Canonical NF-κB Signaling Pathway Research Articles

A20-mediated negative regulation of canonical NF-κB signaling pathway

The nuclear factor kappa B (NF-κB) plays vital role in the immune system by regulating innate and adaptive immunity, development and survival of lymphocytes, and lymphoid organogenesis. All known NF-κB activators converge on the IkappaB kinase (IKK) complex to activate the canonical and non-canonical NF-κB pathways. The IKK complex contains two catalytic subunits (IKKα and IKKβ) and a regulatory subunit NEMO/IKKγ that regulates the canonical NF-κB pathway, whereas IKKα regulates the non-canonical pathway. The process of IKKα activation and its role in the regulation of canonical NF-κB activation remain elusive. The canonical pathway is rapidly activated and produces a potent inflammatory response to bacterial and viral infections as well as different types of stress; however, uncontrolled NF-κB activation can lead to autoimmune diseases and cancers. Therefore, to keep the inflammatory response in check, elaborate negative regulatory mechanisms operate to terminate NF-κB activation at multiple levels by de novo synthesis of NF-κB inhibitory proteins, and orchestration of protein ubiquitination and deubiquitination. The NF-κB target genes, IκBα and A20, play critical roles in termination of the active canonical NF-κB pathway. In this review, we discuss our recent findings describing a novel function for IKKα in nucleating the ubiquitin-editing enzyme A20 complex, a major negative regulator of canonical NF-κB signaling. Consistently with an inhibitory function of IKKα, it is targeted by the human T-cell leukemia virus 1 (HTLV-1) oncoprotein, Tax, to prevent assembly of the A20 complex to maintain persistent NF-κB activation that promotes transformation and survival of virus-transformed cells.

Noncanonical Nuclear Factor Kappa B (NF-κB) Signaling and Potential for Therapeutics in Sepsis

NF-κB signaling plays a central role in the pathophysiology of severe sepsis and septic shock. Despite tremendous and missed efforts, novel therapeutics for severe sepsis and septic shock are still needed. Many drugs have been designed to target the canonical NF-κB signaling pathway with limited success, potentially due to the nonspecificity of the drugs for other kinases and the interaction of canonical signaling with other pathways. Here, we review the canonical and noncanonical signaling pathways of NF-κB, the cross talk and negative regulation of the two pathways, and the potential for therapeutics arising from the noncanonical NF-κB pathway in relation to the pathophysiology of septic shock.

Phenotype profiling of primary testicular diffuse large B‐cell lymphomas

Primary testicular diffuse large B-cell lymphomas (tDLBCL) are rare neoplasms with few comprehensive studies conducted so far. We aimed to systematically characterize the phenotype of tDLBCL. Forty-five patients from three Swiss hospitals diagnosed with tDLBCL between 1972 and 2009 were reviewed and included in this study. A tissue microarray was assembled, and the protein expression profiles were assessed by immunohistochemistry and fluorescence in situ hybridization for the C-MYC locus. All tDLBCL expressed CD79a, followed by CD20 (98% of cases) and CD19 (93%). One case expressed the germ cell marker OCT4 and showed a rearrangement of C-MYC. Eighty-two per cent of cases showed active STAT signalling by expression of either pSTAT1 or pSTAT3 or both, but not pSTAT5. The p53 was overexpressed in 10% of cases, but p21 staining (∆p21/p53) did not suggest the presence of TP53 mutations. tDLBCL had a median MIB1 labelling index of 40%, and only 6% of cases appeared to have C-MYC rearrangements. Most cases were of the non-germinal centre type (77%), and showed as expected for this cell of origin B-cell lymphoma 2 (BCL2) rearrangements only in 4% and amplifications in 15% of cases, whereas BCL6 was rearranged in 48% of cases. CXCR4 was expressed in 52% of cases, and high CXCR4 expression was of prognostic significance for progression-free survival (p = 0.003). Because 84% of cases expressed nuclear p50, the canonical NF-κB signalling pathway seems to be active. Multimarker phenotyping is important for lineage determination of tDLBCL. Occasionally, tDLBCL can express germ cell markers like OCT4, and they have active STAT signalling mediated through pSTAT1 and pSTAT3 and active canonical NF-κB signalling. tDLBCL are of non-germinal centre/post-germinal centre cell origin and not hyperproliferative. TP53 mutations are unlikely, and C-MYC as well as BCL2 rearrangements are rare, whereas BCL6 is commonly rearranged. CXCR4 might prove to be the first protein-based prognostic marker for tDLBCL, inciting studies in larger cohorts corroborating these findings.

