Expression Of NF-κB Target Genes Research Articles

C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in β-cells

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic β-cells. Pro-inflammatory cytokines are early mediators of β-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in β-cells, but the role for CHOP overexpression in cytokine-induced β-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating β-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting β-cell death: (1) CHOP directly contributes to cytokine-induced β-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

Defective nuclear IKKα function in patients with ectodermal dysplasia with immune deficiency (180.19)

Abstract Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB-regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.

Abstract 2216: SUMOylated CPAP contributes to NF-kB activation in cancer cells

Abstract Nuclear factor kappa-B (NF-κB) is a transcription factor which involves in hepatocellular carcinoma (HCC). The development of therapeutic drugs to specifically target NF-κB signaling pathway is imperative to achieve. Centrosomal and P4.1-associated protein (CPAP) is a novel co-activator of NF-κB. The effects of CPAP on NF-κB activation pathway were determined by over-expression and knockdown approaches. CPAP increased the expression of NF-κB target genes by enhancing the transcriptional activaty of NF-κB. Through recruiting more IkappaB kinase beta (IKKα) to the inactivated NF-κB complex, CPAP augmented the phosphorylation of inhibitor of NF-κB (IαBβ) and NF-κB. Chromatin immunoprecipitation assay further showed that CPAP is recruited to IL-8 and ICAM-1 promoter under TNF-α treatment. CPAP was covalently modified by SUMO-1 (small ubiquitin-related modifier-1) protein at the C-terminal region. SUMO-deficient CPAP mutants lost the co-activator activity on NF-κB activation pathway and failed to enter the nucleus upon TNF-α treatment. By immunohistochemistry (IHC) analysis, CPAP was detected in the nucleus of HBV-associated HCC. Activation of NF-κB was synergically augmented by CPAP overexpression in HBx-expressing Hep3B (Hep3BX) and HepG2 (HepG2X) cells lines. SUMOylation of CPAP could modulate the activity of NF-κB, and CPAP may serve as a target for therapeutic purpose in HBV-associated HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2216. doi:1538-7445.AM2012-2216

Abstract 1753: Prospective stratification using gene expression arrays in a randomised trial of R-CHOP +/− Bortezomib in diffuse large B-cell lymphoma (DLBL): The UK NCRI REMoDL-B study, ISRCTN 51837425

Abstract DLBL subtypes are recognised by patterns of gene expression, corresponding to Germinal Center (GCB) or activated peripheral blood (ABC) B-cells. They differ in frequency of mutation in the B-cell receptor signalling pathway and levels of expression of NF-kB target genes. Results of treatment with standard (R-CHOP) therapy are inferior for ABC type, and this study tests the use of bortezomib to downregulate NF-kB and reverse the deficit. This trial is the first to use gene expression profiling (GEP) to stratify cases into GCB and ABC at entry, with adaptive statistical design to analyse the outcome by subtype at predefined timepoints. Patients (pts) newly diagnosed with DLBL undergo staging and commence R-CHOP. During the first 3 week cycle formalin-fixed paraffin-embedded (FFPE) tissue undergoes extraction of messenger RNA for GEP of 24 000 probe sets using Illumina cDNA-mediated Annealing, Selection, extension, and Ligation (DASL®) array at the central laboratory. Cases are allocated to GCB, ABC or other type before the 2nd cycle. Thereafter, all pts are randomized to continue conventional R-CHOP or to add bortezomib 1.3mg/m2 days 1 + 8. The study will randomize a minimum 260 ABC type, to detect a difference in progression free survival (PFS) of 10% with bortezomib, with two-sided significance 5% & 90% power. The design allows closure of randomization for GCB cases if 6 month PFS is <80% after 55 receive R-CHOP-B. A second analysis for futility in GCB is planned after 73 treated with R-CHOP-B are followed for one year: if PFS is <85%, further exploration of bortezomib in this group is not warranted. Prior to trial opening, FFPE material from 200 pts with DLBL was analysed by DASL® protocol. Of 27 genes in a published (LLMPP) classification, 20 are present on the platform. Array data were standardised by converting to z-score. Four decision tree machine learning tools were trained on LLMPP data and combined into a meta-classifier using the Vote scheme with average of probabilities. This showed DLBL reproducibly classified using this platform with outcomes similar to those previously reported. Additional analyses of DNA extracted from tumour material will detect mutations in NF-kB pathway genes by combining the Fluidigm Access Array™ PCR System with next generation sequencing. Since trial opening in 2011, samples have been analysed from 39 pts. 30 (77%) biopsies were successfully classified; 3 cases had poor quality of RNA and in 6 there was insufficient tumour in the block. Cases classified include both surgical and needle core biopsies. Maximum laboratory turnround time was 12 working days. This study demonstrates it is possible to carry out GEP at diagnosis in a multicenter trial. It will address the relationship between response to treatment and molecular biomarkers, and serve as a model for future study design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1753. doi:1538-7445.AM2012-1753

