the DMSO (vehicle) treated cells. Rate of degradation was measured by actinomycin treatment of both these groups followed by hourly RNA isolation and qPCR over 12 hours. MYC is a known mRNA binding target of IGF2BP1. BTYNB was also shown to decrease the expression of MYC mRNA by accelerating its degradation. Simultaneously there was an increase in the expression of MYC pre-mRNA indicating a possible for IGF2BP1 in splicing of MYC RNA. In the NF-κB signalling pathway, we had previously identified that NFAT5 expression was not altered significantly after IGF2BP1 knockout even though it binds to IGF2BP1. BTYNB followed by actinomycin treatment demonstrated a dramatic increase in the rate of degradation of NFAT5 implying that IGF2BP1 prevents the degradation of NFAT5 transcript and directly influences its stability. Interestingly, the rates of degradation of other targets like IL6ST and GADD45 were not altered. These findings suggest a heterogeneous influence of IGF2BP1 on the stability of its target transcripts. Some of the transcripts seem to be directly stabilised by IGF2BP1 while others may be influenced indirectly. Taken together, these findings suggest a role of IGF2BP1 in multiple phases of the RNA synthesis and degradation warranting further studies to elucidate role of IGF2BP1 in each of these steps. IGF2BP1 appears to have a critical role in preventing the degradation and stabilizing MYC and NFAT5 transcripts.

TRIM55 promotes noncanonical NF-κB signaling and B cell-mediated immune responses by coordinating p100 ubiquitination and processing.

The ubiquitination-dependent processing of NF-κB2 (also known as p100) is a critical step in the activation of the noncanonical NF-κB pathway. We investigated the molecular mechanisms regulating this process and showed that TRIM55 was the E3 ubiquitin ligase that mediated the ubiquitination of p100 and coordinated its processing. TRIM55 deficiency impaired noncanonical NF-κB activation and B cell function. Mice with a B cell-specific Trim55 deficiency exhibited reduced germinal center formation and antibody production. These mice showed less severe symptoms than those of control mice upon the induction of a systemic lupus-like disease, suggesting B cell-intrinsic functions of TRIM55 in humoral immune responses and autoimmunity. Mechanistically, the ubiquitination of p100 mediated by TRIM55 was crucial for p100 processing by VCP, an ATPase that mediates ubiquitin-dependent protein degradation by the proteasome. Furthermore, we found that TRIM55 facilitated the interaction between TRIM21 and VCP as well as TRIM21-mediated K63-ubiquitination of VCP, both of which were indispensable for the formation of the VCP-UFD1-NPL4 complex and p100 processing. Together, our results reveal a mechanism by which TRIM55 fine-tunes p100 processing and regulates B cell-dependent immune responses in vivo, highlighting TRIM55 as a potential therapeutic target for lupus-like disease.

Expression and Activity of the NF-κB Subunits in Chronic Lymphocytic Leukaemia: A Role for RelB and Non-Canonical Signalling.

Canonical NF-κB signalling by p65 (RelA) confers chemo-resistance and poor survival in chronic lymphocytic leukaemia (CLL). The role of non-canonical NF-κB signalling (leading to RelB and p52 subunit activation) in CLL is less understood, but given its importance in other B-cell tumour types, we theorised that RelB and p52 may also contribute to the pathology of CLL. DNA binding activity of all five NF-kB subunits, p65, p50, RelB, p52, and c-Rel, was quantified using ELISA and correlated to ex vivo chemoresistance, CD40L-stimulated signalling (to mimic the lymph node microenvironment), and clinical data. Importantly, we show for the first time that high basal levels of RelB DNA binding correlate with nuclear RelB protein expression and are associated with del(11q), ATM dysfunction, unmutated IGHV genes, and shorter survival. High levels of nuclear p65 are prevalent in del(17p) cases (including treatment-naïve patients) and also correlate with the outcome. CD40L-stimulation resulted in rapid RelB activation, phosphorylation and processing of p100, and subsequent CLL cell proliferation. These data highlight a role for RelB in driving CLL cell tumour growth in a subset of patients and therefore strategies designed to inhibit non-canonical NF-κB signalling represent a novel approach that will have therapeutic benefit in CLL.

