key: cord-0855402-55lftxc7
authors: Verhelst, Kelly; Verstrepen, Lynn; Carpentier, Isabelle; Beyaert, Rudi
title: IκB kinase ɛ (IKKɛ): A therapeutic target in inflammation and cancer
date: 2013-04-01
journal: Biochem Pharmacol
DOI: 10.1016/j.bcp.2013.01.007
sha: d62fe4f429fc162677afe80b8e3ae386c16246de
doc_id: 855402
cord_uid: 55lftxc7

The innate immune system forms our first line of defense against invading pathogens and relies for a major part on the activation of two transcription factors, NF-κB and IRF3. Signaling pathways that activate these transcription factors are intertwined at the level of the canonical IκB kinases (IKKα, IKKβ) and non-canonical IKK-related kinases (IKKɛ, TBK1). Recently, significant progress has been made in understanding the function and mechanism of action of IKKɛ in immune signaling. In addition, IKKɛ impacts on cell proliferation and transformation, and is thereby also classified as an oncogene. Studies with IKKɛ knockout mice have illustrated a key role for IKKɛ in inflammatory and metabolic diseases. In this review we will highlight the mechanisms by which IKKɛ impacts on signaling pathways involved in disease development and discuss its potential as a novel therapeutic target.

Our first line of defense against viral and bacterial attacks relies on the innate immune system, which detects non-self products by means of specific surface, endosomal or cytosolic receptors. For example, bacterial lipopolysaccharide (LPS) is sensed by Toll-like receptor (TLR)-4 on the cell surface, whereas viral RNA is sensed by endosomal TLR3 or cytosolic retinoid acid-inducible gene (RIG-I) [1] . A major output from these receptors is the activation of transcription factors belonging to the nuclear factor-kappa B (NF-kB) and interferon (IFN) regulatory factor (IRF) family, which control the expression of multiple immune regulatory genes. In this review we will focus on p50/p65 NF-kB and IRF3, whose activation involves specific members of the inhibitor of kB (IkB) kinase (IKK) family. The canonical IKKs, IKKa and IKKb, form a complex with the adaptor protein NEMO (also known as IKKg), which has a regulatory role. This IKK complex is required for proper NF-kB signaling in response to multiple proinflammatory stimuli such as TNF and TLR ligands. IKKa and IKKb are Ser/Thr kinases that phosphorylate the NF-kB inhibitor protein IkBa, resulting in its Lys48-linked polyubiquitination and subsequent proteasomal degradation. This allows NF-kB to translocate to the nucleus and bind to specific DNA elements [2] . IKKa and IKKb contain an N-terminal catalytic kinase domain (KD), a more central leucine zipper (LZ) and helix loop helix (HLH) domain, and a C-terminal NEMO-binding domain (NBD) (Fig. 1) . The major impact of the IKKs on NF-kB signaling inspired many researchers to search intensively for IKK-related kinases. Based on sequence similarities with IKKa and IKKb, two IKK-related kinases, TANK binding kinase 1 (TBK1) and IKKe (also known as IKK-inducible or IKK-i), were discovered. IKKe and TBK1 are known as the non-canonical IKKs and expand the IKK family to four members [3, 4] . The kinase domain of IKKe shares 33% and 31% amino acid identity with the corresponding domains in IKKa and IKKb, respectively, and 67% with TBK1 [5] . Furthermore, TBK1 and IKKe have a similar domain composition as the canonical IKKs. IKKb and the non-canonical IKKs also share an ubiquitin-like domain (ULD), which is required for optimal kinase activity [6] . Importantly, IKKe and TBK1 lack a NBD and do not interact with NEMO, but each forms similar complexes with other specific scaffolding proteins (see Section 4) .

Although IKKe and TBK1 were originally classified as IKKs based on their ability to phosphorylate IkBa upon overexpression, studies with IKKe and TBK1 deficient cells revealed that these kinases are dispensable for IkBa phosphorylation [7, 8] . Instead, both kinases were shown to contribute to LPS-and virus-induced phosphorylation of IRF3 and IRF7, allowing their homodimerization, nuclear import, and activation of type I IFN genes (IFN-a and IFN-b) [9] . IKKe and TBK1 are similar in their ability to activate IRF3 and IRF7 and their ability to phosphorylate the IkBa inhibitor of NF-kB, but they present some differences that may be of importance. For instance, deletion of the TBK1 gene leads to embryonic lethality at day 15 due to TNF-induced apoptosis in the A B S T R A C T The innate immune system forms our first line of defense against invading pathogens and relies for a major part on the activation of two transcription factors, NF-kB and IRF3. Signaling pathways that activate these transcription factors are intertwined at the level of the canonical IkB kinases (IKKa, IKKb) and non-canonical IKK-related kinases (IKKe, TBK1). Recently, significant progress has been made in understanding the function and mechanism of action of IKKe in immune signaling. In addition, IKKe impacts on cell proliferation and transformation, and is thereby also classified as an oncogene. Studies with IKKe knockout mice have illustrated a key role for IKKe in inflammatory and metabolic diseases. In this review we will highlight the mechanisms by which IKKe impacts on signaling pathways involved in disease development and discuss its potential as a novel therapeutic target.