Abstract 2214: COUP-TFII suppresses NFκB activation in endocrine-resistant breast cancer cells

Abstract COUP-TFII is an orphan nuclear receptor that can function as either a transcriptional activator or repressor. We have previously shown that COUP-TFII expression is reduced in tamoxifen-resistant LCC9 breast cancer cells and that overexpression of COUP-TFII restored the ability of tamoxifen to inhibit cell proliferation. An increase in NFκB activity and p65/RELA level has been reported in endocrine-resistant breast cancer cells. Because NFκB activation results in increased expression of anti-apoptotic and pro-proliferative genes, increased NFκB signaling promotes the survival of cancer cells. The goal of the current study was to further elucidate the mechanism by which COUP-TFII enables cells to maintain endocrine sensitivity and to determine if COUP-TFII modulates NFκB activity in tamoxifen-resistance. To examine whether COUP-TFII can suppress NFκB activity, MCF-7 (tamoxifen-sensitive, TAM-S) and LCC9 (tamoxifen-resistant, TAM-R) breast cancer cells were transfected with a NFκB luciferase reporter and treated with TNFα to activate NFκB. Cotransfection with COUP-TFII reduced NFκB activity in both TAM-S and TAM-R cell lines. Knockdown of COUP-TFII with siRNA led to an increase in p65 expression; conversely, COUP-TFII overexpression led to a suppression of p65 expression. COUP-TFII overexpression also reduced TNFα-induced endogenous NFκB target gene expression. Preliminary data also suggests that suppression of NFκB activation by COUP-TFII leads to a reduction in cell viability in MCF-7 and LCC9 cells in response to tamoxifen and TNFα treatment. This indicates a novel role for COUP-TFII in suppression of NFκB activity which may explain the ability of COUP-TFII to promote tamoxifen-sensitivity. COUP-TFII may be both a useful biomarker to predict tamoxifen-sensitivity as well as a target to restore endocrine sensitivity to resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2214. doi:1538-7445.AM2012-2214

The Steroid and Xenobiotic Receptor Negatively Regulates B-1 Cell Development in the Fetal Liver

The steroid and xenobiotic receptor (SXR) (also known as pregnane X receptor or PXR) is a broad-specificity nuclear hormone receptor that is well known for its role in drug and xenobiotic metabolism. SXR is activated by a wide variety of endobiotics, dietary compounds, pharmaceuticals, and xenobiotic chemicals. SXR is expressed at its highest levels in the liver and intestine yet is found in lower levels in other tissues, where its roles are less understood. We previously demonstrated that SXR(-/-) mice demonstrate elevated nuclear factor (NF)-κB activity and overexpression of NF-κB target genes and that SXR(-/-) mice develop lymphoma derived from B-1 lymphocytes in an age-dependent manner. In this work, we show that fetal livers in SXR(-/-) mice display elevated expression of NF-κB target genes and possess a significantly larger percentage of B-1 progenitor cells in the fetal liver. Furthermore, in utero activation of SXR in wild-type mice reduces the B-1 progenitor populations in the embryonic liver and reduces the size of the B-1 cell compartment in adult animals that were treated in utero. This suggests that activation of SXR during development may permanently alter the immune system of animals exposed in utero, demonstrating a novel role for SXR in the generation of B-1 cell precursors in the fetal liver. These data support our previous findings that SXR functions as a tumor suppressor in B-1 lymphocytes and establish a unique role for SXR as a modulator of developmental hematopoiesis in the liver.

