NF-κB Dimers Research Articles

Targeting IκB Proteins for HIV Latency Activation: the Role of Individual IκB and NF-κB Proteins

Latently infected cell reservoirs represent the main barrier to HIV eradication. Combination antiretroviral therapy (cART) effectively blocks viral replication but cannot purge latent provirus. One approach to HIV eradication could include cART to block new infections plus an agent to activate latent provirus. NF-κB activation induces HIV expression, ending latency. Before activation, IκB proteins sequester NF-κB dimers in the cytoplasm. Three canonical IκBs, IκBα, IκBβ, and IκBε, exist, but the IκB proteins' role in HIV activation regulation is not fully understood. We studied the effects on HIV activation of targeting IκBs by single and pairwise small interfering RNA (siRNA) knockdown. After determining the relative abundance of the IκBs, the relative abundance of NF-κB subunits held by the IκBs, and the kinetics of IκB degradation and resynthesis following knockdown, we studied HIV activation by IκB knockdown, in comparison with those of known HIV activators, tumor necrosis factor alpha (TNF-α), tetradecanoyl phorbol acetate (TPA), and trichostatin A (TSA), in U1 monocytic and J-Lat 10.6 lymphocytic latently infected cells. We found that IκBα knockdown activated HIV in both U1 and J-Lat 10.6 cells, IκBβ knockdown did not activate HIV, and, surprisingly, IκBε knockdown produced the most HIV activation, comparable to TSA activation. Our data show that HIV reactivation can be triggered by targeting two different IκB proteins and that IκBε may be an effective target for HIV latency reactivation in T-cell and macrophage lineages. IκBε knockdown may offer attractive therapeutic advantages for HIV activation because it is not essential for mammalian growth and development and because new siRNA delivery strategies may target siRNAs to HIV latently infected cells.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-κB) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream κB binding sites in RAW 264.7 macrophage cell lines was repressed when NF-κB activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-κB subunits. Therefore, transcription of aa-nat driven by NF-κB dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-κB in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Piperlongumine inhibits proliferation and survival of Burkitt lymphoma in vitro

Piperlongumine (PL), a pepper plant alkaloid from Piper longum, kills solid tumor cells in a highly selective, potent fashion. To evaluate whether PL may have similar effects on malignant blood cells, we determined the efficacy with which PL inhibits the B-lymphocyte derived neoplasm, Burkitt lymphoma (BL). Low micromolar concentrations of PL (IC50=2.8μM×8.5μM) curbed growth and survival of two EBV+ BL cell lines (Daudi, Raji) and two EBV BL cell lines (Ramos, DG-75), but left normal peripheral blood B-lymphocytes unharmed. PL-dependent cytotoxicity was effected in part by reduced NF-κB and MYC activity, with the former being caused by inhibition of IκBα degradation, nuclear translocation of p65, and binding of NF-κB dimers to cognate DNA sequences in gene promoters. In 4 of 4 BL cell lines, the NF-κB/MYC-regulated cellular target genes, E2F1 and MYB, were down regulated, while the stress sensor gene, GADD45B, was up regulated. The EBV-encoded oncogene, LMP-1, was suppressed in Daudi and Raji cells. Considering that NF-κB, MYC and LMP-1 play a crucial role in the biology of many blood cancers including BL, our results provide a strong preclinical rationale for considering PL in new intervention approaches for patients with hematologic malignancies.

The Transcriptional Specificity of NF-κB Dimers Is Coded within the κB DNA Response Elements

SUMMARYNuclear factor κB (NF-κB) regulates gene expression by binding to specific DNA elements, known collectively as κB sites, that are contained within the promoters/enhancers of target genes. We found that the identity of the central base pair (bp) of κB sites profoundly affects the transcriptional activity of NF-κB dimers. RelA dimers prefer an A/T bp at this position for optimal transcriptional activation (A/T-centric) and discriminate against G/C-centric κB sites. The p52 homodimer, in contrast, activates transcription from G/C-centric κB sites in complex with Bcl3 but represses transcription from the A/T-centric sites. The p52:Bcl3 complex binds to these two classes of κB sites in distinct modes, permitting the recruitment of coactivator, corepressor, or both coactivator and corepressor complexes in promoters that contain G/C-, A/T-, or both G/C- and A/T-centric sites. Therefore, through sensing of bp differences within κB sites, NF-κB dimers modulate biological programs by activating, repressing, and altering the expression of effector genes.