ß 2013 Elsevier Inc. All rights reserved. liver [7] , whereas IKKe deficient mice are viable [9] . The reason for these different phenotypes is still unclear but may in part reflect the differential expression and use of TBK1 and IKKe in various cell types (see also section 2). Moreover, TBK1 and IKKe may have nonredundant functions in other signaling pathways than those controlling IRF and canonical NF-kB activity. For example, recently published work demonstrated a specific role for TBK1 as a negative regulator of non-canonical NF-kB signaling in B cells stimulated with BAFF or anti-CD40, which was associated with the inducible TBK1 mediated phosphorylation of the kinase NIK, leading to its degradation [10] . Although B cells express both TBK1 and IKKe, NIK phosphorylation was highly specific for TBK1. Since TBK1 and IKKe share the same substrate consensus phosphorylation motif [11] , substrate specificity is likely driven by recruitment of TBK1 or IKKe to discrete signaling complexes, possibly involving specific scaffolding proteins (see Section 4) . Given the ever-growing number of TBK1 and IKKe interaction partners and substrates [12] , localized kinase activation and substrate modification could be an effective strategy to confer TBK1 or IKKe specific functions.

While IKKa, IKKb and TBK1 are constitutively expressed in most cell types, basal IKKe expression is only observed in specific tissues (pancreas, thymus and spleen) and cell types (T-cells and peripheral blood leukocytes). However, in other cell types (e.g. fibroblasts) IKKe is rapidly upregulated by cytokines (e.g. TNF, IL-1, IL-6, IFN-g), microbial products (e.g. LPS, viral RNA), and phorbol esters (PMA), and has therefore also been called inducible IKK or IKK-i [12, 13] . The promoter of the IKKe gene contains two putative NF-kB binding sites, of which only one was shown to be functionally active [14] , as well as seven STAT3-binding sites, of which two are active [15] . Whether the increase in IKKe expression induces kinase activation is currently still unclear. Recently, two IKKe splice variants, missing 25 amino acids (IKKe-sv1) or 13 amino acids (IKKe-sv2) at their C-terminal end, were detected in human peripheral blood mononuclear cells [16] . While these splice variants are ubiquitously expressed at the mRNA level, both protein isoforms are selectively upregulated by TNF stimulation or virus infection. IKKe is frequently overexpressed in a number of human cancers, in particular breast, ovarian and pancreatic cancer and has been implicated in tumorigenesis (see Section 6) . Although IKKe and TBK1 show many overlapping activities that are relevant to innate immunity and inflammation, the more restricted expression of IKKe is of particular interest. In this review we will therefore focus on IKKe and mainly refer to TBK1 for reasons of comparison.

The identification of TBK1 and IKKe as IkBa kinases has always been very controversial and the source of many debates. Evidence is based primarily on the observation that overexpression of IKKe or TBK1 in cultured cells leads to phosphorylation of IkBa, be it only at one phosphoacceptor site (Ser36 or Ser32, respectively), driving increased IkBa turnover [13] . However, both IKKe and TBK1 deficient MEF cells display normal IkBa degradation in response to TNF, IL-1 or LPS, contradicting the overexpression data [7, 8] . Nevertheless, TNF-, IL-1-or LPS-induced NF-kB dependent gene expression is abrogated in the absence of IKKe [7, 8] . Therefore it is believed that IKKe influences NF-kB signaling and concomitant gene expression downstream of IkBa [12] (Fig. 2) . In this context, several studies support a role for IKKe-mediated Ser468 and Ser536 phosphorylation of the p65 NF-kB subunit in the expression of a specific subset of NF-kB target genes in response to pro-inflammatory signals and viral infection [12, [17] [18] [19] . It has also been shown that activated p65 (phosphorylated on Ser468 or Ser536) serves as a docking site to bring IKKe-enzymatic activity to kB-containing inflammatory gene promoters in the nucleus, enabling IKKe to phosphorylate adjacent c-Jun and initiate nuclear receptor corepressor clearance [20] . These results suggest that p65-recruitment of IKKe to promoters that exhibit AP1 and kB sites in close proximity (e.g. inos, cxcl2, cxcl9, cxcl10, ccl4, tnfaip3) may regulate their activation by initiating corepressor turnover. Besides p65, IKKe/TBK1 also targets c-Rel NF-kB, leading to its nuclear accumulation and activation of NF-kB dependent gene expression [21] .

