NF-κB Reporter Cell Line Research Articles

Pivotal role of AKR1B1 in pathogenesis of colitis associated colorectal carcinogenesis

Identifying the target linking inflammation and oxidative stress to aggravate the disease progression will help to prevent colitis associated carcinogenesis. Since AKR1B1 overexpression is observed in inflammatory diseases and various cancers, we have investigated the role of AKR1B1 in colitis-associated colon carcinogenesis with the aid of epalrestat and its potent analogue NARI-29 (investigational molecule) as pharmacological probes. A TNF-α inducible NF-κB reporter cell line (GloResponse™ NF-κB-RE-luc2P HEK293) and dextran sodium sulfate (DSS) and 1,2 dimethyl hydrazine (DMH))-induced mouse model was used to investigate our hypothesis in vitro and in vivo. Clinically, an increased expression of AKR1B1 was observed in patients with ulcerative colitis. Our in vitro and in vivo findings suggest that the AKR1B1 modulated inflammation and ROS generation for the progression of colitis to colon cancer. AKR1B1 overexpression was observed in DSS+DMH-treated mice colons. Moreover, we could observe histopathological changes like immune cell infiltration, aberrant crypt foci, and tumour formation in DC groups. Mechanistically, we have witnessed modulation of the IKK/IκB/NF-κB and Akt/FOXO-3a/DR axis, increased inflammatory cytokines, increased expression of proliferative markers, Ki-67 and PCNA, and accumulation of β-catenin in the colon epithelium. However, pharmacological inhibition of AKR1B1 using NARI-29 or EPS has reversed the clinical, histopathological, and molecular alterations induced by DSS+DMH, confirming the obvious role of AKR1B1 in the promotion of colitis-associated carcinogenesis. In conclusion, our findings suggest that AKR1B1 targeted therapy could be a promising strategy for preventing CA-CRC and NARI-29 could be developed as a potent AKR1B1 inhibitor.

Reduced proinflammatory activity of outer membrane vesicles of Tannerella forsythia treated with quorum sensing inhibitors.

Outer membrane vesicles (OMVs) of bacteria harbor physiologically active molecules, and quorum sensing inhibitors (QSIs) are expected to regulate bacterial virulence. In this study, we analyzed the proinflammatory activity of OMVs of the periodontal pathogen Tannerella forsythia treated with d-arabinose and d-galactose as QSIs, which inhibit the biofilm formation of periodontal pathogens and autoinducer 2 activity. Compared to OMVs of nontreated T. forsythia (TF OMVs), OMVs released from QSI-treated T. forsythia, designated TF ara-OMVs and TF gal-OMVs, showed reduced production of TNF-α, IL-1β, IL-6, and IL-8 in THP-1 monocytes through decreased activation of NF-κB/MAPKs. Using a human NF-κB reporter cell line and bone marrow-derived macrophages from TLR2-/- mice, TF ara-OMVs and TF gal-OMVs showed less activation of TLR2 than TF OMVs. These results demonstrated that QSIs provide a dual advantage against bacterial infection by inhibiting bacterial biofilm formation and generating OMVs with reduced proinflammatory activity.

Abstract 358: LTB and LTBR mediates alternative Nf-kB activation through NIk and RELB/NF-kB2 to promote cell migration of HNSCC