Oroxylin A, a classical natural product, shows a novel inhibitory effect on angiogenesis induced by lipopolysaccharide

BackgroundThere is an obvious relationship among angiogenesis and inflammation. From previous study, we learn that oroxylin A possesses anti-angiogenic activity in vitro and in ovo. It also has an inhibitory effect on inflammation. But whether oroxylin A suppresses the inflammation-induced angiogenesis is still unknown. Our present study focuses on the role of oroxylin A in targeting LPS-induced angiogenesis, inflammatory and related pathways. MethodsThe effects of oroxylin A on angiogenesis were investigated by transwell assay, tube formation assay, rat aortic ring assay and chorioallantoic membrane (CAM) model. Western blotting analysis was used to detect the expression of certain proteins. ResultsWe found that oroxylin A inhibited LPS-induced migration and tube formation of human umbilical vein endothelial cells (HUVECs), as well as microvessel sprouting from rat aotric ring in vitro and the angiogenesis of chicken chorioallantoic membrane (CAM) model in ovo. The results also indicated that oroxylin A could inhibit the expression of LPS acceptor toll-like receptor 4 (TLR4) and the activities of its downstream mitogen-activated protein kinases (MAPKs), including reducing expressions of the phosphorylation of JNK, p38, and ERK. Moreover, oroxylin A prevented NF-κB dimers from translocating to the nucleus. ConclusionsTaken together, oroxylin A can suppress the angiogenesis induced by LPS and it may affect the LPS/TLR4 signaling pathway.

Double-edged sword: a p53 regulator mediates both harmful and beneficial effects in experimental acute kidney injury

Acute kidney injury triggers activation of innate immune responses and of proapoptotic programs such as the p53 pathway. Mulay et al. examine the effects of blocking murine double minute-2 (mdm2), a negative regulator of p53, using a novel chemotherapeutic agent, nutlin-3a, in mouse ischemia-reperfusion injury. Their results indicate that mdm2 promotes renal regeneration by limiting p53-mediated apoptosis but also enhances early inflammation by facilitating DNA binding of nuclear factor-κB independently of p53.

IKKβ–I-κBɛ–c-Rel/p50: a new axis of NF-κB activation in lung epithelial cells

Cigarette smoke (CS), a major risk factor for developing lung cancer, is known to activate transcriptional activator nuclear factor kappa B (NF-κB). However, the underlying mechanism of this activation remains unclear because of conflicting reports. As NF-κB has a pivotal role in the generation and maintenance of malignancies, efforts were targeted towards understanding its activation mechanism using both ex vivo and in vivo studies. The results show that CS-induced NF-κB activation mechanism is different from that of other pro-inflammatory signals such as lipopolysaccharide (LPS). The NF-κB dimer that translocates to the nucleus upon stimulation with CS is predominantly composed of c-Rel/p50 and this translocation involves degradation of I-κBɛ and not I-κBα. This degradation of I-κBɛ depends on IKKβ activity, which preferentially targets I-κBɛ. Consistently, CS-activated form of IKKβ was found to be different from that involved in LPS activation as neither Ser177 nor Ser181 of IKKβ is crucial for CS-induced NF-κB activation. Thus, unlike other pro-inflammatory stimulations where p65 and I-κBα have a central role, the predominantly active signaling cascade in CS-induced NF-κB activation in the lung epithelial cells comprises of IKKβ–I-κBɛ–c-Rel/p50. Thus, this study uncovers a new axis of NF-κB activation wherein I-κBɛ and c-Rel have the central role.