Next to their role in NF-kB signaling, TBK1 and IKKe are also implicated in IRF3 and IRF7 signaling in response to viral infection that is sensed by a diversity of receptors, such as TLR3 and RIG-I, leading to the production of type I IFNs [12] (Fig. 2 ). While IRF3 is constitutively expressed in the cytoplasm of many cells, IRF7 needs to be transcriptionally upregulated. Both IKKe and TBK1 directly phosphorylate IRF3 and IRF7 at their C-termini, resulting in their homoand heterodimerization and translocation to the nucleus. Mass spectrometry pinpointed Ser386, Ser396 and Ser402 in IRF3 as redundant phosphorylation sites for IKKe upon viral infection of innate immune cells [22] . IKKe deficient MEF cells showed no change in IRF3 activation [9] , whereas TBK1 deficient MEF cells showed reduced IRF3 activation upon TLR3 and TLR4 triggering [9, 23, 24] . However, IRF3 activation was completely abolished in TBK1 and IKKe double deficient MEFs [9] . IKKe overexpression, but not its kinase dead mutant, could restore IRF3 activation in TBK1 deficient cells, suggesting redundancy between both kinases [24] . Although IKKe and TBK1 seem to exert overlapping functions in IRF3 activation, mechanistic differences in the activation of TBK1 and IKKe following virus infection have been observed (e.g. mitochondrial localization of IKKe versus cytoplasmic localization of TBK1) [25] . Moreover, partially different substrates have been reported for both kinases. It is interesting to note that the TLR adaptor protein MyD88 was recently shown to abrogate TLR3-induced IFN production by preventing IKKe but not TBK1mediated IRF3 phosphorylation [25] , indicating that both kinases can also be differentially regulated.

Although IKKe/TBK1 can be activated by several inflammatory stimuli, their activation by TLR3, TLR4 and RIG-I receptors has been best documented [26, 27] . Receptor stimulation induces the recruitment of specific adaptor proteins to the receptor (TRIF for TLR3 and TLR4, MAVS for RIG-I). Subsequently, these adaptors interact with the E3 ubiquitin ligase TRAF3 to activate IRF3, or with the E3 ubiquitin ligase TRAF6 and RIP1 kinase to activate NF-kB. Autoubiquitination of TRAF3 is needed for the recruitment and activation of IKKe and TBK1 [28] . At later time points, the presence of IKKe in the MAVS complex eventually leads to the release of TRAF3, with the shutdown of IFN signaling as a consequence [29] .

At the protein level, IKKe (and TBK1) activity is regulated by phosphorylation at a single site (Ser172) in its activation loop [30] , in contrast to IKKa and IKKb, which require phosphorylation at two sites (IKKa: Ser176 and Ser180; IKKb: Ser177 and Ser181) for their function. Although overexpression studies indicated a role for autophosphorylation of IKKe/TBK1 at Ser172 [13, 31] , activation of the endogenous kinases does not only involve autophosphorylation. Indeed, cells treated with the TBK1 and IKKe inhibitor BX795 still showed phosphorylation of TBK1 and IKKe at Ser172 in response to poly(I:C), LPS, TNF and IL-1 [32] , suggesting the involvement of other kinases. Clark and co-workers could show that Ser172 phosphorylation of IKKe/TBK1 is impaired in IKKa deficient cells treated with an IKKe inhibitor [31] , indicating IKKa as a potential IKKe kinase. In line with these observations, TNFinduced activation of IKKe/TBK1 seems to be mediated solely by the canonical IKKs [31] . Interestingly, IKKe/TBK1 activation by the canonical IKK pathway only results in NF-kB activation and not IRF3 activation.

Similar to the canonical kinases IKKa and IKKb, which require the formation of a complex with the adaptor protein NEMO, also TBK1 and IKKe activation involves specific adaptor proteins. So far, three scaffold proteins have been shown to bind IKKe/TBK1 and to promote IKKe/TBK1-mediated phosphorylation of IRF3 and IRF7: NAK associated protein 1 (NAP1), TNF receptor-associated factor (TRAF) family member-associated NF-kB activator (TANK) and similar to NAP and TBK1 adaptor (SINTBAD) [30, [33] [34] [35] . Therefore, it is an attractive idea that different scaffold proteins might assemble distinct TBK1 and IKKe complexes under distinct conditions, providing signaling specificity. Whether or not these scaffold proteins assemble hetero-or homodimers of IKKe and TBK1 is currently still unknown.

In contrast to the positive regulation of NF-kB dependent gene expression by IKKe/TBK1, the latter have also been described to negatively regulate NF-kB activation in response to TNF or IL-1 stimulation. More specifically, IKKe/TBK1 was shown to directly phosphorylate the canonical IKKs outside their activation loop, restricting their activity [31] (Fig. 2) . Interestingly, this negative regulation of the canonical IKKs by IKKe/TBK1 was impaired in TANK-deficient macrophages [36] . Using these cells it was also shown that TANK is required for IKKe/TBK1 and NF-kB activation in response to LPS via both the MyD88-and TRIF-dependent pathways, and mediates the interaction of the non-canonical IKKs with the canonical IKKs. The latter most likely involves the binding of TANK to the canonical IKK adaptor protein NEMO [37] . Together, these findings demonstrate a key role for the IKKe/TBK1 adaptor protein TANK in enabling the canonical and non-canonical IKKs to regulate each other.