Hsp90 inhibition suppresses LPS‐mediated NFkB target gene expression without affecting NFkB nuclear translocation in human lung microvascular endothelial cells

Heat shock protein 90 (hsp90) regulates the stability and function of several kinases, including IKB kinases (IKKs). We investigated the mechanisms by which hsp90 inhibitors prevent induction of NFkB‐dependent genes. The hsp90 inhibitor, 17‐AAG, significantly suppressed lipopolysaccharide (LPS)‐mediated IKK‐beta activation and phosphorylation of IKB‐alpha. However, 17‐AAG did not restore IKB‐alpha protein level and was unable to block p65 nuclear translocation and total DNA binding, as reflected by ELISA, in LPS‐stimulated human lung microvascular endothelial cells (HLMVEC). Conversely, prolonged 17‐AAG treatment (>16h) blocked LPS‐mediated induction of NFkB regulated genes, IKB‐alpha, IL‐8 and MMP‐9. Interestingly, binding of p65 to the IKB‐alpha promoter, as reflected by ChIP assay, was reduced by 17‐AAG. Selective pharmacologic inhibition of several key steps in the toll‐like receptor 4 (TLR‐4) signaling pathway revealed that the regulation of NFkB target genes by LPS is dependent on the cAMP‐dependent kinase (PKA) and MAPK pathways. Thus, LPS stimulated p65 (Ser276) phosphorylation in a PKA‐dependent manner; this was significantly inhibited by 17‐AAG and by IKK inhibitors (IKK16 and BAY 11‐7082). We conclude that hsp90 inhibition suppresses NFkB signaling and target gene expression by affecting p65 post‐translational modifications. (Supported by NIH grants: HL093460 and HL101902)

A new lupane-type triterpene from the seeds of Panax ginseng with its inhibition of NF-κB

A new lupane-triterpene, 3β-cis-feruloyloxy-16β-hydroxylup-20(29)-ene (1) were isolated from the ethyl acetate extract of Panax ginseng seeds along with the known compound, 3β-transferuloyloxy-16β-hydroxylup-20(29)-ene (2). Compound 2 was isolated from this plant for the first time. Their chemical structures were determined by mass spectroscopy and one-dimensional and two-dimensional magnetic resonance spectra. The bioactive effects of these compounds on TNF-α-induced NF-κB transcription were evaluated in transfected HepG2 cells. Effects on the expression of NF-κB target genes were also examined using a reverse transcription-polymerase chain reaction. Both compounds 1 and 2 were inhibited NF-κB activity in HepG2 cells by decreasing the cellular concentrations of inflammatory factors iNOS and COX2.

Inflammasome-Activated Caspase 7 Cleaves PARP1 to Enhance the Expression of a Subset of NF-κB Target Genes

Caspase 1 is part of the inflammasome, which is assembled upon pathogen recognition, while caspases 3 and/or 7 are mediators of apoptotic and nonapoptotic functions. PARP1 cleavage is a hallmark of apoptosis yet not essential, suggesting it has another physiological role. Here we show that after LPS stimulation, caspase 7 is activated by caspase 1, translocates to the nucleus, and cleaves PARP1 at the promoters of a subset of NF-κB target genes negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes. These findings propose an apoptosis-independent regulatory role for caspase 7-mediated PARP1 cleavage in proinflammatory gene expression and provide insight into inflammasome signaling.

