Receptor proximal kinases in NF-κB signaling as potential therapeutic targets in cancer and inflammation

Biochemical Pharmacology 92 (2014) 519–529

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Research update

Receptor proximal kinases in NF-kB signaling as potential therapeutic targets in cancer and inflammation Lynn Verstrepen a,b, Rudi Beyaert a,b,* a b

Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 September 2014 Accepted 30 October 2014 Available online 7 November 2014

Many signaling pathways leading to activation of transcription factors and gene expression are characterized by phosphorylation events mediated by specific kinases. The transcription factor NF-kB plays a key role in multiple cellular processes, including immune signaling, inflammation, development, proliferation and survival. Dysregulated NF-kB activation is associated with autoimmunity, chronic inflammation and cancer. Activation of NF-kB requires IkB kinase (IKK)a or b, the activity of which is regulated via phosphorylation by specific IKK kinases and by autophosphorylation. Receptor specificity is further obtained by the use of multiple upstream receptor proximal kinases. We review the identities of several IKK regulatory kinases as well as the proposed molecular mechanisms. In addition, we discuss the potential for therapeutic targeting of some of these kinases in the context of inflammatory diseases and cancer. ß 2014 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: MLN-0415 (PubChem CID: 56603702) SPC-839 (PubChem CID: 9820526) IMD-0354 (PubChem CID: 5081913) 5Z-7-ozozeanol (PubChem CID: 9863776) KRX-0401 (PubChem CID: 148177) MK-2206 (PubChem CID: 46930998) Sotrastaurin (PubChem CID: 10296883) KN-93 (PubChem CID: 5312122) Y27632 (PubChem CID: 448042) Fasudil (PubChem CID: 3547) Keywords: Therapy Kinase NF-kB Inflammation Cancer

Abbreviations: Aly, alymphoplasia; BAFFR, B-cell activating factor receptor; cIAP, cellular inhibitor of apoptosis protein; Bcl10, B-cell lymphoma/leukemia 10; CaMKII, Ca2+/calmodulin-dependent kinase II; CARD, caspase recruitment domain; CARMA1, CARD-containing MAGUK protein 1; CRISPR, clustered regulatory interspaced short palindromic repeats; DLBCL, diffuse large B-cell lymphoma; ERK, extracellular signal-regulated kinase; GSK-3, glycogen synthase kinase 3; IEC, intestinal epithelial cell; IFN, interferon; IkB, inhibitor of kB; IKK, IkB kinase; IKKK, IKK kinase; IL-1, interleukin-1; IL1R, IL-1 receptor; IRAK, IL-1 receptor-associated kinase; IRF, interferon-regulatory factor; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; LTbR, lymphotoxin b receptor; MAGUK, membrane-associated guanylate kinase; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1; MAP3K, mitogen-activated protein kinase kinase kinase; MCP-1, monocyte chemotactic protein 1; MEF, mouse embryonic fibroblast; MEKK, MAPK/ ERK kinase kinase; MKK4, mitogen-activated protein kinase kinase 4; mTOR, mammalian target of rapamycin; MyD88, myeloid differentiation primary response gene 88; NF-kB, nuclear factor kB; NEMO, NF-kB essential modulator; NIK, NF-kB inducing kinase; OVA, ovalbumine; PBMC, peripheral blood mononuclear cells; PDK1, 3-posphoinositide-dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PK, pharmacokinetic; PKB, protein kinase B; PKC, protein kinase C; RANK, http://dx.doi.org/10.1016/j.bcp.2014.10.017 0006-2952/ß 2014 Elsevier Inc. All rights reserved.

receptor activator of NF-kB; RIG-I, retinoic acid-inducible gene 1; RING, really interesting new gene; ROCK, Rho-associated kinase; Ser, serine; STAT1, signal transducer and activator of transcription 1; TAB, TAK1-binding protein; TAK1, TGFb-activated kinase 1; TANK, TRAF family member-associated NF-kB activator; TBK1, TANK-binding kinase 1; TCR, T-cell receptor; TGFb, transforming growth factor b; Thr, threonine; TLR, Toll-like receptor; TNF, tumor necrosis factor; TNFR1, TNF receptor 1; TRAF, TNF receptor associated factor; VSV, vesicular stomatitis virus. * Corresponding author. E-mail address: [email protected] (R. Beyaert).