In analogy to NEMO, one can expect that the mechanisms driving IKKe/TBK1 activation most likely require different posttranslational modifications of their scaffold proteins. Indeed, IKKe/ TBK1 dependent phosphorylation and Lys63-linked polyubiquitination of TANK have already been described in response to LPS stimulation [33] , but their significance is still unclear.

Multiple negative regulatory mechanisms are in place to avoid excessive IKKe/TBK1 activity and IFN production. For example, the deubiquitinase DUBA reverses TRAF3 ubiquitination, disconnecting TRAF3 from its substrates TBK1 and IKKe [38] . Also the deubiquitinase CYLD negatively regulates RIG-I induced interferon production by deubiquitinating RIG-I and TBK1/IKKe [39, 40] . Conversely, IKKe directly phosphorylates CYLD at Ser418, hereby decreasing its deubiquitinating potential but increasing the IKKeinduced cell transformation [41] (Fig. 3 ) (see Section 6) . Others have shown that the ubiquitin-editing enzyme A20, in conjunction with Tax1 binding protein 1 (TAX1BP1), antagonizes Lys63polyubiquitination of TBK1 and IKKe. Surprisingly, A20-mediated inhibition of TBK1/IKKe Lys63-polyubiquitination was independent of its DUB function [42] . Recently, the E3 ubiquitin ligases TRIP and DTX4 have been shown to negatively regulate IRF3 activation by inducing TBK1 Lys48-polyubiquitination and proteosomal degradation [43, 44] . DTX4 showed weak or no activity against IKKe, while the activity of TRIP against IKKe was not studied. Interestingly, also many viruses themselves interfere with the IRF pathway and antiviral IFN production at the level of IKKe and/or TBK1. For example, specific viral components disturb the interaction of IKKe/TBK1 with other signaling proteins (e.g. Ebola virus VP35 protein [45] , M protein of Corona virus [46] , Hepatitis B or C virus [47, 48] ). Alternatively, viral proteins may act as alternative substrates for IKKe and TBK1, thereby targeting these kinases for degradation (e.g. Borna disease virus P protein, paramyxovirus V proteins [49, 50] ). These virus adaptations further emphasize the importance of the non-canonical kinases in antiviral signaling.

IKKe activity has been linked to the pathology of inflammatory diseases such as rheumatoid arthritis (RA) [51] [52] [53] . For instance, IKKe is constitutively expressed and phosphorylated in synovial intimal lining of RA patients, resulting in uncontrolled IRF3-driven production of proinflammatory mediators such as IFN-b, matrix metalloproteinases and chemokines [52] . Further supporting a major contribution of IKKe to the pathogenesis of RA is the finding that mice deficient in IKKe show less synovial inflammation in a passive K/BxN arthritis model due to lower expression of inflammatory mediators [51] . Furthermore, IKKe single nucleotide polymorphisms have been associated with the early stages of RA [54] , and genome wide associated studies revealed IKKe as a susceptibility locus for systemic lupus erythematosus (SLE), in which type I IFNs play a crucial role [55] . Computational analysis of predicted protein-protein interactions also pinpointed IKKe as a potential therapeutic target in psoriasis [56] .

IKKe has also been implicated in pulmonary inflammation. In this context, IKKe deficient mice showed fewer infiltrating neutrophils and diminished expression of proinflammatory cytokines and chemokines after intranasal administration of IL-17. Surprisingly, in this model IKKe deficiency did not affect NF-kB activation, but did reduce MAP kinase signaling [57] . More specifically, IKKe was shown to be responsible for phosphorylation of the IL-17 receptor adaptor Act-1, thereby activating the TRAF2/ TRAF5 pathway leading to increased MAP kinase activation and leaving the TRAF6/NF-kB axis emanating from Act-1 undisturbed. Increased MAP kinase activation led to increased chemokine mRNA stability, contributing to increased chemokine production and neutrophil infiltration into the lungs. A role for IKKe in the regulation of IL-17 responses is also illustrated by the recent finding that IKKe can promote the AKT-mTOR signaling pathway, which mediates IL-1 induced Th17 maintenance, by phosphorylating and inactivating GSK3a [58] . Taken together, many studies indicate an important role of IKKe in the pathophysiology of inflammatory diseases, mainly by regulating NF-kB, IFN and IL-17 responses.

IKKe might also be a potential target to treat inflammatory pain. In inflammatory pain models (hind paw inflammation evoked by injection of zymosan or formalin), IKKe-deficient mice exhibited a significantly reduced nociceptive behavior in comparison with wild type mice, indicating that IKKe contributes to the development of inflammatory hyperalgesia [59] . At the same time, the provoked NF-kB activation in nociceptive neurons (neurons involved in perception of pain) was reduced, as reflected by lower inflammatory gene expression. Of interest, this process is independent of type I IFN responses, suggesting that IKKe is promoting inflammatory hyperalgesia merely by activating the NF-kB pathway.