MicroRNA 15a, Inversely Correlated to PKCα, Is a Potential Marker to Differentiate between Benign and Malignant Renal Tumors in Biopsy and Urine Samples

NF-κB signal transduction is a potential therapeutic target in many malignant tumors. We have recently shown, in malignant renal proximal tumor cells, that a transcription complex, consisting of NF-κB p65 and mitogen-activated protein kinase p38α, joined by protein kinase C (PKC) α, transmigrates into the nucleus. There, PKCα suppresses the nuclear release of primary microRNA (pri-miRNA) 15a. Induced by endothelin (ET)-1, a decrease in PKCα levels leads to increased miRNA 15a (miR-15A) expression. An identical system can be identified in renal carcinomas, in which, after nuclear transmigration, PKCα binds directly to pri-miRNA 15a in the nucleus. However, the pattern of PKC isoforms differs between malignant renal cell carcinoma (RCC) and benign oncocytoma. PKCα, a component of the transcription complex in tumors, is up-regulated in benign oncocytoma but down-regulated in RCC. Conversely, miRNA 15a is up-regulated in RCC and down-regulated in oncocytoma. A similar behavior is observed in chromophobe carcinoma, whereas differences are less pronounced in papillary RCC (type I): NF-κB target gene expression (ie, ET-1, ET-A and ET-B receptors, vascular cell adhesion molecule-1, IL-6, and fractalkine) is particularly high in malignant RCCs. Up-regulated miRNA 15a can be measured in urine from patients with RCC but is nearly undetectable in oncocytoma, other tumors, and urinary tract inflammation. Thus, the up-regulation of miRNA 15a may be an important marker to help identify malignant clear-cell RCCs in both biopsy and urine samples.

Suppression of the NF-κB Pathway by Diesel Exhaust Particles Impairs Human Antimycobacterial Immunity

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1β, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.

A new synthetic chalcone derivative, 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139), suppresses the Toll-like receptor 4-mediated inflammatory response through inhibition of the Akt/NF-κB pathway in BV2 microglial cells

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IκB and p65/RelA NF-κB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-κB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, and IL-1β, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant-active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IκB kinase (IKK)/NF-κB signaling pathway.

Yangonin Blocks Tumor Necrosis Factor-α–Induced Nuclear Factor-κB–Dependent Transcription by Inhibiting the Transactivation Potential of the RelA/p65 Subunit

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, and apoptosis. In our search for NF-κB inhibitors from natural resources, we identified yangonin from Piper methysticum as an inhibitor of NF-κB activation. In the present study, we demonstrate that yangonin potently inhibits NF-κB activation through suppression of the transcriptional activity of the RelA/p65 subunit of NF-κB. This compound significantly inhibited the induced expression of the NF-κB-reporter gene. However, this compound did not interfere with tumor necrosis factor-α (TNF-α)-induced inhibitor of κBα (IκBα) degradation, p65 nuclear translocation, and DNA-binding activity of NF-κB. Further analysis revealed that yangonin inhibited not only the induced NF-κB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Moreover, yangonin did not inhibit TNF-α-induced activation of p38, but it significantly impaired activation of extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase. We also demonstrated that pretreatment of cells with this compound prevented TNF-α-induced expression of NF-κB target genes, such as interleukin 6, interleukin 8, monocyte chemotactic protein 1, cyclooxygenase-2 and inducible nitric oxide. Taken together, yangonin could be a valuable candidate for the intervention of NF-κB-dependent pathological conditions such as inflammation.

APPL1 regulates basal NF-κB activity by stabilizing NIK

SummaryAPPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here, we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-κB under basal but not TNFα-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate canonical NF-κB signaling. APPL1 synergized with TRAF2 to induce NF-κB activation, and both proteins were necessary for this process and function upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-κB pathway. Importantly, in the canonical pathway, APPL1 appeared to regulate the proper spatial distribution of the p65 subunit of NF-κB in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. By analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-κB target genes that encode cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-κB pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-κB activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-κB activation.