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1. Introduction to NF-kB and its role in inflammation and cancer Nuclear factor kB (NF-kB) is a transcription factor involved in many cellular processes, including immune signaling, inflammation, proliferation, apoptosis and development. The name NF-kB actually refers to a transcription factor family comprised of five members (RelA (also known as p65), RelB, c-Rel, p50 and p52), which form homo- and heterodimers [1]. They are kept inactive in the cytoplasm by the binding of inhibitor of kB (IkB) family members (IkBa, IkBb, IkBg, BCL3, IkBe, p105 and p100) [2], whose activity is regulated mainly by phosphorylation, ubiquitination and proteasomal maturation or degradation upon NF-kB activating stimuli. Most receptors, such as tumor necrosis factor receptor 1 (TNFR1), interleukin-1 receptor (IL1R), Toll-like receptors (TLRs), and T-cell receptors (TCR), activate the canonical NF-kB pathway (Fig. 1), which requires IkB kinase (IKK)b and its scaffolding protein IKKg (also called NF-kB essential modulator, NEMO). IKKb phosphorylates IkBa, marking it for ubiquitination and proteasomal degradation, thus releasing NF-kB to go to the nucleus. Some receptors, such as CD40, B-cell activating factor receptor (BAFFR), lymphotoxin b receptor (LTbR), receptor activator of NF-kB (RANK) and TNFR2, can activate an alternative (also known as noncanonical) NF-kB pathway (Fig. 1), which is independent of IKKb/ NEMO but requires the IKKb-related kinase IKKa. IKKa phosphorylates the IkB protein p100, which associates primarily with RelB. p100 phosphorylation results in its processing to p52 and the nuclear translocation of p52/RelB to the nucleus [3]. Canonical NFkB signaling is the major pathway in immunity and inflammation, while the non-canonical pathway is more associated with lymphoid organogenesis. Dysregulation of NF-kB activation has been linked to several autoimmune diseases, such as rheumatoid arthritis, inflammatory

bowel disease, multiple sclerosis, systemic lupus erythematosus and type I diabetes [1]. Moreover, in several cancers, NF-kB is constitutively activated and drives tumor cell survival and proliferation [2]. Moreover, chronic exposure to inflammatory signals in the tumor microenvironment can lead to NF-kB activation in malignant cells, further stimulating their growth. For many years the NF-kB signaling pathway has attracted much interest because of the possibility of targeting it for the treatment of inflammatory diseases and cancer. NF-kB activation can be targeted at multiple levels, including binding of NF-kB to DNA, IkBa degradation and IKK or upstream kinase activation. Most efforts have gone to the development of small-compound IKKb inhibitors, but no IKKb inhibitors have made it to the clinic so far. Considering the central role of NF-kB and IKKb in normal immune responses, this is not surprising. Multiple receptor-specific signaling pathways converge on IKKb. Therefore, interfering more upstream in the NF-kB signaling cascade is expected to be much more specific and to cause fewer side effects. Signaling upstream of IKKb is quite complex and involves multiple protein–protein interactions and posttranslational modifications [4]. We review several kinases that have been reported to regulate IKK activity, the underlying molecular mechanisms, and the potential for therapeutic intervention. 2. IkB kinases (IKKs): Where everything converges 2.1. Function and activation of IKKa and IKKb The IKK family consists of four members, IKKa, IKKb, IKKe (also known as IKKi) and TANK-binding kinase 1 (TBK1). IKKe and TBK1 are best known for their role in the activation of the family of interferon-regulatory factor (IRF) transcription factors, namely the direct phosphorylation and activation of IRF3 and IRF7 [5]. These

Fig. 1. Canonical versus non-canonical NF-kB signaling. Receptor-induced NF-kB activation primarily involves two signaling pathways that regulate different NF-kB family members. The canonical pathway requires the activity of NEMO and IKKb, whereas the non-canonical pathway involves IKKa. See text for more details. (P, phosphorylation).