Abstract Nuclear factor-κB (NF-κB) includes a family of NF-κB/REL transcription factors that regulate key inflammatory and cancer genes, and which are aberrantly activated in head and neck squamous cell carcinomas (HNSCC). Lymphotoxin beta/Lymphotoxin beta receptor (LTβ/LTβR) can mediate activation of the “alternative” NF-κB pathway that includes NF-κB-inducing kinase (NIK), Inhibitor-κB kinase α (IKKα), and transcription factors RELB/NF-κB2. While LTβ is expressed in lymph nodes where HNSCC spread, the role of this pathway in pathogenesis of HNSCC is not well studied. Analysis of The Cancer Genome Atlas (TCGA) data reveals that LTβR is amplified or overexpressed in larynx or oral cavity HNSCC, while LTα/β, NIK, (MAP3K14) and RELB are overexpressed in oropharynx and tonsil cancers. Similarly, we observed overexpression of LTβR, NIK, and RELB proteins in subsets of HNSCC cell lines. In UM-SCC46, LTβ enhanced nuclear translocation of RELB/NF-κB2 (p52) subunit proteins, while LTβR siRNA knockdown decreased the expression of its target kinase NIK and RELB/NF-κB2 (p52). NIK knockdown also decreased RELB/NF-κB2 (p52) protein expression, whereas LTβ treatment stabilized NIK, and RELB/NF-κB2 (p52). Functional LTβ-mediated NF-κB activation was examined using a stably transfected NF-κB reporter cell line for β-lactamase/Fret activity. Knockdown of LTβR and NIK decreased NF-κB activity, whereas treatment with LTβ partially restored it. Treatment with a NIK inhibitor (1, 3[2H, 4H]-Iso-Quinoline Dione) reduced the protein expression of NIK, IKKα, and RELB/NF-κB2 (p52) in the cytoplasm and/or in the nucleus, and blocked LTβ induced translocation of RELB to nucleus. NIK and RELB knockdown by siRNA or using NIK inhibitor slowed HNSCC migration. Knockdown of NIK inhibited the LTβ-induced expression of MET, and SERPINE1 genes involved in migration, and knockdown of MET by siRNA inhibited the migration of HNSCC cell lines. Our findings show that LTβ/LTβ receptor promotes activation of the alternative NIK-IKKa-RELB/NF-κB2 pathway, to enhance MET-mediated cell migration in HNSCC. This may help explain why HNSCC exhibit migration and metastasis to local lymph nodes, where LTβ is expressed. The constitutive and LTβ induced activation of NIK and downstream NF-κB alternate pathway proteins, RELB and NF-κB2/p52 could be therapeutic targets in HNSCC. (Supported by NIDCD intramural projects ZIA-DC-000016, 74) Citation Format: Rita Das, Jamie Coupar, Anthony saleh, Paul E. Clavijo, Zhong Chen, Carter VanWaes. LTB and LTBR mediates alternative Nf-kB activation through NIk and RELB/NF-kB2 to promote cell migration of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2017-358

Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans

Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection.

Pharmacogenomics of Scopoletin in Tumor Cells.

Drug resistance and the severe side effects of chemotherapy necessitate the development of novel anticancer drugs. Natural products are a valuable source for drug development. Scopoletin is a coumarin compound, which can be found in several Artemisia species and other plant genera. Microarray-based RNA expression profiling of the NCI cell line panel showed that cellular response of scopoletin did not correlate to the expression of ATP-binding cassette (ABC) transporters as classical drug resistance mechanisms (ABCB1, ABCB5, ABCC1, ABCG2). This was also true for the expression of the oncogene EGFR and the mutational status of the tumor suppressor gene, TP53. However, mutations in the RAS oncogenes and the slow proliferative activity in terms of cell doubling times significantly correlated with scopoletin resistance. COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression resulted in a set of 40 genes, which all harbored binding motifs in their promoter sequences for the transcription factor, NF-κB, which is known to be associated with drug resistance. RAS mutations, slow proliferative activity, and NF-κB may hamper its effectiveness. By in silico molecular docking studies, we found that scopoletin bound to NF-κB and its regulator IκB. Scopoletin activated NF-κB in a SEAP-driven NF-κB reporter cell line, indicating that NF-κB might be a resistance factor for scopoletin. In conclusion, scopoletin might serve as lead compound for drug development because of its favorable activity against tumor cells with ABC-transporter expression, although NF-κB activation may be considered as resistance factor for this compound. Further investigations are warranted to explore the full therapeutic potential of this natural product.