Double Plant Homeodomain (PHD) Finger Proteins DPF3a and -3b Are Required as Transcriptional Co-activators in SWI/SNF Complex-dependent Activation of NF-κB RelA/p50 Heterodimer

We have previously shown that DPF2 (requiem/REQ) functions as a linker protein between the SWI/SNF complex and RelB/p52 NF-κB heterodimer and plays important roles in NF-κB transactivation via its noncanonical pathway. Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-κB reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-κB dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-κB heterodimer transactivation induced by TNF-α stimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-α. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-α treatment correlate well with the viral transcriptional activation levels. Additionally, re-ChIP experiments showed DPF3a/b and the SWI/SNF complex associate with RelA on the endogenous IL-6 promoter after TNF-α treatment. In conclusion, our present data indicate that by linking RelA/p50 to the SWI/SNF complex, DPF3a/b induces the transactivation of NF-κB target gene promoters in relatively inactive chromatin contexts.

NF‐κB and chromatin: ten years on the path from basic mechanisms to candidate drugs

Release of nuclear factor κΒ (NF-κB) dimers from the inhibitors of NF-κΒ (IκBs) and their subsequent nuclear translocation are only the initial events leading to the induction of NF-κB-regulated genes. Once in the nucleus, NF-κB dimers must gain access to their cognate sites in target genes. While some sites are found in a constitutively accessible state, many others are associated with nucleosomal histones in a manner that prevents NF-κB binding. Binding to such sites requires specific chromatin remodeling events driven by functionally cooperating transcription factors. Ten years of research on the complex interplay between chromatin and NF-κB led to some major successes, most notably the identification of the specific sequence features or trans-acting factors controlling the state of accessibility of κB sites, as well as the dissection of the mechanisms and players involved in the opening of occluded sites. Moreover, attempts at identifying mechanism-based compounds that inhibit the activation of selected subsets of NF-κB-dependent genes acting on chromatin-regulated transitions are starting to give initial promising results in preclinical tests.

Dimer‐specific regulatory mechanisms within the NF‐κB family of transcription factors

A fundamental feature of the transcriptional response to an nuclear factor-κB (NF-κB)-inducing stimulus is that the response is highly selective and limited to the activation of only a subset of potential NF-κB target genes. One major contributor to selectivity of the response is likely to be the capacity of different NF-κB dimers to regulate different sets of target genes. The NF-κB family of transcription factors consists of five proteins, RelA, c-Rel, RelB, p50, and p52, which assemble into several homodimeric and heterodimeric species. Studies of mutant mouse strains have revealed that each family member, and perhaps each dimer, carries out unique functions in regulating transcription in cells of the immune system and in many other cell types. Dimer-specific functions can be conferred by selective protein-protein interactions with other transcription factors, coregulatory proteins, and chromatin proteins. Unique DNA-binding specificities and affinities make additional contributions to selectivity of the response, with growing evidence that some NF-κB dimers can adopt different conformations and thereby function differently when bound to different DNA sequences. Despite significant advances, our knowledge remains limited and many years of additional work will be needed to fully understand how the dimer-specific functions of NF-κB contribute to transcriptional selectivity.

Upregulation of MHC class I expression following dengue virus infection: the mechanism at the promoter level

Evaluation of: Othman A, Abd Rahman N, Yusof R. Induction of MHC class I HLA-A2 promoter by dengue virus occurs at the NFκB binding domains of the class I regulatory complex. Virus Res. 163(1), 238–245 (2011).Unlike many other viruses that downregulate MHC class I expression on infected cell membranes, flaviviruses were reported to upregulate the MHC class I expression. Dengue virus was shown to induce HLA class I expression; however, the precise transcriptional mechanism that is used by the virus remains unclear. This article assessed the findings of a recently published report describing the mechanism used by dengue virus to induce HLA-A2 expression and characterizing the transcription factors that are involved. The study showed that p50/p65 and p65/65 NF-κB dimers bind to the class I regulatory complex within the HLA-A2 promoter. This finding and its significance for the design of possible antiviral therapeutic agents are discussed in this article.