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kinases are believed to have no direct role in NF-kB signaling, but crosstalk with IKKa/IKKb has been reported in cells stimulated with TNF, IL-1 or TLR ligands [6,7]. IKKb directly phosphorylates IkBa and is a central kinase in canonical NF-kB signaling, at which multiple receptor-specific NFkB signaling pathways converge. Together with IKKa and the adaptor protein NEMO, IKKb is part of the so-called IKK complex. Although IKKa coexists with IKKb in the same complex, IKKa is mainly implicated in non-canonical NF-kB signaling, in which it directly phosphorylates the NF-kB family member p100 in the cytoplasm. Moreover, IKKa is also found in the nucleus, where it has multiple functions [4,8]. Besides IkBa and p100, several other NF-kB regulatory proteins have been shown to serve as substrates for IKKa and IKKb, including other members of the IkB family, NFkB subunits, co-factors, histone modifying enzymes and other signaling molecules [4,8]. In addition, IKK kinases have important functions beyond NF-kB signaling (summarized in Fig. 2). The kinase activity of IKKa/b critically depends on the dual phosphorylation of S176/S180 (IKKa) or S177/S181 (IKKb) in the IKK activation loop [4]. Recent crystallographic studies of IKKb dimers shed more light on its activation mechanism [9–11]. When the activation loop is not phosphorylated, the IKK complex adopts a closed conformation incapable of substrate binding [9]. This explains why phosphorylation is needed for their activation. In non-activated IKKb dimers, the neighboring kinase domains cannot interact, but upon activation the kinase domains interact, potentially leading to IKKb autophosphorylation in trans [10,11]. However, many things are still unknown. For example, it is not clear if the kinase domains are positioned in the same way in the in vivo more relevant IKKa/b heterodimer. Also, different models have been proposed to explain IKKb dimer activation: IKKb autophosphorylation, IKKb phosphorylation by upstream kinases, or a combination of both (see below). 2.2. Therapeutic targeting of IKKs: Too much to be good? Because NF-kB activation is dysregulated in many diseases [1,2], the NF-kB signaling pathway was believed to be the Holy Grail for novel therapies in the field of cancer and chronic inflammation. Because of the central role of IKKb in NF-kB signaling, most research efforts of academic institutes and pharmaceutical companies focused on the development of small-compound IKK inhibitors. In the past 15 years, a number of structurally diverse natural and synthetic small molecules that block the NF-kB signaling pathway in cultured cells have been identified. These molecules showed significant therapeutic activity in several murine models of chronic inflammation and cancer. For a complete overview we refer to [12–14]. However, despite these promising results in animal models, only a few drugs made it to clinical trials, and there are still no approved agents that directly target IKKs (Table 1). In 2006, MLN-0415 from Millennium was the first small-molecule IKKb inhibitor to enter human trials. Although a single ascending-dose trial was successfully completed, MLN-0415 testing was later terminated due to a nonspecified unfavorable safety profile in the multiple ascendingdose study. Compound SPC-839, an ATP competitive reversible IKKb inhibitor originally developed by Celgene and advanced into a phase I trial for hematological malignancies by Merck Serono, was terminated in 2008 for portfolio repositioning reasons. IMD-0354 and IMD-0560 are salicylanilides developed by the Institute of Medicinal Molecular Design Inc., and a phase I clinical trial of a topical formulation of IMD-0354 for treatment of atopic dermatitis has been successfully completed. However, whether IKKb is the true molecular target of these compounds has been questioned. The most advanced pharmacological IKKb targeting agent is presumably the Sanofi-Aventis SAR113945, which