Herpes simplex virus 1 DNA polymerase processivity factor UL42 inhibits TNF-α-induced NF-κB activation by interacting with p65/RelA and p50/NF-κB1

Herpes simplex virus 1 (HSV-1) is the archetypal member of the alphaherpesvirus with a large genome encoding over 80 viral proteins, many of which are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrated that the HSV-1 UL42 protein, a DNA polymerase processivity factor, was a novel antagonism of the canonical NF-κB signaling pathway. UL42 was shown to significantly suppress TNF-α mediated NF-κB activation. Co-immunoprecipitation experiment revealed that UL42 bound to the NF-κB subunits p65 and p50. Fluorescence microscopy demonstrated that UL42 abolished nuclear translocation of p65 and p50 upon TNF-α-stimulation. But the inhibiting capacity of UL42 2R/2A (R279A, R280A) and UL42 3R/3A (R113A, R279A and R280A) mutants were less than wild type UL42. Also UL42 bound to the Rel homology domain of the NF-κB subunit p65 and p50. Notably, the N-terminal of UL42 was sufficient to interact with p65 and p50 and abolished NF-κB reporter gene activity. Thus, it was first time we demonstrated that HSV-1 UL42 appeared to prevent NF-κB-dependent gene expression by retaining p65 and p50 in the cytoplasm, and UL42-dependent transcriptional activation were inherently coupled to promote HSV-1 lytic replication, which also may contribute to immune evasion and pathogenesis of HSV-1.

PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

Synthetic peroxisome proliferator-activated receptor (PPAR) agonists are used to treat dyslipidemia and insulin resistance. In this study, we examined molecular mechanisms that explain differential effects of a PPARα agonist (fenofibrate) and a PPARγ agonist (rosiglitazone) on macrophages during obesity-induced atherogenesis. Twelve-week-old mice with combined leptin and LDL-receptor deficiency (DKO) were treated with fenofibrate, rosiglitazone or placebo for 12 weeks. Only rosiglitazone improved adipocyte function, restored insulin sensitivity, and inhibited atherosclerosis by decreasing lipid-loaded macrophages. In addition, it increased interleukin-1 receptor-associated kinase-3 (Irak3) and decreased monocyte chemoattractant protein-1 (Mcp1) expressions, indicative of a switch from M1 to M2 macrophages. The differences between fenofibrate and rosiglitazone were independent of Pparγ expression. In bone marrow-derived macrophages (BMDM), we identified the rosiglitazone-associated increase in adiponectin as cause of the increase in Irak3. Interestingly, the deletion of Irak3 in BMDM (IRAK3−/− BMDM) resulted in activation of the canonical NFκB signaling pathway and increased Mcp1 protein secretion. Rosiglitazone could not decrease the elevated Mcp1 secretion in IRAK3−/− BMDM directly and fenofibrate even increased the secretion, possibly due to increased mitochondrial reactive oxygen species production. Furthermore, aortic extracts of high-fat insulin-resistant LDL-receptor deficient mice, with lower adiponectin and Irak3 and higher Mcp1, showed accelerated atherosclerosis. In aggregate, our results emphasize an interaction between PPAR agonist-mediated increase in adiponectin and macrophage-associated Irak3 in the protection against atherosclerosis by PPAR agonists.

Abstract 1135: Chemical Proteomics effort identifies PKN1 as a key player in the canonical NF-κB signaling pathway.