Interleukin-34-NF-κB signaling aggravates myocardial ischemic/reperfusion injury by facilitating macrophage recruitment and polarization

Macrophage infiltration and polarization are integral to the progression of heart failure and cardiac fibrosis after ischemia/reperfusion (IR). Interleukin 34 (IL-34) is an inflammatory regulator related to a series of autoimmune diseases. Whether IL-34 mediates inflammatory responses and contributes to cardiac remodeling and heart failure post-IR remains unclear. IL-34 knock-out mice were used to determine the role of IL-34 on cardiac remodeling after IR surgery. Then, immunofluorescence, flow cytometry assays, and RNA-seq analysis were performed to explore the underlying mechanisms of IL-34-induced macrophage recruitment and polarization, and further heart failure after IR. By re-analyzing single-cell RNA-seq and single-nucleus RNA-seq data of murine and human ischemic hearts, we showed that IL-34 expression was upregulated after IR. IL-34 knockout mitigated cardiac remodeling, cardiac dysfunction, and fibrosis after IR and vice versa. RNA-seq analysis revealed that IL-34 deletion correlated negatively with immune responses and chemotaxis after IR injury. Consistently, immunofluorescence and flow cytometry assays demonstrated that IL-34 deletion attenuated macrophage recruitment and CCR2+ macrophage polarization. Mechanistically, IL-34 deficiency repressed both the canonical and noncanonical NF-κB signaling pathway, leading to marked reduction of P-IKKβ and P-IκBα kinase levels; downregulation of NF-κB p65, RelB, and p52 expression, which drove the decline in chemokine CCL2 expression. Finally, IL-34 and CCL2 levels were increased in the serum of acute coronary syndrome patients, with a positive correlation between circulating IL-34 and CCL2 levels in clinical patients. In conclusion, IL-34 sustains NF-κB pathway activation to elicit increased CCL2 expression, which contributes to macrophage recruitment and polarization, and subsequently exacerbates cardiac remodeling and heart failure post-IR. Strategies targeting IL-34-centered immunomodulation may provide new therapeutic approaches to prevent and reverse cardiac remodeling and heart failure in clinical MI patients after percutaneous coronary intervention. This study was supported by the National Nature Science Foundation of China (81670352 and 81970327 to R T, 82000368 to Q F).

IRP1 mediated ferroptosis reverses temozolomide resistance in glioblastoma via affecting LCN2/FPN1 signaling axis depended on NFKB2

The prognosis of glioblastoma (GBM) patients is poor, and temozolomide (TMZ) resistance has become an important obstacle to its treatment effect. A growing number of researches have revealed the special characteristics of iron metabolism in GBM chemosensitivity. Iron regulatory protein 1 (IRP1) is an important protein for maintaining intracellular iron homeostasis. IRP1 has been indicated to have additional vital roles beyond its conventional metabolic activity, but the underlying mechanisms and biological consequences remain elusive. Here, we unprecedentedly demonstrated that amplifying IRP1 signals can reverse TMZ resistance and suppress tumor growth invivo via inhibiting NFKB2 in the noncanonical NF-κB signaling pathway. In addition, we identified that NFKB2 affected TMZ sensitivity of GBM by modulating the expression of LCN2 and FPN1. Taken together, this study established a role for the IRP1/NFKB2 pathway in regulating LCN2/FPN1 signaling axis among the progression of TMZ resistance, suggesting a potential innovative GBM therapeutic strategy.

Bioactive silica nanoparticles target autophagy, NF-κB, and MAPK pathways to inhibit osteoclastogenesis

Spherical 50 nm silica-based nanoparticles (SiNPs) promote healthy bone homeostasis and maintenance by supporting bone forming osteoblast lineage cells while simultaneously inhibiting the differentiation of bone resorbing osteoclasts. Previous work demonstrated that an intraperitoneal injection of SiNPs in healthy mice - both young and old - increased bone density and quality, suggesting the possibility that SiNPs represent a dual action therapeutic. However, the underlying mechanisms governing the osteoclast response to SiNPs have yet to be fully explored and defined. Therefore, the goals of this study were to investigate the cellular and molecular mechanisms by which SiNPs inhibit osteoclastogenesis. SiNPs strongly inhibited RANKL-induced osteoclast differentiation within the first hours and concomitantly inhibited early transcriptional regulators such as Nfatc1. SiNPs simultaneously stimulated expression of autophagy related genes p62 and LC3β dependent on ERK1/2 signaling pathway. Intriguingly, SiNPs were found to stimulate autophagosome formation while inhibiting the autophagic flux necessary for RANKL-stimulated osteoclast differentiation, resulting in the inhibition of both the canonical and non-canonical NF-κB signaling pathways and stabilizing TRAF3. These results suggest a model in which SiNPs inhibit osteoclastogenesis by inhibiting the autophagic machinery and RANKL-dependent functionality. This mechanism of action defines a novel therapeutic strategy for inhibiting osteoclastogenesis.