Abstract 1724: Integrated functional RNAi screening and structural genomics identify inverse co-modulators of TP53 family and NF-κB transitional activation as potential therapeutic targets in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide with a 50-60% mortality rate. Deregulation of p53 family members in HNSCC occurs in over 90% of cases, preventing transcription of growth arrest and apoptosis genes. Conversely, members of the NF-κB/REL family are aberrantly activated in about ∼70% of cases, and drive expression of pro-proliferation, inflammation, angiogenesis, and therapeutic resistance genes. The function of different TP53 and NF-κB family members are inversely modulated within two major subsets of HNSCC, suggesting that common molecules and pathways coordinate this modulation. To test this hypothesis, we have developed HNSCC stable cell lines that report transcriptional activation of TP53 or NF-κB individually through a β-lactamase reporter. Whole genome siRNA screening in our NF-κB reporter cell line has been performed. Screening was performed by 56-hour knockdown with siRNAs, followed by stimulation with TNF-α. The results have revealed known and novel targets that maintain oncogenic NF-κB signaling in HNSCC. Validating the assay, most structural components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, scored highly in the screen. We integrated this functional genomics data with structural mutation and expression data from TCGA to prioritize genes that are frequently deregulated in HNSCC specimens from patients. Several co-receptors that may support NF-κB signaling were identified such as WNT9/FZD6, NOTCH2, and TGFβR2. Our primary novel finding was the identification of genes that regulate mitosis, such as CDC2, CDC7, CHEK1, WEE1 and CALM2. Several components of the kinetochore were also identified including NDC80, PLK1,TTK and AURKA. To validate our screening data, individual assays were performed with two siRNAs for a panel of 27 genes from the pathways mentioned above. >95% of siRNAs tested validated our screening data. Genes scoring well in our analysis were also assayed in our p53 reporter cell line using cisplatin and TNF-α as stimuli. Genes that inversely activate p53 were identified. To further investigate the relationship between kinetochore function and NF-κB activation, we treated cells with various G2/M inhibitors such as docetaxel and showed decreased NF-κB signaling. Future experiments are underway to determine the signaling mechanism that connects the kinetochore to NF-κB regulators. Experiments are also ongoing to determine if aberrant activation of NF-κB can serve as a biomarker for sensitivity to cell cycle inhibitors. Citation Format: Anthony Saleh, Shaleeka Cornelius, Scott Martin, Pinar Ormanoglu, Hui Cheng, Rita Das, Xinping Yang, Zhong Chen, Carter Van Waes. Integrated functional RNAi screening and structural genomics identify inverse co-modulators of TP53 family and NF-κB transitional activation as potential therapeutic targets in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2015-1724

Abstract 5253: LTβ receptor and NIK signaling activates the alternative NF-kB pathway in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinomas (HNSCC) are highly inflammatory and preferentially metastasize to lymph nodes. NF-κB, a key target and regulator of inflammatory and cancer genes is constitutively activated in HNSCC, but the upstream regulation, especially for activation of alternative pathway involving LTβ/LTβR is not clear. We hypothesized that LTβ/LTβR mediated alternative NF-kB pathway signaling is an important mechanism for activation of downstream target genes, and promotion of malignant and metastatic phenotypes in HNSCC. In this study, we have found that LTβ and LTβR are overexpressed in subsets of HNSCC (UM-SCC) tissues and cell lines. In UM-SCC cell lines, LTβ mainly activated the alternative NF-kB pathway, enhancing nuclear translocation of RELB and NF-κB2/p52, in contrast to TNFα induced activation of classical NF-κB pathway. Knockdown of LTβR by Si RNA decreased LTβR mRNA by quantitative RT-PCR and protein expression by Western blots. Knockdown of LTβR decreased its target kinase NIK, and downstream NF-κB subunits RELB and NF-κB2/p52 protein expression. Knockdown of NIK protein decreased RELB and p52 protein expression, while LTβ treatment stabilized NIK, RELB and NF-κB2/p52 expression. LTB mediated NF-κB activity was examined by transient luciferase reporter assay or using stably transfected NF-κB reporter cell line through beta lactamase Fret Blazer activity. Knockdown of LTBR and NIK functionally decreased NF-κB reporter activity, while treatment of LTB partially restored the NF-κB reporter activity. Our data have shown that constitutive and LTβ induce activation of NIK and downstream NF-κB alternate pathway proteins, RELB and NF-kB2/p52 in HNSCC. LTβ and NIK activation of the alternative NF-κB pathway may contribute to the malignant phenotype and metastasis of HNSCC. Citation Format: Rita Das, Tsu-Fang Cheng, Jamie coupar, Anthony Saleh, Xingping Yang, jialing Zhang, shaleeka Cornelius, Carter Van Waes, zhong Chen. LTβ receptor and NIK signaling activates the alternative NF-kB pathway in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2015-5253

The Carbomer-Lecithin Adjuvant Adjuplex Has Potent Immunoactivating Properties and Elicits Protective Adaptive Immunity against Influenza Virus Challenge in Mice.