Abstract Deregulation of NF-κB signaling pathway has been identified as one of the key drivers of many hematological malignancies. Activation of the NF-κB pathway in these cells can happen either through activating mutations within the pathway, loss of function mutations of inhibitory molecules or through interaction with the stroma that secrete specific cytokines. The strategies for blocking the NF-κB pathway activation have so far focused on NF-κB pathway kinase inhibitors or the more general proteasome inhibitors. However, the adverse events associated with available NF-κB pathway modulators in pre-clinical and clinical settings have limited their general therapeutic use. To discover new targets and modulators of this key survival pathway, we performed a cellular phenotypic screen that led to the identification of a novel class of small molecules that inhibit the activation of the canonical NF-κB pathway. The compounds showed potent inhibition of NF-κB activation by multiple receptors including the TNF, antigen and BAFF/ APRIL receptors. Structure activity relationship analysis for this novel class of molecules identified pairs of stereoisomers wherein the cis isoform showed greater than 100 fold activity than the trans isoform. Detailed interrogation of the mechanism of action of the active cis isomers using biochemical and cellular pathway analysis revealed that the compounds are potent inhibitors of RHO associated kinases, only weakly inhibit IKK1 and IKK2 and strongly inhibit the induction of phosphorylated IkBα upon stimulation. However, the differential activity of the stereoisomers was not seen for the inhibition of the RHO associated kinases, suggesting the inhibition of NF-κB pathway was due to modulation of other targets. This raises the possibility of identifying novel targets associated with this compound. Using in-lysate affinity chemical proteomics with the active and in-active enantiomer, we have identified PKN1 as a key efficacy target for the inhibitors. Knockdown of PKN1 using genetic tools was sufficient to abrogate the response of cells to TNFα stimulation implicating a critical role for PKN1 in the activation of NF-κB pathway. Our findings therefore present an opportunity to develop novel NF-κB inhibitors for the tumors that depend on this key pathway for survival. Citation Format: Katherine Tang, Michael Lampa, Tieu-Binh Le, Matthieu Barrague, Ronald Tomlinson, Brittain Scott, Tahir Majid, Marion Dorsch, Hong Cheng, Christoph Lengauer, Carlos Garcia-Echeverria, Francisco Adrian, Mikhail Levit, Balin Zhang, Kin Yu, Ivan Cornella-Taracido, Lakshmi Srinivasan. Chemical Proteomics effort identifies PKN1 as a key player in the canonical NF-κB signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2013-1135

Gold nanoparticles induce transcriptional activity of NF-κB in a B-lymphocyte cell line.

Gold nanoparticles (Au-NPs) have been designated as superior tools for biological applications owing to their characteristic surface plasmon absorption/scattering and amperometric (electron transfer) properties, in conjunction with low or no immediate toxicity towards biological systems. Many studies have shown the ease of designing application-based tools using Au-NPs but the interaction of this nanosized material with biomolecules in a physiological environment is an area requiring deeper investigation. Immune cells such as lymphocytes circulate through the blood and lymph and therefore are likely cellular components to come in contact with Au-NPs. The main aim of this study was to mechanistically determine the functional impact of Au-NPs on B-lymphocytes. Using a murine B-lymphocyte cell line (CH12.LX), treatment with citrate-stabilized 10 nm Au-NPs induced activation of an NF-κB-regulated luciferase reporter, which correlated with altered B lymphocyte function (i.e. increased antibody expression). TEM imaging demonstrated that Au-NPs can pass through the cellular membrane and therefore could interact with intracellular components of the NF-κB signaling pathway. Based on the inherent property of Au-NPs to bind to -thiol groups and the presence of cysteine residues on the NF-κB signal transduction proteins IκB kinases (IKK), proteins specifically bound to Au-NPs were extracted from CH12.LX cellular lysate exposed to 10 nm Au-NPs. Electrophoresis identified several bands, of which IKKα and IKKβ were immunoreactive. Further evaluation revealed activation of the canonical NF-κB signaling pathway as evidenced by IκBα phosphorylation at serine residues 32 and 36 followed by IκBα degradation and increased nuclear RelA. Additionally, expression of an IκBα super-repressor (resistant to proteasomal degradation) reversed Au-NP-induced NF-κB activation. Altered NF-κB signaling and cellular function in B-lymphocytes suggests a potential for off-target effects with in vivo applications of gold nanomaterials and underscores the need for more studies evaluating the interactions of nanomaterials with biomolecules and cellular components.