Dysregulated NF-κB Pathway in Peripheral Mononuclear Cells of Alzheimers Disease Patients

Diagnosis and therapeutic strategies in Alzheimer's disease (AD) might greatly benefit of the present multidisciplinary approach for studying the molecular pathogenesis of the disorder. Gene expression profile at peripheral level could be a promising tool for pathogenic studies as well as for early diagnosis of AD. A dysregulated inflammatory response, as well as other systemic disorders, have been described in AD. Therefore, we investigated the expression, at peripheral level, of a number of genes involved in the inflammatory, oxidative stress and proliferative response of a well defined, small cohort of sporadic AD patients. Firstly, the mRNA expression of inflammatory, stress and proliferation/ differentiation genes were evaluated, using SuperArray, in mitogen-stimulated peripheral blood mononuclear cells (PBMC) from a group of 12 well-characterized, sporadic AD patients with various levels of dementia, by comparison with aged-matched controls. Real-time RT-PCR confirmed the trend of alteration in 16 genes out of the 36 supposed to be dysregulated in AD patients, by the preliminary screening. The expression level of the NFKB1(p105/50Kd) gene was significantly higher in AD with respect to adult age-matched controls (AA) and was related to the Mini-Mental State Examination (MMSE) score of the same patients. In addition, the expression of various NF-κB target genes and of both NF-κBp50 and NF-κBp65 DNA-binding activity were increased in PBMC from AD patients in comparison with those from AA. Our results suggest that NF-κB activation at peripheral blood cell level could be a potential new hallmark of AD progression and sustain a rationale to more deeply investigate the therapeutic potential of specific NF-κB inhibitors in AD.

Defective nuclear IKKα function in patients with ectodermal dysplasia with immune deficiency

Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB-regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.

The Bruton’s Tyrosine Kinase (BTK) Inhibitor PCI-32765 Inhibits Growth of ABC DLBCL Tumors In Vivo and in Vitro by Preventing Activation of Pro-Survival NF-κB pathways

Abstract 4969Bruton’s tyrosine kinase (BTK), a Tec family kinase essential to signaling via the B-cell Receptor (BCR) has emerged as a very promising drug target in lymphoma. PCI-32765, a potent and selective covalently-bound small molecule inhibitor of BTK (Honigberg, Proc. Natl Acad. Sci. USA 107:13075, 2010), has demonstrated promising clinical activity in a number of Phase I and II clinical trials in patients with chronic lymphocytic leukemia (CLL) (Byrd et al., ASCO 2011 Annual Meeting Abstracts, JCO29 (15): 8012, 2010) and several subtypes of B-cell Non-Hodgkin’s Lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL). Many cell lines derived from B-cell malignancies do not show in vitro growth inhibition by BTK inhibitors, perhaps because most established B-cell lines no longer depend on BCR stimulation for survival. However, some cell lines derived from the aggressive ABC (activated B-cell) subtype of DLBCL such as OCI-Ly10 and TMD8 are very sensitive to BTK inhibition by PCI-32765 (Davis et al, Nature463: 88, 2010). We show here that both of these cell lines are potently and irreversibly inhibited by PCI-32765 (EC50 continuous exposure < 10 nM and EC50 following a 1-hr pulse < 50 nM). Interestingly, 2 other ABC-DLBCL cell lines, OCI-Ly3 and HBL-1, in which the NF-κB pathway is activated by a mutation in CARD11 which is downstream of BTK, are not inhibited by PCI-32765. Both sensitive and resistant cells express Btk at similar levels, and the active site of BTK is fully occupied in the presence of low concentrations of the inhibitor as shown using a fluorescently-labeled affinity probe. We show that the sensitive TMD8 and OCI-Ly10 cells have chronically active BCR signaling to NF-κB which is dose-dependently inhibited by PCI-32765, as measured by comparing the phosphorylation and nuclear relocation of NF-κB p65 subunit. These cells also show inhibition of other major signaling pathways downstream of the BCR, including p-ERK, p-AKT, p-PLCγ and intracellular calcium flux, ultimately leading to NF-κB activation. The non-responsive OCI-Ly3 cells do not show inhibition of NF-κB p65 phosphorylation or nuclear relocation, although Btk is present and active, as shown by the probe assay and inhibition of p-AKT by PCI-32765. Furthermore, whole genome expression analysis reveals inhibition of mRNA expression of several NF-κB target genes in OCI-Ly10, particularly c-Myc, NF-κB subunits and several chemokines and cytokines, leading to downregulation of STAT3 signaling, NFAT signaling, cell cycle and cytokine-chemokine signaling pathways, none of which was observed in OCI-Ly3 cells. In vivo, PCI-32765 dose-dependently inhibited tumor growth in mice carrying OCI-Ly10 (and TMD8, ongoing) xenografts. Analysis of these tumors exhibited dose-dependent occupancy of the BTK active site as well as inhibition of signaling pathways. These results demonstrate the mechanistic basis of PCI-32765 inhibitory activity in ABC-DLBCL and support the ongoing clinical investigation of the therapeutic use of PCI-32765 in patients with this devastating disease. Disclosures:Balasubramanian:Pharmacyclics: Employment, Equity Ownership. Crowley:Pharmacyclics: Employment, Equity Ownership. Sirisawad:Pharmacyclics: Employment, Equity Ownership. Thiemann:Pharmacyclics: Employment, Equity Ownership. Chen:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.