Finally, IKKe has been reported as an important link between inflammation and obesity. Obese mice have major risks to develop inflammatory or metabolic diseases such as type 2 diabetes [60] . In fact, obesity mimics a permanent low-grade inflammatory condition due to dietary fatty acid recognition by TLR4 [61] or hypoxia [62] . Elevated NF-kB activity in obese mice drives increased IKKe expression in the liver, adipocytes and adipose tissue macrophages [63, 64] . Furthermore, mice deficient in IKKe were found to be protected from high fat diet-induced obesity and showed less chronic liver inflammation, hepatic steatosis and insulin resistance. These events are regulated by changes in the expression of regulatory proteins and enzymes that are involved in glucose and lipid metabolism, and by a decrease in the production of proinflammatory cytokines and proteins involved in insulin resistance. Transfection of cultured adipocytes and hepatoma cells with IKKe induced similar changes, suggesting a direct role for IKKe in the regulation of hepatic inflammation. However, contradictory results were reported by another group, showing no difference between IKKe knockout and wild type mice in a model of high fat dietinduced obesity and insulin resistance [65] . Therefore, more work is needed to define the precise role of IKKe in the development of metabolic diseases.

IKKe has been associated with the initiation and progression of multiple cancers and might function as an oncogene for malignant transformation. For instance, several breast cancer cell lines and $30% of primary human breast tumors express high levels of IKKe [66] . Increased IKKe expression is due to an up till now unknown mutation regulating IKKe transcript levels or to an amplification of the 1q32 region comprising the IKBKE locus [67, 68] . Ectopic expression of IKKe in immortalized mammary epithelial cells at levels found in human cancer cells renders them tumorigenic, confirming that the allele amplified in breast cancer specimens is transforming [67] . High IKKe expression is often associated with the accumulation of c-Rel and p65 NF-kB subunits in the nucleus (e.g. in primary breast tumors), and IKKe silencing in several breast cancer cell lines was shown to reduce NF-kB activation and cell proliferation [69] . Similarly, introducing a kinase inactive IKKe mutant (IKKe K38A) in breast cancer cells, which exerts a dominant negative effect on endogenous IKKe, reduced NF-kB dependent gene expression (e.g. cyclin D1 and RelB) [70] , demonstrating the necessity of IKKe catalytic activity. Recently, this was further confirmed by demonstrating the requirement of IKKe-mediated phosphorylation at Ser418 of the tumor suppressor CYLD, which prevents its deubiquitinating activity on NF-kB signaling proteins such as TRAF2 and NEMO [41, 71] (Fig. 3) . Moreover, IKKe also activates the E3 ubiquitin ligase TRAF2 by direct phosphorylation at Ser11, resulting in increased TRAF2 ubiquitination [71] . Together these events lead to enhanced NF-kB activation, which promotes survival, transformation and proliferation of mammary epithelial cells (Fig. 3) . Furthermore, IKKe (as well as TBK1) may contribute to enhanced NF-kB activity and tumorigenesis by directly phosphorylating NF-kB p65 (as described above) or by phosphorylating Akt, which then phosphorylates and activates p65 [72, 73] . In addition, elevated IKKe levels are also associated with STAT1 activation in different primary tumors and cell lines derived from a diversity of cancers, like lung and breast carcinoma [15, 74, 75] , which may also contribute to the oncogenic activities of IKKe. As IRF3 suppression did not affect IKKe-induced cell transformation, the oncogenic potential of IKKe seems to be independent of its IRF3 signaling function [67] .

More recently, human ovarian cancer cell lines and primary tumors were also shown to have elevated levels and activity of IKKe, which was associated with a lower overall survival rate [76, 77] . Furthermore, alterations of IKKe were associated with late-stage and high-grade tumors, suggesting a role of IKKe in ovarian tumor progression rather than in tumor initiation. Finally, IKKe was also described as an oncogene in prostate and oesophageal squamous cell carcinoma with increased levels of different NF-kB family members [78] [79] [80] [81] , and in clear cell renal cell carcinoma [82] . Overexpression of IKKe in tumor cells induces cell survival, cell transformation and proliferation by different mechanisms involving IKKe mediated phosphorylation of specific substrates. IKKe can either directly or indirectly (via Akt phosphorylation and activation) phosphorylate NF-kB (p65), leading to increased NF-kB dependent gene expression. IKKe also phosphorylates and inactivates the tumor suppressor CYLD, preventing CYLD from deubiquitinating specific substrates in the NF-kB signaling pathway. In addition, phosphorylation of TRAF2 activates its E3 ubiquitin ligase activity. Both CYLD and TRAF2 phosphorylation thus increase ubiquitin-dependent NF-kB signaling. IKKe also directly phosphorylates STAT1, increasing its gene activating potential.