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entered phase II clinical trials for local treatment of osteoarthritis. However, the trial was recently discontinued because it did not meet the primary endpoints. Altogether, the potential of IKKb inhibition as a therapeutic strategy remains unrealized and previous optimism for IKK as a target has cooled down completely. Given the central role of IKKb in innate and adaptive immunity, its chronic, systemic inhibition may not be feasible. More innovative targeting strategies are emerging for selective delivery to tissues, disease types, or cell type. Pro-drugs may be used to improve the selectivity of the drug for certain cell types or processes that are the intended target. For example, the Institute of Medicinal Molecular Design Inc. completed a phase I study with IMD-2560, which is a pro-drug of IMD-0560. In addition, they initiated a phase II study with the IKKb inhibitor IMD-1041, which is a prodrug of IMD-0354, for the treatment of chronic obstructive pulmonary disorder. Similarly, Chroma Therapeutics is developing pro-drug IKKb inhibitors (thiophenecarboxamides) that incorporate esterase-sensitive motifs that can be hydrolyzed only inside monocytes and macrophages. The coming years will tell if IKKb can still be exploited as a target by further optimizing targeting strategies. 3. IkB kinase kinases (IKKKs): Highways to IKK activation 3.1. Function and activation of IKKKs Several kinases can directly phosphorylate and activate IKKs (referred to as IKK kinases (IKKKs) hereafter) (Fig. 3). The best known IKKK is TGFb-activated kinase 1 (TAK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K) 7 or MAPK/ERK Kinase Kinase (MEKK) 7, a serine/threonine kinase that acts as an essential component of the IKK and MAPK signaling cascades evoked by many pro-inflammatory stimuli. TAK1 activation involves its recruitment to upstream K63-polyubiquitinated signaling proteins (e.g. IRAK1 and MyD88 in the IL-1 signaling pathway), which is mediated by the TAK1-binding proteins (TAB) 2 and TAB3, which form a complex with TAK1 [15]. Ubiquitin binding of TAB2/TAB3 leads to TAK1 conformational changes, which induce its autophosphorylation. TAK1 also binds TAB1, which has been suggested to shield TAK1 from negative regulatory phosphatases. K63-polyubiquitinated substrates were recently shown to be further modified by M1-linked polyubiquitin chains, leading to K63/M1-linked hybrid ubiquitin chains [16]. Whereas the TAB2/3 components of the TAK1 complex bind to K63 polyubiquitin, the NEMO component of the IKK complex binds to M1 polyubiquitin. The formation of K63/M1polyubiquitin hybrids may therefore provide an elegant mechanism for colocalizing the two complexes, facilitating the TAK1catalyzed activation of IKKa and IKKb. Activation of IKKa and IKKb requires phosphorylation of the activation loops of these kinases at Ser176 and Ser180 (IKKa) or Ser 177 and Ser 181 (IKKb) [2]. TAK1, immunoprecipitated from HeLa cells, has been reported to specifically phosphorylate both Ser residues and activate IKKa and IKKb in vitro [15], supporting a role for TAK1 as an IKKK. However, it was recently reported that the activation of IKKb induced by IL-1, TNF and TLR ligands involves phosphorylation only of Ser177 by TAK1 [17]. This phosphorylation primes IKKb for subsequent autophosphorylation of Ser181. How TAK1 obtains specificity for Ser177 in living cells, whereas in vitro it phosphorylates both Ser177 and Ser181, remains to be solved. Perhaps the positioning of TAK1 and IKKb at K63/M1 hybrid polyubiquitin chains creates a platform that directs TAK1 catalytic activity specifically to Ser177. TAK1 is not a universal activator of the IKK complex and its action may depend on the stimulus and cellular context. This is illustrated by studies on cells derived from mice with TAK1

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Fig. 2. Pleiotropic activities of IKK kinases. IKKa and IKKb regulate multiple cellular processes. Proteins phosphorylated by IKKs are indicated.