Inhibitory kappa B Kinases as targets for pharmacological regulation.

The inhibitory kappa B kinases (IKKs) are well recognized as key regulators of the nuclear factor kappa B (NF-κB) cascade and as such represent a point of convergence for many extracellular agents that activate this pathway. The IKKs generally serve to transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes but also play a key role in the pathogenesis of a number of human diseases. Therefore, the catalytic IKKs represent attractive targets for intervention with small molecule kinase inhibitors. IKK isoforms are assembled as variable multi-subunit IKK complexes that regulate not only NF-κB dimers, but also protein substrates out-with this cascade. Consequently, close consideration of how these individual complexes transduce extracellular signals and more importantly what impact small molecule inhibitors of the IKKs have on functional outcomes are demanded. A number of adenosine triphosphate (ATP)-competitive IKKβ-selective inhibitors have been developed but have demonstrated a lack of activity against IKKα. A number of these chemicals have also exhibited detrimental outcomes such as cellular toxicity and immuno-suppression. The impact of small molecule inhibitors of IKK catalytic activity will therefore be reappraised, examining the advantages and potential disadvantages to this type of intervention strategy in the treatment of diseases such as arthritis, intestinal inflammation and cancer. Furthermore, we will outline some emerging strategies, particularly the disruption of protein-protein interactions within the IKK complex, as an alternative route towards the development of novel pharmacological agents. Whether these alternatives may negate the limitations of ATP-competitive molecules and potentially avoid the issues of toxicity will be discussed.

Bindarit

The activation of nuclear factor (NF)κB pathway and its transducing signaling cascade has been associated with the pathogenesis of many inflammatory diseases. The central role that IκBα and p65 phosphorylation play in regulating NFκB signalling in response to inflammatory stimuli made these proteins attractive targets for therapeutic strategies. Although several chemical classes of NFκB inhibitors have been identified, it is only for a few of those that a safety assessment based on a comprehensive understanding of their pharmacologic mechanism of action has been reported. Here, we describe the specific anti-inflammatory effect of bindarit, an indazolic derivative that has been proven to have anti-inflammatory activity in a variety of models of inflammatory diseases (including lupus nephritis, arthritis and pancreatitis). The therapeutic effects of bindarit have been associated with its ability to selectively interfere with monocyte recruitment and the "early inflammatory response," although its specific molecular mechanisms have remained ill-defined. For this purpose, we investigated the effect of bindarit on the LPS-induced production of inflammatory cytokines (MCP-1 and MCPs, IL-12β/p40, IL-6 and IL-8/KC) in both a mouse leukaemic monocyte-macrophage cell line and bone marrow derived macrophages (BMDM). Bindarit inhibits the LPS-induced MCP-1 and IL-12β/p40 expression without affecting other analyzed cytokines. The effect of bindarit is mediated by the downregulation of the classical NFκB pathway, involving a reduction of IκBα and p65 phosphorylation, a reduced activation of NFκB dimers and a subsequently reduced nuclear translocation and DNA binding. Bindarit showed a specific inhibitory effect on the p65 and p65/p50 induced MCP-1 promoter activation, with no effect on other tested activated promoters. We conclude that bindarit acts on a specific subpopulation of NFκB isoforms and selects its targets wihtin the whole NFκB inflammatory pathway. These findings pave the way for future applications of bindarit as modulator of the inflammatory response.