Contrary Functions of NF-κB Signaling in Anti-Cancer Treatment Outcome

Abstract 1652Nuclear factor-κB (NF-κB) transcription factors are involved in cancer-relevant processes such as suppression of apoptosis, growth promotion, enhanced migration and invasiveness, although their actual role as oncogenic or tumor-suppressive activities remains controversial. Moreover, NF-κB-mediated suppression of apoptosis has been linked to chemoresistance. Interestingly, cellular senescence, a terminal cell-cycle arrest initiated via DNA-damaging chemotherapy as well, and known to improve long-term outcome, is associated with the massive induction of secretable NF-κB target genes, which, in turn, potentially reinforce the senescence phenotype.In this study, primary Eμ-myc transgenic mouse lymphomas as a well established model for human aggressive B-NHL, and information from human diffuse large B-cell lymphomas (DLBCL) were used in a cross-species approach to identify oncogenic networks in which chemotherapy-activated NF-κB signaling no longer mediates resistance but promotes therapy-induced senescence (TIS) and contributes to the outcome of anti-cancer treatment.Primary Myc-driven lymphoma cells, stably bcl2-transduced to block apoptosis, were exposed to the DNA-damaging chemotherapeutic agent adriamycin (ADR) to induce TIS. Gene set enrichment analysis of microarray-based gene expression profiles from drug-senescent vs. untreated cells found NF-κB target genes strongly skewed towards the TIS group, and multiplex ELISA-based analysis detected significantly higher DNA binding activities for the NF-κB family subunits p50, p52, p65 (RelA) and RelB in senescent cells.Inactivation of NF-κB by stable expression of the NF-κB super-repressor IκBα-δN (SR) lowered expression levels of NF-κB target genes in ADR-treated lymphomas. Matched pairs of individual primary lymphomas differing only in their SR status displayed compromised senescence induction in vivo when expressing the SR, indicating that TIS depends on intact NF-κB function. To assess the contribution of endogenous NF-κB signaling to long-term outcome, we grouped primary Myc-lymphomas by their NF-κB activity levels as “NF-κB low” (NL) or “NF-κB high” (NH). ADR-treated NH, but not NL lymphomas presented selective vulnerability to the SR moiety. Recapitulating the clinical outcome of patients suffering from DLBCL, around 60% of the mice harboring Eμ-myc lymphomas achieved long-term remissions, while the remaining 40% encountered a relapse after chemotherapy. Relapsing Myc-lymphomas exhibited substantially higher expression of the NF-κB targets IκBα and bcl2, reminiscent of activated B-cell-like (ABC) DLBCL, the clinically inferior subtype characterized by constitutively active NF-κB signaling. In contrast, germinal center B-cell-like (GCB) DLBCL rarely possess activating NF-κB mutations, but frequently develop in the context of a t(14;18) translocation that drives Bcl2 overexpression independent of NF-κB. Induced NF-κB target gene expression and increased TIS induction after overexpression of an NF-κB-activating CARD11 mutant suggested that higher NF-κB activity may contribute to treatment outcome via TIS promotion. Indeed, stratifying a large dataset of untreated GEP and corresponding clinical data after immunochemotherapy from 233 DLBCL patients by a 63-gene NF-κB expression signature (Shaffer-AL et al, Immunol Rev, 2006; Lenz-G et al, N Eng J Med, 2008), confirmed for the subset of GCB patients with above-median Bcl2 expression – the group whose genetic features we modeled before in the mouse – a significantly superior progression-free survival.In essence, our “cross-species” investigations demonstrate opposing roles of NF-κB in treatment outcome and have important ramifications for clinical trials that aim at inhibiting NF-κB signaling in DLBCL. Disclosures:No relevant conflicts of interest to declare.