A noncanonical NF-kB - β-catenin signaling axis in dendritic cells instructs gut inflammation

Abstract Dendritic cells (DCs) balance intestinal homeostasis against inflammatory insults. In particular, the noncanonical NF-kB pathway, which activates RelB:p52 heterodimers, has been implicated in DC differentiation. However, if DC-intrinsic noncanonical signaling modulates DC function in the inflamed gut remains unclear. Here we report that genetic ablation of the noncanonical signaling in DCs accumulated regulatory T cells in the lamina propria and associated lymph nodes, restraining inflammation in the colitogenic gut. Our mechanistic investigation revealed that an impaired RelB:p52 gene function, rather unexpectedly, augmented the abundance of active β-catenin via impairment of the β-catenin destruction complex. In turn, β-catenin transcriptionally upregulated Raldh2, imparting a retinoic acid gene signature and tolerogenic functions to noncanonical NF-kB-deficient DCs. Indeed, mice possessing DC-intrinsic noncanonical signaling defects exhibited an increased frequency of β-catenin +DCs in the gut and resilience to chemical induced experimental colitis. Taken together, we chart a novel crosstalk between noncanonical NF-kB signaling and the β-catenin pathway that instructs tolerogenic DC functions and modulates inflammation in the gut. National Institute of Immunology Core funding and BT/PR36631/BRB/10/1862/2020, Department of Biotechnology (DBT).

Proliferation, Plasticity, and Migration: Exploring the synergistic relationship between eosinophils and TH2 cells in development of HES-like syndrome

Abstract Aberrancies in eosinophil proliferation, activation and chemotaxis can lead to acute eosinophilic leukemia, paraneoplastic eosinophilia associated with T cell lymphoma and carcinomas, and Hypereosinophilic Syndrome (HES), respectively. HES is an idiopathic disease affecting humans and veterinary patients characterized by persistent eosinophilia (>1,500 cells/uL) and eosinophilic infiltration of solid organs leading to increased morbidity and mortality. Previous studies have demonstrated the role of NF-κB inducing kinase, NIK, an upstream regulator of the noncanonical NF-κB signaling pathway, in development of a HES-like syndrome due to aberrant TH2 polarization in the Nik−/−murine model. However, the intricacies of eosinophilic granulopoiesis, eosinophil/neutrophil plasticity, and chemotactic ability has not been assessed during development of HES-like syndrome in Nik−/−mice. Here we show that loss of NIK potentially enhances eosinophilic chemotaxis to CCL11, enhances eosinophilic/neutrophilic plasticity, and affects granulopoiesis in the bone marrow and spleen beginning at the level of eosinophilic metamyelocytes, despite potentially decreasing the proliferative potential of these cells. Taken together, these results suggest that NIK and the noncanonical NF-κB signaling pathway plays a role in dictating the hematopoietic and chemotactic potential of eosinophils and highlights a synergistic relationship with TH2 cells and their respective cytokines during development of HES-like syndrome in Nik−/−mice. Overall, these findings warrant further study to better characterize the synergistic relationship described above to better understand development of HES and HES-like diseases. Virginia-Maryland College of Veterinary Medicine and the Institute for Critical Technology and Applied Science

FGFR4 and EZH2 inhibitors synergistically induce hepatocellular carcinoma apoptosis via repressing YAP signaling

BackgroundHepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide, but current treatment options remain limited and cause serious life-threatening side effects. Aberrant FGFR4 signaling has been validated as an oncogenic driver of HCC, and EZH2, the catalytic subunit of the PRC2 complex, is a potential factor that contributes to acquired drug resistance in many tumors, including HCC. However, the functional relationship between these two carcinogenic factors, especially their significance for HCC treatment, remains unclear. In this study, we systematically evaluated the feasibility of a combination therapy targeting FGFR4 and EZH2 for HCC.MethodsRNA sequencing data of patients with Liver hepatocellular carcinoma (LIHC) from The Cancer Genome Atlas (TCGA) were analyzed to determine FGFR4 and EZH2 expression and their interaction with prognosis. Moreover, the HCC cell lines, zebrafish/mouse HCC xenografts and zebrafish HCC primary tumors were treated with FGFR4 inhibitor (Roblitinib) and/or EZH2 inhibitor (CPI-169) and then subjected to cell proliferation, viability, apoptosis, and tumor growth analyses to evaluate the feasibility of combination therapy for HCC both in vitro and in vivo. Furthermore, RNA-Seq was performed in combination with ChIP-Seq data analysis to investigate the critical mechanism underlying the combination treatment with Roblitinib and CPI-169.ResultsEZH2 accumulated through the non-canonical NF-kB signaling in response to FGFR4 inhibitor treatment, and the elevated EZH2 levels led to the antagonism of HCC against Roblitinib (FGFR4 inhibitor). Notably, knockdown of EZH2 sensitized HCC cells to Roblitinib, while the combination treatment of Roblitinib and CPI-169 (EZH2 inhibitor) synergistically induced the HCC cell apoptosis in vitro and suppressed the zebrafish/mouse HCC xenografts and zebrafish HCC primary tumors development in vivo. Moreover, Roblitinib and CPI-169 synergistically inhibited HCC development via repressing YAP signaling.ConclusionsCollectively, our study highlighted the potential of the therapeutic combination of FGFR4 and EZH2 inhibitors, which would provide new references for the further development of clinical treatment strategies for HCC.