Tumour necrosis factor alpha down-regulates the expression of peroxisome proliferator activated receptor alpha (PPARα) in human hepatocarcinoma HepG2 cells by activation of NF-κB pathway

Peroxisome proliferator activated receptor-alpha (PPARα) plays a major role in the regulation of lipid and glucose homeostasis, and inflammatory responses. The objectives of the study were to systematically investigate the effects of TNF-α and its regulatory pathway on PPARα expression in HepG2 cells using Real-Time RT-PCR and western blot analysis. Here, TNF-α suppressed PPARα mRNA expression in a dose- and time-dependent manner at the level of gene transcription. Pre-treatment of cells with 10μM of Wedelolactone for 2h was sufficient to restore PPARα expression to basal levels and also affected the expression of PPARα-regulated genes. This study also demonstrated that TNF-α represses PPARα expression by augmenting the activity of canonical NF-κB signalling pathway. This was shown by the abrogation of TNF-α-mediated PPARα down-regulation, after both p65 and p50 were knocked down via siRNA. The IKK contributes to IκBα degradation and mediates inducible phosphorylation of p105 at Ser933. Surprisingly, phosphorylation of p65 at Ser468 and Ser536 were severely abrogated with Wedelolactone inhibition, suggesting that Ser468 and Ser536, but not Ser276, may mediate the TNF-α inhibitory action on PPARα gene expression. These results suggest that TNF-α might, at least in part, suppress PPARα expression through activation of IKK/p50/p105/p65 pathway. Furthermore, phosphorylation of p65 at Ser468 and Ser536 may play a crucial role in the mechanism that limits PPARα production in the human HepG2 cells.

Proinflammatory effects of malondialdehyde in lymphocytes

Diabetes is an inflammatory disease promoted by alterations in immune cell function. Animal study indicates that T cells are important mediators of inflammation in diabetes. Lipid peroxidation by reactive oxygen species leads to the formation of highly reactive malondialdehyde (MDA), and extensive MDA is found in diabetes. However, the biological functions of MDA have not been studied yet. We hypothesized that increased MDA, as in diabetes, can regulate inflammatory cytokines via specific signaling pathways. This could then result in increased lymphocyte activation and skewing a particular inflammatory subset thereby exacerbates diabetes complications. Commercial cytokine antibody and RT(2)-PCR array profiling were performed with Jurkat T cells grown with or without MDA. Ingenuity pathways analysis (IPA) and pharmacological inhibitors were used for networks and signaling pathway identification, respectively. For validation, real-time PCR, RT-PCR, and Western blots were performed. MDA induced significant increases in 47 key proinflammatory molecules such as IL-25, IL-6, IL-8, ICAM-1, and light mRNA in Jurkat T cells and primary peripheral blood lymphocytes (PBLCs). A significant 2-fold increase in serum MDA also correlated the increased IL-25 and IL-8 mRNA in PBLCs of diabetic patients. Pharmacological inhibitor studies showed that MDA induced its effect via p38MAPK and protein kinase C pathways. Furthermore, IPA uncovered 5 groups of inflammatory networks and placed our candidate genes in canonical IL-6 and NF-κB signaling pathways and also suggested 5 toxic lists and 3 major toxic functions, namely cardiotoxicity, hepatotoxicity, and nephrotoxicity. These new results suggest that MDA can promote lymphocyte activation via induction of inflammatory pathways and networks.

The NS1 Protein of Influenza A Virus Blocks RIG-I-Mediated Activation of the Noncanonical NF-κB Pathway and p52/RelB-Dependent Gene Expression in Lung Epithelial Cells

Influenza A virus (IAV) infection of epithelial cells activates NF-κB transcription factors via the canonical NF-κB signaling pathway, which modulates both the antiviral immune response and viral replication. Since almost nothing is known so far about a function of noncanonical NF-κB signaling after IAV infection, we tested infected cells for activation of p52 and RelB. We show that the viral NS1 protein strongly inhibits RIG-I-mediated noncanonical NF-κB activation and expression of the noncanonical target gene CCL19.