The continued discovery and development of adjuvants for vaccine formulation are important to safely increase potency and/or reduce the antigen doses of existing vaccines and tailor the adaptive immune response to newly developed vaccines. Adjuplex is a novel adjuvant platform based on a purified lecithin and carbomer homopolymer. Here, we analyzed the adjuvant activity of Adjuplex in mice for the soluble hemagglutinin (HA) glycoprotein of influenza A virus. The titration of Adjuplex revealed an optimal dose of 1% for immunogenicity, eliciting high titers of HA-specific IgG but inducing no significant weight loss. At this dose, Adjuplex completely protected mice from an otherwise lethal influenza virus challenge and was at least as effective as the adjuvants monophosphoryl lipid A (MPL) and alum in preventing disease. Adjuplex elicited balanced Th1-/Th2-type immune responses with accompanying cytokines and triggered antigen-specific CD8+ T-cell proliferation. The use of the peritoneal inflammation model revealed that Adjuplex recruited dendritic cells (DCs), monocytes, and neutrophils in the context of innate cytokine and chemokine secretion. Adjuplex neither triggered classical maturation of DCs nor activated a pathogen recognition receptor (PRR)-expressing NF-κB reporter cell line, suggesting a mechanism of action different from that reported for classical pathogen-associated molecular pattern (PAMP)-activated innate immunity. Taken together, these data reveal Adjuplex to be a potent and well-tolerated adjuvant with application for subunit vaccines.

Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway.

Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.

Identification of a NFκB inhibitory peptide from tryptic β-casein hydrolysate

Several bioactive peptides are encrypted within the sequence of major milk proteins, requiring enzymatic proteolysis for release and activation. The present study aimed at the identification of potential anti-inflammatory activities in tryptic hydrolysates of bovine β-casein. Inflammatory processes involve in most cases an activation of Nuclear factor Kappa-light-chain enhancer of activated B cells (NFκB), which is a pro-inflammatory transcription factor of several genes. Hence, a NFκB reporter cell line was established, and TNF-α mediated activation of NFκB was used as a measurement. Bovine β-casein (β-CN) was hydrolysed by trypsin and fractionated by ultrafiltration. Total proteolysate as well as the fraction containing peptides between 1 and 5kDa showed an inhibitory effect in the cell-based assay, while the fraction containing molecules smaller than 1kDa did not. This anti-inflammatory effect was ascribed to a group of large, hydrophobic peptides, which were identified using LC–MS. The main peptide was synthesised and showed a significant anti-inflammatory effect in HEKnfkb-RE-cells. Thus, for the first time, a casein-derived peptide having an anti-inflammatory effect in vitro has been identified.

A functional genomics screen for microRNA regulators of NF-kappaB signaling.

BackgroundThe nuclear factor-KappaB (NF-κB) pathway is conserved from fruit flies to humans and is a key mediator of inflammatory signaling. Aberrant regulation of NF-κB is associated with several disorders including autoimmune disease, chronic inflammation, and cancer, making the NF-κB pathway an attractive therapeutic target. Many regulatory components of the NF-κB pathway have been identified, including microRNAs (miRNAs). miRNAs are small non-coding RNAs and are common components of signal transduction pathways. Here we present a cell-based functional genomics screen to systematically identify miRNAs that regulate NF-κB signaling.ResultsWe screened a library of miRNA mimics using a NF-κB reporter cell line in the presence and absence of tumor necrosis factor (+/- TNF). There were 9 and 15 hits in the -TNF and +TNF screens, respectively. We identified putative functional targets of these hits by integrating computational predictions with NF-κB modulators identified in a previous genome-wide cDNA screen. miR-517a and miR-517c were the top hits, activating the reporter 86- and 126-fold, respectively. Consistent with these results, miR-517a/c induced the expression of endogenous NF-κB targets and promoted the nuclear localization of p65 and the degradation of IκB. We identified TNFAIP3 interacting protein1 (TNIP1) as a target and characterized a functional SNP in the miR-517a/c binding site. Lastly, miR-517a/c induced apoptosis in vitro, which was phenocopied by knockdown of TNIP1.ConclusionsOur study suggests that miRNAs are common components of NF-κB signaling and miR-517a/c may play an important role in linking NF-κB signaling with cell survival through TNIP1.