NF-κB/p52 augments ETS1 binding genome-wide to promote glioma progression

Gliomas are highly invasive and chemoresistant cancers, making them challenging to treat. Chronic inflammation is a key driver of glioma progression as it promotes aberrant activation of inflammatory pathways such as NF-κB signalling, which drives cancer cell invasion and angiogenesis. NF-κB factors typically dimerise with its own family members, but emerging evidence of their promiscuous interactions with other oncogenic factors has been reported to promote transcription of new target genes and function. Here, we show that non-canonical NF-κB activation directly regulates p52 at the ETS1 promoter, activating its expression. This impacts the genomic and transcriptional landscape of ETS1 in a glioma-specific manner. We further show that enhanced non-canonical NF-κB signalling promotes the co-localisation of p52 and ETS1, resulting in transcriptional activation of non-κB and/or non-ETS glioma-promoting genes. We conclude that p52-induced ETS1 overexpression in glioma cells remodels the genome-wide regulatory network of p52 and ETS1 to transcriptionally drive cancer progression.

119 Noncanonical NF-κB signaling is essential for development of chronic contact hypersensitivity (CHS)

119 Noncanonical NF-κB signaling is essential for development of chronic contact hypersensitivity (CHS)

Inhibition of TANK-binding kinase1 attenuates the astrocyte-mediated neuroinflammatory response through YAP signaling after spinal cord injury.

TANK-binding kinase 1 (TBK1) is involved in regulating the pathological process of a variety of inflammatory diseases in the central nervous system. However, its role and underlying molecular mechanisms in spinal cord injury (SCI) remain largely unknown. We employed the TBK1 inhibitor amlexanox (ALX) to address this question. An in vivo clip-compressive SCI model and in vitro lipopolysaccharide (LPS)-induced astrocyte inflammation model were established to examine the effects of TBK1 inhibition on the expression of proinflammatory cytokines. In this study, we found that TBK1 and TBK1-medicated innate immune pathways, such as TBK1/IRF3 and noncanonical NF-κB signaling, were activated in astrocytes and neurons after SCI. Furthermore, inhibition of TBK1 by ALX alleviated neuroinflammation response, reduced the loss of motor neurons, and improved the functional recovery after SCI. Mechanistically, inhibition of TBK1 activity promoted the activation of the noncanonical NF-κB signaling pathway and inhibited p-IRF3 activity in LPS-induced astrocytes, and the TBK1 activity was required for astrocytic activation through yes-associated protein (YAP) signaling after SCI and in LPS-induced astrocytes inflammation model. TBK1-medicated innate immune pathway in astrocytes through YAP signaling plays an important role in the pathogenesis of SCI and inhibition of TBK1 may be a potential therapeutic drug for SCI.

NFKB2 haploinsufficiency identified via screening for IFN-α2 autoantibodies in children and adolescents hospitalized with SARS-CoV-2–related complications

Autoantibodies against type I IFNs occur in approximately 10% of adults with life-threatening coronavirus disease 2019 (COVID-19). The frequency of anti-IFN autoantibodies in children with severe sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is unknown. We quantified anti-type I IFN autoantibodies in a multicenter cohort of children with severe COVID-19, multisystem inflammatory syndrome in children (MIS-C), and mild SARS-CoV-2 infections. Circulating anti-IFN-α2 antibodies were measured by a radioligand binding assay. Whole-exome sequencing, RNA sequencing, and functional studies of peripheral blood mononuclear cells were used to study any patients with levels of anti-IFN-α2 autoantibodies exceeding the assay's positive control. Among 168 patients with severe COVID-19, 199 with MIS-C, and 45 with mild SARS-CoV-2 infections, only 1 had high levels of anti-IFN-α2 antibodies. Anti-IFN-α2 autoantibodies were not detected in patients treated with intravenous immunoglobulin before sample collection. Whole-exome sequencing identified a missense variant in the ankyrin domain of NFKB2, encoding the p100 subunit of nuclear factor kappa-light-chain enhancer of activated B cells, aka NF-κB, essential for noncanonical NF-κB signaling. The patient's peripheral blood mononuclear cells exhibited impaired cleavage of p100 characteristic of NFKB2 haploinsufficiency, an inborn error of immunity with a high prevalence of autoimmunity. High levels of anti-IFN-α2 autoantibodies in children and adolescents with MIS-C, severe COVID-19, and mild SARS-CoV-2 infections are rare but can occur in patients with inborn errors of immunity.