High levels of IKKe coincide with resistance to well established chemotherapeutics [83] . This is also the case for elevated NF-kB activation, which is known to contribute to cancer cell survival and to reduce sensitivity to chemotherapeutic agents and ionizing radiation [84] . Unfortunately, breast cancer cells in which IKKe was inactivated retained their resistance to chemotherapeutics despite a lowered NF-kB activation, suggesting that additional mechanisms must be involved in the regulation of cell death [69, 77, 85] . Finally, IKKe also accumulates in subnuclear promyelocytic leukemia (PML) bodies upon genotoxic stress, where it undergoes SUMOylation, leading to its activation and ultimately resulting in NF-kB mediated anti-apoptotic responses [86] . In this respect, it is worthwhile to mention that several glioma cell lines and human primary glioma tissues exhibit elevated levels of IKKe and are less sensitive to DNA damage-induced apoptosis [87] . Suppression of IKKe, however, renders the cells more sensitive, confirming its prosurvival function.

The role of IKKe in the development of inflammatory and metabolic diseases, as well as cancer, indicate the potential of IKKe as a therapeutic target. So far, only few small molecule inhibitors of IKKe have been described. BX-795 was the first IKKe inhibitor on the market and suppresses TBK1 and IKKe activity at nanomolar concentrations in vitro [32] . The specificity of BX-795 is however an issue since it was originally developed as an inhibitor of 3-phosphoinositide-dependent protein kinase 1 (PDK1) and only later found to also inhibit IKKe [88] . Moreover, BX-795 shows off-target effects towards several other kinases, including JNK and p38 MAP kinases [31, 32] . A modified version of BX-795, MRT67307, no longer inhibits JNK or p38 MAP kinases, but still interferes with the activity of TBK1 and PDK1 [31, 32] . More recently, a series of azabenzimidole derivatives and 2,4diamino-5-cyclopropyl pyrimidines with improved kinase selectivity and drug-like properties were described [88, 89] . However, these compounds still inhibit both IKKe and TBK1, with a slightly higher potency against TBK1. In mice, the pyrimidines significantly inhibited LPS-induced release of IFN-b, although toxicity was observed at higher doses [88, 89] . It is worth mentioning that also a number of naturally occurring compounds (polyphenoles such as (À)-epigallocatechin-3-gallate, lutolin, quercetin) with anti-inflammatory properties have been shown to target TBK1 [90, 91] . Activities against IKKe have not been described, but in general the specificity of these polyphenoles is low and most of them have multiple targets.

The development of more specific and better IKKe inhibitors may be enabled by the recent elucidation of the substrate specificity of IKKe and TBK1 [11] . These studies demonstrated that the consensus phosphorylation motif of IKKe differs from that of IKKb, but is identical to that of TBK1, suggesting that the development of IKKe inhibitors that do not target TBK1 may be very difficult. Nevertheless, high-throughput screening using a specific IKKe/TBK1 substrate peptide resulted in several lead molecules that showed selectivity for either IKKe or TBK1 [11] . However, as IKKe and TBK1 show many overlapping functions in oncogenic and inflammatory signaling pathways, it is likely that the therapeutic effectiveness of IKKe specific inhibitors may be hampered by the redundant activity of TBK1. Therefore, at least in some cases inhibition of the activity of both kinases may be a better therapeutic approach. Finally, as overexpression of IKKe is linked with tumorigenesis, therapeutic approaches that would reduce IKKe expression to normal levels may be a valid alternative. Therefore, more fundamental research on the molecular mechanisms that regulate IKKe expression may further boost the development of IKKe targeting drugs.

The potential value of IKKe as therapeutic target for antiinflammatory or anti-cancer therapies requires further investigation into the mechanisms and pathways involved. The pharmaceutical industry has been very active in the development of IKKa/ IKKb inhibitors as novel anti-inflammatory agents, but so far with limited success. Since NF-kB mediates a number of physiological functions, non-selective and complete inhibition of the NF-kB pathway may lead to serious side-effects. This is also illustrated by the fact that p65-, IKKa-, IKKb-, or NEMO-deficient mice die during embryonic development or perinatally. Compounds that more selectively repress the activation of NF-kB in response to specific receptors or the expression of only a specific subset of NF-kB dependent genes would be associated with fewer side effects. Therefore, IKKe could well be such a target with great clinical value. This is supported by the fact that IKKe knockout mice, in contrast to IKKa or IKKb knockout mice, are viable and fertile. Since TBK1 knockout mice also die embryonically, the challenge will be to develop IKKe specific inhibitors that do not target TBK1. In this context, it will also be important to further determine the relative contribution of IKKe and TBK1 in different pathways (e.g. NF-kB versus IRF) and pathophysiological processes. Modulation of IKKe or TBK1 expression and activity in distinct cell types or tissues by means of conditional knockout mice will therefore be of high value. In addition, knowledge of the different IKKe substrates and the physiological relevance of their phosphorylation may help to predict efficiency or side effects of IKKe inhibitors.