PPARγ activation by baicalin suppresses NF-κB-mediated inflammation in aged rat kidney

Baicalin, a herb-derived flavonoid compound, has beneficial activities, including the modulation of oxidative stress and inflammation. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor that plays an important role in regulating nuclear factor-κB (NF-κB)-induced age-related inflammation. We investigated the anti-inflammatory action of baicalin, which depends on its ability to activate PPARγ, and subsequently to suppress NF-κB. We examined baicalin-treated kidney tissue from 24-month-old Fischer 344 aged rats (10 or 20 mg/kg/day for 10 days) and baicalin-fed mice (10 mg/kg/day for 3 days) for in vivo investigations, and used endothelial YPEN-1 cells for in vitro studies. In the baicalin-fed aged rats, there was a marked enhancement of both nuclear protein levels and DNA binding activity of PPARγ, and a decreased expression of NF-κB target genes (VCAM-1, IL-1β, and IL-6) compared with non-baicalin-fed aged rats. Furthermore, to confirm the anti-inflammatory action of PPARγ activated by baicalin, we used lipopolysaccharide (LPS)-treated cells and mice. The results showed that baicalin induced PPARγ-selective activation in YPEN-1 cells, and that the effects of baicalin were blocked by the PPARγ receptor antagonist, GW9662. In addition, baicalin treatment prevented RS generation, NF-κB activation and the expression of pro-inflammatory genes, whereas it increased PPARγ expression in LPS-treated cells and mouse kidney. Our data suggest that baicalin-induced PPARγ expression reduced age-related inflammation through blocking pro-inflammatory NF-κB activation. These results indicate that baicalin is a novel PPARγ activator and that this agent may have the potential to minimize inflammation.

Notch is an essential upstream regulator of NF-κB and is relevant for survival of Hodgkin and Reed–Sternberg cells

A major pathogenetic mechanism in classical Hodgkin lymphoma (cHL) is constitutive activation of canonical nuclear factor-κB (NF-κB) p50/p65 signaling, controlling lymphoma cell proliferation and survival. Recently, we demonstrated that aberrant Notch1 activity is a negative regulator of the B cell program in B cell-derived Hodgkin and Reed-Sternberg (HRS) cells. Despite abundant evidence for a complex context-dependent cross talk between Notch and NF-κB signaling in hematopoietic cells, it is unknown whether these pathways interact in HRS cells. Here, we show that Notch-signaling inhibition in HRS cells by the γ-secretase inhibitor (GSI) XII results in decreased alternative p52/RelB NF-κB signaling, interfering with processing of the NF-κB2 gene product p100 into its active form p52. As a result, expression of Notch and NF-κB target genes is reduced, and survival of HRS cells is impaired. Stimulation of alternative NF-κB signaling in the Hodgkin cell line L540cy by activation of the CD30 receptor rescued GSI-mediated loss of cell viability and apoptosis induction. Our data reveal that Notch is an essential upstream regulator of alternative NF-κB signaling and indicate cross talk between both the pathways in HRS cells. Therefore, we suggest that targeting the Notch pathway is a promising therapeutic option in cHL.