A novel NEDD8-binding protein modulates NF-κB signaling pathway

In canonical NF-κB signaling pathway, a ubiquitin ligase called SCF (Skp1, Cul1, F-box protein) complex is essential for I-κB degradation. The activity of the SCF complex is positively regulated by a post-translational modification of Cul1 subunit with a ubiquitin-like protein NEDD8. Like ubiquitin, NEDD8 possesses evolutionary conserved Lys residues on its surface, and forms poly-NEDD8 chain in vivo and in vitro [1,2]. Despite the importance of the NEDD8 modification in all eukaryotic cells, little is known about the function of poly-NEDD8 chain. To elucidate the function of the poly-NEDD8 chain in vivo, we screened poly-NEDD8 chain binding proteins (PNBPs) using a yeast two-hybrid system. Of the identified PNBPs, PNBP1 was identical to a gene present in non-HLA celiac disease and rheumatoid arthritis risk loci [3]. PNBP1 interacted with NEDD8, NEDD8-conjugating enzyme Ubc12 and Cul1. PNBP1 strongly associated with wild-type Cul1, but not its NEDDylation defective Cul1(K720R) mutant, suggesting that the interaction is mediated in part through NEDD8. Furthermore, PNBP1 promoted NEDDylation of Cul1 in an in vitro reconstitution assay. These activities were dependent on RING-finger domain of PNBP1. Finally, knockdown of PNBP1 led to reduction of the NF-κB activation, suggesting that PNBP1 is an important modulator of the NF-κB signaling pathway.

An RNA Interference Screen Identifies the Deubiquitinase STAMBPL1 as a Critical Regulator of Human T-Cell Leukemia Virus Type 1 Tax Nuclear Export and NF-κB Activation

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein actively shuttles between the nucleus, where it interacts with transcriptional and splicing regulatory proteins, and the cytoplasm, where it activates NF-κB. Posttranslational modifications of Tax such as ubiquitination regulate its subcellular localization and hence its function; however, the regulation of Tax trafficking and NF-κB activation by host factors is poorly understood. By screening a deubiquitinating (DUB) enzyme small interfering RNA (siRNA) library, we identified the metalloprotease STAM-binding protein-like 1 (STAMBPL1) as a positive regulator of Tax-mediated NF-κB activation. Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant, enhanced Tax-mediated NF-κB activation, whereas silencing of STAMBPL1 with siRNA impaired Tax activation of both the canonical and noncanonical NF-κB signaling pathways. STAMBPL1 regulated Tax-induced NF-κB signaling indirectly by controlling Tax nuclear/cytoplasmic transport and was required for DNA damage-induced Tax nuclear export. Together, these results reveal that the deubiquitinase STAMBPL1 is a key regulator of Tax trafficking and function.

Potential roles of the NFκB and glutathione pathways in mature human erythrocytes.

Anucleated erythrocytes were long considered as oxygen-transporting cells with limited regulatory functions. Components of different nuclear signaling pathways have not been investigated in those cells, yet. Surprisingly, we repeatedly found significant amounts of transcription factors in purified erythrocyte preparations, i.e. nuclear factor κB (NFκB), and major components of the canonical NFκB signaling pathway. To investigate the functional role of NFκB signaling, the effects of the preclinical compounds Bay 11-7082 and parthenolide on the survival of highly purified erythrocytes were investigated. Interestingly, both inhibitors of the NFκB pathway triggered erythrocyte programmed cell death as demonstrated by enhanced phospholipid scrambling (phosphatidylserine exposure) and cell shrinkage. Anucleated erythrocytes are an ideal cellular model allowing the study of nongenomic mechanisms contributing to suicidal cell death. As NFκB inhibitors might also interfere with the anti-oxidative defense systems of the cell, we measured the levels of reduced glutathione (GSH) after challenge with the inhibitors. Indeed, incubation of erythrocytes with Bay 11-7082 clearly decreased erythrocyte GSH levels. In conclusion, the pharmacological inhibitors of the NFκB pathway Bay 11-7082 and parthenolide interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression. Besides affecting erythrocyte survival, NFκB inhibition and induction of erythrocyte phosphatidylserine exposure may influence blood clotting. Future studies will be aimed at discriminating between NFκB-dependent and NFκB-independent GSH-mediated effects of Bay 11-7082 and parthenolide on erythrocyte death.