The relationship between the canonical and non-canonical IKKs, and other signaling pathways, is also an open line of investigation. In particular the negative regulatory effect of IKKe/ TBK1 on the canonical IKKs could suggest that inhibition of IKKe/ TBK1 may actually have a pro-inflammatory effect. In this context, a more detailed understanding of the molecular mechanisms involved in the counter-regulation in the IKK family could be helpful. Many of the players that are involved (e.g. TANK, NEMO) in the connection between IKK and IKK-related kinases are phosphorylated or modified by different types of ubiquitin chains, but the consequence of these modifications is not fully clear. IKKe is also believed to form distinct complexes with different adaptor proteins (TANK, NAP1, SINTBAD), which could perform different functions. Future studies on the existence and function of IKKe complexes consisting of distinct IKKe adaptor proteins are therefore of high interest as it may allow more selective IKKe targeting. Such studies may also help to explain why some stimuli (e.g. TNF, IL-1, IL-17) activate IKKe/TBK1 without inducing the phosphorylation of IRF3, whereas other stimuli (e.g. LPS, viral RNA) do activate this pathway via IKKe/TBK1. The strong inducibility of IKKe expression and its overexpression in multiple tumors is also worthwhile to examine. What are the upstream signals that can regulate IKKe expression? What determines IKKe stability? Is IKKe overexpression sufficient to be oncogenic? In this context, generating (e.g. mammary) tissue specific IKKe transgenic mice may be very informative.

In summary, the generation and exploitation of IKKe specific inhibitors and conditional knockout/knockin/transgenic mice of IKKe and its regulators or substrates is likely to give answers to many of the above mentioned questions. Eventually, this could lead to the use of IKKe specific inhibitors for the successful treatment of autoimmunity, obesity, diabetes and certain cancers. G0089.10, 3G023611, G028712N, G046612N, G016413N,  G027413N ), the 'Foundation Against Cancer', the 'Strategic Basic Research' programme of the IWT, and the 'Hercules', 'GOA', and 'Group-ID MRP' initiatives of Ghent University. LV holds a FWO postdoctoral fellowship.

Pattern recognition receptors: sentinels in innate immunity and targets of new vaccine adjuvants

NF-kappaB: where did it come from and why

IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex

NAK is an IkappaB kinase-activating kinase

RalB GTPasemediated activation of the IkappaB family kinase TBK1 couples innate immune signaling to tumor cell survival

Involvement of the ubiquitin-like domain of TBK1/IKK-i kinases in regulation of IFN-inducible genes

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription

IKKi/ IKKepsilon plays a key role in integrating signals induced by pro-inflammatory stimuli

The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection

The kinase TBK1 controls IgA class switching by negatively regulating noncanonical NF-kappaB signaling

Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKepsilon

The IKK-related kinases: from innate immunity to oncogenesis

IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IkappaB kinases

Genomic structure and functional characterization of the promoter region of human IkappaB kinase-related kinase IKKi/ IKKvarepsilon gene

IKBKE is induced by STAT3 and tobacco carcinogen and determines chemosensitivity in non-small cell lung cancer

Novel splice variants of human IKKepsilon negatively regulate IKKepsilon-induced IRF3 and NF-kB activation

IKKepsilon modulates RSV-induced NF-kappaB-dependent gene transcription

Phosphorylation of NF-kappaB p65 at Ser468 controls its COMMD1-dependent ubiquitination and target gene-specific proteasomal elimination

Specification of the NF-kappaB transcriptional response by p65 phosphorylation and TNF-induced nuclear translocation of IKK epsilon

Transcriptional integration of TLR2 and TLR4 signaling at the NCoR derepression checkpoint

Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKK epsilon

Systematic characterization by mass spectrometric analysis of phosphorylation sites in IRF-3 regulatory domain activated by IKK-i

IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts

Differential requirement for TANK-binding kinase-1 in type I interferon responses to toll-like receptor activation and viral infection

Absence of MyD88 Results in Enhanced TLR3-Dependent Phosphorylation of IRF3 and Increased IFN-{beta} and RANTES Production

Pathogen recognition by the innate immune system

Emerging role of ubiquitination in antiviral RIG-I signaling

Beyaert RTAX1BP1. a ubiquitin-binding adaptor protein in innate immunity and beyond

A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response

Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated

Novel crosstalk within the IKK family controls innate immunity

Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation

Lipopolysaccharide-mediated interferon regulatory factor activation involves TBK1-IKKepsilon-dependent Lys(63)-linked polyubiquitination and phosphorylation of TANK/I-TRAF

Signaling to NF-kappaB by Toll-like receptors

a novel component of innate antiviral immunity, shares a TBK1-binding domain with NAP1 and TANK

The TRAF-associated protein TANK facilitates cross-talk within the IkappaB kinase family during Toll-like receptor signaling

Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases

DUBA: a deubiquitinase that regulates type I interferon production

The tumour suppressor CYLD is a negative regulator of RIG-I-mediated antiviral response

Regulation of IkappaB kinase-related kinases and antiviral responses by tumor suppressor CYLD

Phosphorylation of the tumor suppressor CYLD by the breast cancer oncogene IKKepsilon promotes cell transformation

TAX1BP1 and A20 inhibit antiviral signaling by targeting TBK1-IKKi kinases

NLRP4 negatively regulates type I interferon signaling by targeting the kinase TBK1 for degradation via the ubiquitin ligase DTX4

TRAF-interacting protein (TRIP) negatively regulates IFN-beta production and antiviral response by promoting proteasomal degradation of TANK-binding kinase 1