deficiency in specific cell types, which revealed differences in NF-kB signaling (impaired, normal or enhanced) depending on the cell type and stimulus used (for overview see [18]). For example, TAK1 seems to be dispensable for NF-kB signaling in TCRstimulated effector T cells [19] and even negatively regulates LPS-induced IKK activation in mouse neutrophils [20]. Therefore, other IKKKs may be responsible for IKK activation in certain

conditions. In this context, IL-1-induced NF-kB activation has been reported to involve both MEKK3 (also known as MAP3K3) and TAK1 [21]. MEKK3-deficient cells show reduced NF-kB activation upon TNF, IL-1 and TLR4 triggering, and MEKK3 directly phosphorylates the IKKs in vitro [22,23]. However, MEKK3 deficiency did not completely abrogate NF-kB activation, so parallel mechanisms might indeed exist.

Table 1 IKKb inhibitors with clinically interesting properties. Potential benefit

Drawback

Clinical experience

Company

References

Potential as treatment for multiple inflammatory disorders including rheumatoid arthritis and multiple sclerosis. Potential as veterinary drug Potential as therapy for haematological malignancies Potential as topical application for treatment of atopic dermatitis. In vitro evidence for therapeutical benefits in chronic lymphocytic leukemia (CCL). Evidence from mouse models for therapeutical benefits in: - Chronic asthma - Non-small cell lung cancer - Breast cancer - Diabetes

Safety issues with multiple dose treatment

Phase I clinical trial terminated due to unfavourable safety profile. Is currently tested in dogs with multicentric lymphoma Phase I clinical trial stopped due to portfolio repositioning reasons Phase I clinical trial for topical formulation successfully completed. Phase II clinical trial planned

Millennium Pharmaceuticals Inc. Purdue University

[1]

Developed by Celgene, licensed to Merck Serono Institute of Medicinal Molecular Design Inc.

[1]

Phase II clinical trial for local treatment of osteoarthritis discontinued because it did not meet the primary endpoints Phase I clinical trial completed

Sanofi-Aventis

www.sanofi.fi [1,7]

Institute of Medicinal Molecular Design Inc.

[1]

Institute of Medicinal Molecular Design Inc.

[1] www.immd.co.jp

Chroma Therapeutics, Astrazeneca, GlaxoSmithKline, Pfizer

[1,7]

SPC-839 IMD-0354

SAR113945

IMD-2560

IMD-1041

Thiophenecarboxamides

Pro-drug of IMD-0560, might improve selectivity. Treatment of rheumatoid arthritis and osteoporosis Pro-drug of IMD-0354, might improve selectivity. Treatment of chronic obstructive pulmonary disorder Pro-drugs of which some were modified with esterase-sensitive motifs that can only be hydrolysed inside monocytes and macrophages improving selectivity Evidence from animal models to be beneficial in: - Arthritis - Asthma

Not clear if IKKb is true molecular target. Multiple targets reported

Phase II clinical trial

[1] [2] [3] [4] [5] [6]

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Inhibitor MLN-0415

[1] E.R.R. Young. IKKb as a therapeutic intervention point for diseases related to inflammation. In: J.I. Levin and S. Laufer, editors. Anti-inflammatory drug discovery, Cambridge: RSC Publishing; 2012, p. 255-96. [2] Kanduri M, Tobin G, Aleskog A, Nilsson K, Rosenquist R. The novel NF-kappaB inhibitor IMD-0354 induces apoptosis in chronic lymphocytic leukemia. Blood Cancer J 2011;1:e12. [3] Ogawa H, Azuma M, Muto S, Nishioka Y, Honjo A, Tezuka T, et al. IkappaB kinase beta inhibitor IMD-0354 suppresses airway remodelling in a Dermatophagoides pteronyssinus-sensitized mouse model of chronic asthma. Clin Exp Allergy 2011;41:104-15. [4] Gomez-Cabrero A, Wrasidlo W, Reisfeld R. NF-kB inhibitor IMD-0354 targets human non-small cell lung cancer stem cells and combined with chemotherapy reduces multidrug resistance. J Cancer Sci Ther 2013;5:12. [5] Gomez-Cabrero A, Wrasidlo W, Reisfeld RA. IMD-0354 targets breast cancer stem cells: a novel approach for an adjuvant to chemotherapy to prevent multidrug resistance in a murine model. PLoS One 2013;8:e73607. [6] Lennikov A, Hiraoka M, Abe A, Ohno S, Fujikawa T, Itai A, et al. IkappaB kinase beta inhibitor IMD-0354 beneficially suppresses retinal vascular permeability in streptozotocin-induced diabetic mice. Invest Ophthalmol Vis Sci 2014;55:6365-73. [7] Llona-Minguez S, Baiget J, Mackay SP. Small-molecule inhibitors of IkappaB kinase (IKK) and IKK-related kinases. Pharm Pat Anal 2013;2:481-98.