Dietary chlorophyllin inhibits the canonical NF-κB signaling pathway and induces intrinsic apoptosis in a hamster model of oral oncogenesis

Chlorophyllin, a water-soluble, semi-synthetic derivative of the ubiquitous green pigment chlorophyll is shown to exert potent anticarcinogenic effects. In the present study, we investigated the chemopreventive effects of chlorophyllin on 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by analyzing the expression of NF-κB family members and markers of intrinsic apoptosis. Dietary administration of chlorophyllin (4mg/kg bw) suppressed the development of HBP carcinomas by inhibiting the canonical NF-κB signaling pathway by downregulating IKKβ, preventing the phosphorylation of IκB-α, and reducing the expression of nuclear NF-κB. Inactivation of NF-κB signaling by chlorophyllin was associated with the induction of intrinsic apoptosis as evidenced by modulation of Bcl-2 family proteins, enforced nuclear localization of survivin, upregulation of apoptogenic molecules, activation of caspases, and cleavage of PARP. The results of the present study demonstrate that chlorophyllin inhibits the development of DMBA-induced HBP carcinogenesis by targeting NF-κB and the intrinsic apoptotic pathway. Thus, dietary agents such as chlorophyllin that simultaneously target divergent pathways of cell survival and cell death are novel candidates for cancer chemoprevention.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Abstract 1841 Introduction:The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods:We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results:We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions:We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures:Hideshima:Acetylon: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

Crosstalk between the canonical NF-κB and Notch signaling pathways inhibits Pparγ expression and promotes pancreatic cancer progression in mice

The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-κB pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of κB kinase 2 (Ikk2), a component of the canonical NF-κB signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-α stimulated canonical NF-κB signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-α stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Pparγ. Thus, crosstalk between Tnf-α/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.

The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors

The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-κB signaling pathway, acting as both a repressor of transactivation of the NF-κB subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase-dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.

Sexual differentiation of the gonadotropin surge release mechanism: A new role for the canonical NfκB signaling pathway

Sex differences in luteinizing hormone (LH) release patterns are controlled by the hypothalamus, established during the perinatal period and required for fertility. Female mammals exhibit a cyclic surge pattern of LH release, while males show a tonic release pattern. In rodents, the LH surge pattern is dictated by the anteroventral periventricular nucleus (AVPV), an estrogen receptor-rich structure that is larger and more cell-dense in females. Sex differences result from mitochondrial cell death triggered in perinatal males by estradiol derived from aromatization of testosterone. Herein we provide an historical perspective and an update describing evidence that molecules important for cell survival and cell death in the immune system also control these processes in the developing AVPV. We conclude with a new model proposing that development of the female AVPV requires constitutive activation of the Tnfα, Tnf receptor 2, NfκB and Bcl2 pathway that is blocked by induction of Tnf receptor-associated factor 2-inhibiting protein (Traip) in the male.

STAMBPL1 is a deubiquitinating enzyme that regulates HTLV-I Tax subcellular localization and NF-kB activation

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein is predominantly localized in the nuclei where it is associated with transcriptional and splicing regulatory proteins to form the Tax speckled structures (TSS). Tax is also distributed in the Golgi apparatus where it activates the IkB kinase (IKK) complex upstream of NF-κB, an essential transcription factor for Tax-induced immortalization of T lymphocytes. Lysine 63 (K63)-linked polyubiquitination of Tax is necessary for Tax to localize to the Golgi and activate NF-κB, whereas K48-linked polyubiquitination of Tax promotes its degradation in the nucleus. Therefore, post-translational modifications of Tax regulate its subcellular localization and hence its function, however the host factors that regulate Tax localization are unknown. Using a deubiquitinating (DUB) enzyme siRNA library we identified STAM-binding protein-like 1 (STAMBPL1) as a DUB that is required for Tax-mediated NF-κB activation. STAMBPL1 is a member of the JAMM (JAB1/MPN/MOV34 metalloenzyme) family of zinc metalloproteases, several of which have been previously implicated in the regulation of receptor endocytosis and trafficking. Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant, enhances Tax-mediated NF-κB activation and the induction of NF-κB target genes. Silencing of STAMBPL1 with siRNA inhibits Tax activation of both the canonical and noncanonical NF-κB signaling pathways. Furthermore, overexpression of STAMBPL1 enhances the stability of Tax by blocking its proteasomal degradation, whereas siRNA silencing of STAMBPL1 promotes Tax degradation. Confocal microscopy experiments reveal that STAMBPL1 controls the subcellular localization of Tax by promoting its nuclear export and subsequent trafficking to the Golgi where it activates IKK. Together these results suggest that the deubiquitinating enzyme STAMBPL1 regulates the localization and stability of Tax, and consequently the activation levels of NF-κB.