Ebola virus protein VP35 impairs the function of interferon regulatory factor-activating kinases IKKepsilon and TBK-1

Severe acute respiratory syndrome coronavirus M protein inhibits type I interferon production by impeding the formation of TRAF3.TANK.TBK1/IKKepsilon complex

Hepatitis Julkunen I. C virus NS2 protease inhibits host cell antiviral response by inhibiting IKKepsilon and TBK1 functions

Hepatitis Yuan Z. B virus polymerase inhibits RIG-I-and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

Viral targeting of the interferon-{beta}-inducing Traf family member-associated NF-{kappa}B activator (TANK)-binding kinase-1

Synergistic benefit in inflammatory arthritis by targeting I kappaB kinase epsilon and interferon beta

Antiviral gene expression in rheumatoid arthritis: role of IKKepsilon and interferon regulatory factor 3

Interferon beta for rheumatoid arthritis: new clothes for an old kid on the block

Genetic variation in the nuclear factor kappaB pathway in relation to susceptibility to rheumatoid arthritis

A candidate gene study of the type I interferon pathway implicates IKBKE and IL8 as risk loci for SLE

Computational approach to identify enzymes that are potential therapeutic candidates for psoriasis

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation

Inactivation of the Enzyme GSK3alpha by the Kinase IKKi Promotes AKT-mTOR Signaling Pathway that Mediates Interleukin-1-Induced Th17 Cell Maintenance

The protein kinase IKKepsilon is a potential target for the treatment of inflammatory hyperalgesia

Obesity, inflammation, and insulin resistance

Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance

Hypoxia is a potential risk factor for chronic inflammation and adiponectin reduction in adipose tissue of ob/ob and dietary obese mice

The protein kinase IKKepsilon regulates energy balance in obese mice

IKKepsilon: a bridge between obesity and inflammation

Beneficial effects of IKKepsilon-deficiency on body weight and insulin sensitivity are lost in high fat diet-induced obesity in mice

Emerging roles for the non-canonical IKKs in cancer

Integrative genomic approaches identify IKBKE as a breast cancer oncogene

Regulation of IKKepsilon Expression by Akt2 Isoform

Silencing of the IKKepsilon gene by siRNA inhibits invasiveness and growth of breast cancer cells

Inducible IkappaB kinase/IkappaB kinase epsilon expression is induced by CK2 and promotes aberrant nuclear factor-kappaB activation in breast cancer cells

IkB kinase e phosphorylates TRAF2 to promote mammary epithelial cell transformation

IKBKE protein activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and the pleckstrin homology domain to sustain malignant transformation

IkappaB kinase epsilon and TANK-binding kinase 1 activate AKT by direct phosphorylation

Multiple functions of the IKK-related kinase IKKepsilon in interferon-mediated antiviral immunity

The Stat family of transcription factors have diverse roles in mammary gland development

IKK-e coordinates invasion and metastasis of ovarian cancer

Deregulation of IKBKE is associated with tumor progression, poor prognosis, and cisplatin resistance in ovarian cancer

NF-kappaB signalling proteins p50/p105, p52/p100, RelA, and IKKepsilon are over-expressed in oesophageal squamous cell carcinomas

Over-expression of IkappaB-kinase-epsilon (IKKepsilon/IKKi) induces secretion of inflammatory cytokines in prostate cancer cell lines

IkappaB-Kinase-epsilon (IKKepsilon/IKKi/IkappaBKepsilon) expression and localization in prostate cancer tissues

Immunohistochemical analysis of NF-kappaB signaling proteins IKKepsilon, p50/p105, p52/p100 and RelA in prostate cancers

Kinome expression profiling identifies IKBKE as a predictor of overall survival in clear cell renal cell carcinoma patients

IKKepsilon phosphorylation of ERalpha-Ser167 and contribution to tamoxifen resistance in breast cancer

NFkappaB-dependent chemoresistance in solid tumors

Inhibition of the canonical IKK/NF kappa B pathway sensitizes human cancer cells to doxorubicin

SUMOylation-Dependent Localization of IKK epsilon in PML Nuclear Bodies Is Essential for Protection against DNA-Damage-Triggered Cell Death

IKBKE is over-expressed in glioma and contributes to resistance of glioma cells to apoptosis via activating NF-kappaB

Discovery of azabenzimidazole derivatives as potent, selective inhibitors of TBK1/IKKepsilon kinases

Synthesis and structure-activity relationships of a novel series of pyrimidines as potent inhibitors of TBK1/IKKepsilon kinases

Suppression of the TRIFdependent signaling pathway of Toll-like receptors by luteolin

Suppression of MyD88-and TRIF-dependent signaling pathways of Toll-like receptor by (À)-epigallocatechin-3-gallate, a polyphenol component of green tea

Research in the authors' lab is supported by grants from the 'Interuniversity Attraction Poles (IAP-VII, contract P7/32)', the Fund for Scientific Research (FWO)-Flanders (grants G0619.10,