Murine Gammaherpesvirus 68 Evades Host Cytokine Production via Replication Transactivator-Induced RelA Degradation

Cytokines play crucial roles in curtailing the propagation and spread of pathogens within the host. As obligate pathogens, gammaherpesviruses have evolved a plethora of mechanisms to evade host immune responses. We have previously shown that murine gammaherpesvirus 68 (γHV68) induces the degradation of RelA, an essential subunit of the transcriptionally active NF-κB dimer, to evade cytokine production. Here, we report that the immediately early gene product of γHV68, replication transactivator (RTA), functions as a ubiquitin E3 ligase to promote RelA degradation and abrogate cytokine production. A targeted genomic screen identified that RTA, out of 24 candidates, induces RelA degradation and abolishes NF-κB activation. Biochemical analyses indicated that RTA interacts with RelA and promotes RelA ubiquitination, thereby facilitating RelA degradation. Mutations within a conserved cysteine/histidine-rich, putative E3 ligase domain impaired the ability of RTA to induce RelA ubiquitination and degradation. Moreover, infection by recombinant γHV68 carrying mutations that diminish the E3 ligase activity of RTA resulted in more robust NF-κB activation and cytokine induction than did infection by wild-type γHV68. These findings support the conclusion that γHV68 subverts early NF-κB activation and cytokine production through RTA-induced RelA degradation, uncovering a key function of RTA that antagonizes the intrinsic cytokine production during gammaherpesvirus infection.

Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding

The unique DNA-binding properties of distinct NF-κB dimers are known to influence the selective regulation of NF-κB target genes. To gain a stronger appreciation for these dimer-specific differences, we have combined protein-binding microarrays (PBM) and surface plasmon resonance (SPR) to evaluate DNA sites recognized by eight different NF-κB dimers. We observed three distinct binding-specificity classes and provide insight into mechanisms by which dimers might regulate distinct sets of genes. We identified many new non-traditional κB site sequences and highlight an under-appreciated plasticity of NF-κB dimers in recognizing κB sites with a single consensus half-site. This study provides a database that will be of broad utility in efforts to identify NF-κB target sites and uncover gene regulatory circuitry.

Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Primary Myelofibrosis (PMF) Bone Marrow Cells

Abstract 1742Primary myelofibrosis (PMF) is a stem cell–derived hematologic malignancy, characterized by an expansion of one or more myeloid lineage resulting in bone marrow (BM) hypercellularity, magakaryocyte proliferation with atypia, granulocytic proliferation, and reticulin and/or collagen fibrosis. An acquired activating mutation in Janus kinase 2 at codon V617F (JAK2V617F) is detected in BM cells of the majority of patients with PMF. Constitutively activated JAK2 induces phosphorylation and activation of STAT3. Phosphorylated STAT3 forms heterodimers, translocates to the nucleus, binds to DNA, activates STAT3-target genes, and induces production of cytokines that interact with the BM microenvironment. Hematopoietic stroma derived soluble factors provide PMF cells with survival advantage (Manshouri et al. Cancer Res 71: 3831, 2011) and, as reported previously, most of these factors activate NF-κB in a variety of cell types. NF-κB plays an important role in the survival and proliferation of normal and neoplastic cells. In several hematologic malignancies, the NF-κB p65/p50 dimers were found to be activated to variable degrees. The activation of NF-κB is mediated by either the canonical pathway or the alternative pathway. The canonical pathway is typically activated by extracellular signals that activate the β subunit of the IκB kinase (IKK) complex (IKKβ) that induces the phosphorylation and degradation of the NF-κB inhibitor IκBα. Following IκBα degradation, NF-κB heterodimers translocate to the nucleus and bind to DNA. We have recently found that in chronic lymphocytic leukemia (CLL) constitutively activated STAT3 induces the production of unphsophorylated (U) STAT3. U-STAT3 binds to the NF-κB dimers p65/p50 in competition with IκB and the U-STAT3/NF-κB complex shuttles to the nucleus where NF-κB binds to DNA and activates NF-κB-regulated genes (Liu et al. Mol Cancer Res 9: 507, 2011). Because in PMF constitutively activated JAK2 induces phosphorylation of STAT3 and this activated form of STAT3 induces the production of U-STAT3, we wondered whether, like in CLL, U-STAT3 activates NF-κB in PMF. To determine whether NF-κB is constitutively activated in PMF we obtained BM low density cells from untreated patients with PMF. First we studied low-density BM cells of 11 patients with PMF using the electrophoretic mobility shift assay (EMSA). Cells of all samples bound to a p65/NF-κB DNA-labeled probe and the addition of an unlabelled (cold) p65/NF-κB probe attenuated or completely eliminated the binding. Typically, NF-κB-DNA binding appears and disappears due to repeated degradation and re-synthesis of IκB and the consequent activation and inactivation of NF-κB, respectively. Because we found that NF-κB is constitutively activated in all PMF BM samples we hypothesized that, like in CLL cells, activation of NF-κB in PMF cells is induced by an IκB-unrelated mechanism as reported by Yang J et al. (Cancer Res 65:939, 2005). By using immunoprecipitation of two different PMF BM samples we determined that STAT3 binds to the RelA/p65 NF-κB protein, and by using EMSA we found that anti-STAT3, similar to anti- NF-κB p65 antibodies, attenuated the binding of PMF BM cell extract to the NF-κB DNA probe. Taken together, our data suggest that U-STAT3 binds the NF-κB dimers p65/p50 and constitutively activates NF-κB in PMF. Disclosures:No relevant conflicts of interest to declare.