Dangkwisoo-san, an herbal medicinal formula, ameliorates acute lung inflammation via activation of Nrf2 and suppression of NF-κB

Ethnopharmacological relevanceDangkwisoo-san (DS), an herbal medicinal formula, has long been used in Korea for the treatment of inflammatory complications caused by physical trauma. Although the therapeutic effect of DS is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here we sought to elucidate the possible mechanisms of anti-inflammatory activity of DS. Materials and methodsThe water extract of DS was orally fed to C57BL/6 mice for 14 days prior to LPS intranasal instillation for lung inflammation. The effects of DS on lung inflammation were determined by differential cell counting, lung histology, and semi-quantitative RT-PCR of lung sections. The effects of DS on the activities of Nrf2 and NF-κB were assessed by western blotting, semi-quantitative RT-PCR, and luciferase reporter assays in RAW 264.7, an NF-κB reporter cell line, and HEK 293 transfected with an NF-κB reporter construct. ResultsMice that were treated with a water extract of DS showed significant attenuation of lung inflammation induced by intranasal lipopolysaccharide (LPS) compared to control mice treated with vehicle. In vitro experiments show that DS activated Nrf2, an anti-oxidant transcription factor that protects from various inflammatory diseases, and induced Nrf2-regulated genes including GCLC, NQO-1 and HO-1. In addition, DS suppressed NF-κB activity and reduced the production of pro-inflammatory cytokines. Transfection experiment indicates that inhibition of NF-κB likely occurred upstream of IKK complex. Furthermore, DS enhanced the expression of HO-1 and suppressed that of IL-1β and TNF-α in inflamed mouse lungs. ConclusionsThese results suggest that the therapeutic effects of DS are related with suppression of inflammation, which is, at least in part, mediated by activation of anti-inflammatory factor Nrf2 and inhibition of pro-inflammatory factor NF-κB.

Selective Inhibition of Activated B-Cell Lymphoma Cells by a Novel Small Molecule Inhibitor of NF-κB