Pharmacogenetic Approaches Identify ANTXR1 As a Potential Mediator of Response to Therapeutic NF-κB Inhibition in Diffuse Large B Cell Lymphoma

Abstract 234 Background:NF-κB is a family of transcription factors known to play an essential role in the development & survival of lymphocytes. In recent years, it has been clear that aberrant NF-κB activation is a hallmark of various lymphoid malignancies and appears to be associated with chemotherapy resistance and adverse prognosis. The canonical NF-κB pathway is frequently engaged in lymphoid malignancies wherein activated IKK phosphorylates IκB proteins inducing IκB polyubiquination and subsequent proteasomal proteolytic degradation; this allows for release and nuclear translocation of NF-κB dimers to activate target gene transcription.Gene Expression Profiling (GEP) has identified two distinct sub groups of Diffuse Large B cell Lymphoma (DLBCL). While the Activated B cell (ABC) type shows constitutive activation of NF-κB, the role for NF-κB activation in Germinal Center B Cell (GCB) DLBCL is currently unclear. Since NF-κB inhibition has been identified as a therapeutic possibility in DLBCLs, it is important to define the role of this pathway and its modulators. In this study, we sought to investigate key regulators of the NF-κB pathway that might mediate a therapeutic response to IKKβ inhibition of NF-κB in DLBCL including the GCB subtype. Through GEP and Exome Sequencing, we demonstrate that ANTXR1 is a key mediator of response to IKKβ inhibition in DLBCL. Methods/Results:We obtained a novel selective inhibitor of IKKβ, TLX-2001 that has been found to be safe in animal models. IC50 were obtained on 61 cell lines representing various lymphomas including DLBCL (N=25) using cell viability MTT assays. The drug showed efficacy in both ABC and GCB DLBCL cell lines at physiologically achievable concentrations. These results were unsurprising in ABC DLBCLs which are known to depend on NF-κB activation, but the lethality of this selective drug in GCB DLBCLs was unexpected. To better understand the role of individual genes in the response in GCB DLBCLs, gene expression profiling was performed on 61 cell lines using Human Gene 1.0 ST Array. We found that ANTXR1 expression significantly correlated with NF-κB resistance (p = 0.035).Additionally, we sequenced the exomes of DLBCL tumors (N=95) and matched normal tissue (N=34). 95 cases of DLBCLs consisted of 73 cases of primary human DLBCLs and 22 DLBCL cell lines. Whole exome sequencing was performed using the Agilent solution-based system of exon capture to sequence all protein coding exons in the CCDS database. We identified 465 recurrently somatically mutated genes in these DLBCL cases, and found that mutation status of ANTXR1 was associated with high sensitivity to IKKβ inhibition (p =0.015) of NF-κB. Cell lines with non-synonymous mutations in ANTXR1 had over 3-fold lower IC50 (mean= 2.39 μM) compared to cell lines with no mutation in ANTXR1 (mean IC50 = 8.72μM). Discussion/Conclusion:ANTXR1 is the docking receptor for bacillus anthracis toxin, and anti-tumor responses have been observed in mice injected with recombinant engineered anthrax toxin. It is also known as TEM8 and maps to chromosome 2p13.1. Increased levels of TEM8 (tumor endothelial marker 8) have been noted in various malignancies including melanoma. Our data suggest that pharmacogenetic approaches that combine gene expression profiling and whole exome sequencing are useful tools for identifying novel genes that modulate therapeutic responses in lymphoma. Disclosures:No relevant conflicts of interest to declare.