Abstract 2498Diffuse large B-cell lymphomas (DLBCL) account for approximately 40% of lymphomas in adults, with the activated B-cell lymphoma (ABC) subtype being the least curable. ABC lymphoma cells display the phenotype of activated B-cells, which is induced by constitutive activation of the transcription factor Nuclear Factor-kappa B (NF-κB). NF-κB activation in ABC lymphoma can result from several different mutations or abnormal expression of proteins upstream of NF-κB nuclear translocation. No matter the mechanism of NF-κB constitutive activation, the resulting gene expression pattern confers an anti-apoptotic and pro-survival advantage to B-cells, hence driving the oncogenesis and supporting the survival of ABC lymphomas. Therefore, inhibiting NF-κB activity is an attractive strategy for the treatment of ABC lymphomas.Using a cell line with a reporter for NF-κB transcriptional activity in a high throughput screen of 180,000 compounds we discovered 12 compounds that inhibited NF-κB activation, suggesting these compounds negatively regulate NF-κB. Since ABC lines are generally more dependent on NF-κB signalling for cell survival than are the other types of B-cell lymphoma, the hits from the primary screen were tested in a secondary screen for the ability to selectively inhibit the growth of ABC lymphoma cells. Of the 12 hits, two compounds belonging to the same quinolone chemotype were found to selectively inhibit the growth of an ABC line compared to a non-ABC line and normal human peripheral blood mononuclear cells (PBMCs). Three more structurally related quinolones were obtained to investigate a possible limited structure activity relationship for this class of molecules, designated here as Quinolone Inhibitors of NF-κB (QINs). QIN 1 was significantly more potent (Figure A and B) and selective (Figure B and C) relative to other QINs. The limited structure activity relationship suggests that two structural regions of the chemotype may be important for potency and for ABC selectivity, hence providing impetus to further investigate QINs as possible ABC lymphoma drugs.QIN1 is more potent in inhibiting the growth of ABC lines than a known inhibitor of NF-κB activity, a commercially available selective IKK inhibitor (CAS 507475-17-4). Active IKK causes degradation of IκBα, the natural inhibitor of NF-κB, hence IKK inhibitors prevent NF-κB activation by preventing degradation of IκBα. In addition to being more potent, QIN1 retains similar ABC selectivity as the IKK inhibitor (Figure C). QIN1 and the IKK inhibitor both cause apoptosis at a similar rate and both cause G1 arrest in ABC lines at equi-toxic concentrations, initially suggesting similar mechanisms of action. Subsequently, QIN1was found to inhibit NF-κB in several different assays using the IKK inhibitor as a positive control. First, QIN1 inhibited the activation of a transcription based NF-κB reporter cell line in response to LPS, an NF-κB activator. Supporting these results, QIN1 inhibited cytokine release from human PBMCs stimulated with LPS. In addition, QIN1 prevented degradation of IκBα in response to NF-κB activating stimuli, further demonstrating the ability of QIN1 to inhibit NF-κB activity. Finally, QIN1 inhibited nuclear translocation of active NF-κB, thus preventing pro-survival gene expression.The QINs studied here all contain an alpha-beta unsaturated ketone, an electrophilic chemical moiety known to interact with cysteine thiols. Compounds containing similar electrophilic groups are known to bind a cysteine thiol in IKK, preventing its activity and hence inhibiting NF-κB activation. This suggests that this electrophilic moiety in QINs maybe responsible for the NF-κB inhibition observed in these cell lines. The presence of this electrophilic group is not necessarily detrimental to normal cells as we have shown with QIN1 in vitro (Figure A-C) and in vivo (large daily doses of QIN1 cause no observable toxicities in mice). Overall, our data indicate QIN1 inhibits IKK, a kinase immediately upstream of NF-κB nuclear translocation, allowing the majority of ABC lymphomas to be treated irrespective of the upstream mechanism of pathway activation. The selectivity for inhibition of ABC lymphoma cells by QIN1 and our in vitro data showing synergy with an inhibitor of B-cell receptor signalling, which also promotes ABC survival, provides the impetus for further preclinical development of QINs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.

Inhibition of NF-κB Signaling in Human Dendritic Cells by the Enteropathogenic Escherichia coli Effector Protein NleE

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by β-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-β, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer's patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.

Identification of a human NF-kappaB-activating protein, TAB3.

The NF-kappaB pathway plays a critical role in regulating cellular processes such as immune responses, stress responses, apoptosis, proliferation and differentiation, whereas dysfunction of this pathway has been associated with numerous cancer and immune disorders. We have applied our Random Activation of Gene Expression technology to an NF-kappaB reporter cell line to facilitate the discovery of positive regulators of NF-kappaB activation. A small protein expression library, corresponding to approximately 0.1x genome coverage, was generated and screened for clones exhibiting constitutive activation of NF-kappaB. After isolation of cellular clones displaying the relevant phenotypes, we identified two known components of the NF-kappaB pathway and a hypothetical gene that we have designated the human ortholog of Xenopus TAK1-binding protein 3 (TAB3). Overexpression of human TAB3 was found to activate both NF-kappaB and AP-1 transcription factors. Furthermore, the activation of NF-kappaB by TAB3 was blocked by the NF-kappaB inhibitor, SN50, and by expression of dominant-negative forms of tumor necrosis factor alpha-associated factor 6 and transforming growth factor beta-activated kinase. Taken together, these data demonstrate that TAB3 transforming growth factor is a constituent of the NF-kappaB pathway functioning upstream of tumor necrosis factor alpha-associated factor 6/transforming growth factor beta-activated kinase. Interestingly, increased expression of TAB3 was found in some cancer tissues, and its overexpression in NIH 3T3 cells resulted in cellular transformation, thus establishing a causative link between elevated TAB3 expression, constitutive NF-kappaB activation, and oncogenesis.