A Constitutivly Active NFkB Non Canonical Pathway Is Maintained by RelB Repression of TRAF2 to Prevent Autophagy-Mediated Cell Death in Hodgkin Lymphomas

Abstract 428NFkB is a pleitropic transcription factor (TF) known to play major roles in diverse cell processes such as proliferation, apoptosis and inflammation. The REL or NFkB family is comprised of hetero or homo dimeric combinations of five members: RELA (p65), RELB, c-REL, NFKB1 (p50) and NFkB2 (p52).Despite considerable evidence supporting the role of the REL members in the immune system and lymphomagenesis, it has never been accurately established whether specific NFkB dimers control a particular set of target genes that account for the biological functions known to be mediated by these TFs. Furthermore, it is not clear whether NFkB controls different targets in distinct subsets of germinal center (GC) B cell derived lymphomas.We performed loss of function experiments delivering specific shRNAs to knock down each REL member in lymphoma cell lines modeling sequential stages of the GC B cell maturation: Mantle Cell Lymphoma (MCL), Diffuse Large B Cell Lymphoma [DLBCL including ABC-Like (Activated B Cell), GCB-Like (Germinal Center B Cell) and PMBL (Primary Mediastinal B cell Lymphoma)], Burkitt Lymphoma (BL) and Hodgkin Lymphoma (HL). All the NHL subsets showed a dependency on RelA and c-Rel to survive but were largely unaffected by RelB depletion. Only HL cells were dramatically sensitive to RelB knock-down.ChIP-seq analysis uncovered an extensive NFkB network in BJAB (GCB), HBL-1 (ABC), U-H01 (HL) and human primary centroblasts. This data shaped two network architectures: a RelB hierarchical network in HL and a c-rel hierarchy in NHL. Out of 4,581 genes that had relB binding (+/− 2kB TSS) in HL cells, more than 70% were only relB targets in HL as compared to NHL cells.Microarray analysis of these cell lines following RelA, RelB or c-Rel knockdown was merged with our ChIP-seq data. The overlap of RelB peaks and RelB shRNA downregulated targets showed that RelB directly controlled cell cycle regulators and apoptosis inhibitors including CCND3, CDK6, BCL2 and BCLxL. Interestingly, RelB maintained HL cell viability through BCL2 induction since expression of a BCL2 cDNA rescued HL cell lines from the shRNA RelB lethality.Strikingly, the RelB induced BCL2 and BCLxL protein down regulation in HL cells does not kill these cells by predicted apoptotic mechanisms. Morphological and molecular evidence indicated that RelB inhibition lead to cell death by autophagy. Electron microscopy revealed intact nucleus with lack of condensed chromatin and citosolic vacuoles. After RelB inhibition, the well known autophagy marker L3C, is completely degraded meanwhile ATG7 protein expression, a key player in the autophagosomes development, is enhanced. RelB suppressed BCL2/BCL-xL dependent autophagic cell death by maintaining high levels of these two pro-survival molecules interacting with the autophagy executing protein Beclin-1. The interaction is disrupted by the RelB shRNA.We found that RelB is engaged in a positive regulatory loop that keeps the non canonical arm of the NFkB pathway on.Activation of the alternative pathway centers on the modulation of NIK (NFkB inducing kinase). In physiological conditions, NIK undergoes constitutive ubiquitin-dependent degradation through the TRAF-2/TRAF-3/c-IAP-1/c-IAP-2 complex, which keeps its abundance below the threshold required for its function. We found that HL cell lines lack TRAF-2 or TRAF-3 protein expression which leads to stabilization and accumulation of NIK. RelB knock down is followed by up-regulation and/or re-expression of TRAF members and depletion of NIK protein. Remarkably, lymph nodes of HL patient biopsies showed NIK protein citosolic expression by IHC in the Reed-Sternberg Cells, indicating stabilization and accumulation of NIK that accounts for constitutive NFkB-2/p100b processing to release active p52-RelB dimers.In summary, we found that HL is the only GC B cell derived malignancy that relies on RelB for viability, and thus differentiates Hodgkin Lymphomas from non-Hodgkin lymphomas. These data argue that each REL member has specific and unique functions and targets, as we predicted from the lack of similarities in their transactivation domains. Of clinical relevance, HL patient samples reproduced the alternative NFkB pathway molecular alterations that we initially observed in HL cell lines.Furthermore, the unique Hodgkin Lymphoma RelB dependency makes the non canonical NF-kB signaling pathway an attractive therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Murine Gamma Herpesvirus 68 Hijacks MAVS and IKKβ to Abrogate NFκB Activation and Antiviral Cytokine Production

Upon viral infection, mitochondrial antiviral signaling (MAVS) protein serves as a key adaptor to promote cytokine production. We report here that murine gamma herpesvirus 68 (γHV68), a model virus for oncogenic human gamma herpesviruses, subverts cytokine production via the MAVS adaptor. During early infection, γHV68 hijacks MAVS and IKKβ to induce the site-specific phosphorylation of RelA, a crucial subunit of the transcriptionally active NFκB dimer, which primes RelA for the proteasome-mediated degradation. As such, γHV68 efficiently abrogated NFκB activation and cytokine gene expression. Conversely, uncoupling RelA degradation from γHV68 infection promoted NFκB activation and elevated cytokine production. Loss of MAVS increased cytokine production and immune cell infiltration in the lungs of γHV68-infected mice. Moreover, exogenous expression of the phosphorylation- and degradation-resistant RelA variant restored γHV68-induced cytokine production. Our findings uncover an intricate strategy whereby signaling via the upstream MAVS adaptor is intercepted by a pathogen to nullify the immediate downstream effector, RelA, of the innate immune pathway.

Non‐canonical NF‐κB signaling activation and regulation: principles and perspectives

Nuclear-factor κB (NF-κB) transcription factors are activated by a wide variety of stimuli in diverse cell types and control key aspects of immune function and development. Receptor-mediated activation of NF-κB appears to occur through two distinct signaling pathways termed as the canonical and non-canonical NF-κB pathways. Although much work has demonstrated the physiological importance of non-canonical NF-κB signaling to immunity and its involvement in diverse pathologies, such as cancers and autoimmune disease, the architecture and regulation of the pathway is only beginning to be understood. The non-canonical pathway appears to be activated by a select set of receptors within the tumor necrosis factor superfamily, and we discuss the molecular mechanisms that connect ligation of these receptors to pathway activation. It has become increasingly clear that the key regulatory step of the pathway involves modulation of the post-translational degradation of NF-κB-inducing kinase (NIK), the central activating kinase of non-canonical NF-κB signaling. How NIK post-translational stability is controlled before and after receptor ligation is an important aspect of understanding non-canonical NF-κB signaling. Furthermore, how release of NF-κB dimers downstream of the pathway's activation is actually connected to its identified physiological and pathological roles is a key remaining question in the field.