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Fig. 3. Kinase cascades involved in IKK activation. Specific receptors differentially use receptor proximal kinases to directly or indirectly (involving phosphorylation and ubiquitination events) activate IKKKs. The latter then phosphorylate IKKb, which also triggers IKKb autophosphorylation, leading to canonical NF-kB activation. Some IKKKs can also phosphorylate IKKa, leading to non-canonical NF-kB activaton. (P, phosphorylation; Ub, ubiquitination).

Other members of the MAP3K family were put forward as IKKKs, but conclusive data are still lacking. MEKK1 (also known as MAP3K1) phosphorylates IKKa/b in vitro [24]. Moreover, IL-1 activated MEKK1 in Caco2 intestinal epithelial cells, leading to IkBa degradation [25], and MEKK1 silencing inhibited IL-1-induced IKK activation. However, TNF also induced the MEKK1-dependent phosphorylation of MKK4, which in turn phosphorylated IKK [26], raising doubts whether MEKK1 is a true IKKK. Although TNFinduced NF-kB activation was reported to be normal in MEF cells deficient in MEKK2 (also known as MAP3K2) [23], Schmidt and colleagues suggested a role for MEKK2 in the TNF- and IL-1-induced delayed activation of NF-kB [27]. It was suggested that MEKK3 regulates the rapid activation of NF-kB through association with IkBa:NF-kB/IKK, while MEKK2 interacts with IkBb:NF-kB/IKK complexes. This leads to biphasic NF-kB activation, with MEKK2/ 3 regulating specific and temporal gene expression. NIK (also known as MAP3K14) is a MAP3K that is essential for activation of the noncanonical NF-kB pathway, in which NIK directly phosphorylates IKKa in its activation loop, leading to IKKa-mediated NF-kB p100 phosphorylation and processing to p52 [28]. In resting cells, NIK levels are low due to its continuous degradation triggered by the TRAF3-TRAF2-cIAP1/2 ubiquitin ligase complex [3]. Upon stimulation, TRAF3 is degraded, leading to stabilization of NIK and activation of the non-canonical pathway. NIK was first identified as an activator of the canonical NF-kB pathway [29]. However, NIK mutant alymphoplasia (Aly) mice as well as NIK-deficient mice did not show any defects in TNF and IL-1 signaling, excluding an essential role for NIK in canonical NF-kB signaling [30,31]. Later studies, however, suggested a cell-type and stimulus-specific role for NIK in canonical NF-kB signaling [32–35]. For example, NIK-deficient lymphocytes are defective in IkBa degradation and RelA translocation upon CD40L, BAFF or CD70 stimulation, and expression of p100, RelB and cRel, all of which depend on canonical signaling, is lower in the absence of NIK [35]. NIK-mediated activation of the canonical NF-kB pathway may therefore specifically occur in response to receptors that also lead to non-canonical NF-kB signaling, where NIK is recruited together with the classical IKK complex, whereas this is not the case for NIK-independent receptors like TNFR1. Phosphorylation of IKKa by NIK in an IKKa/IKKb heterodimer

complex would then lead to IKKa-mediated phosphorylation of IKKb and activation of the canonical NF-kB pathway [35,36]. NIK has also been shown to induce IKKb autophosphorylation [37]. Although NIK has been reported to be dispensable for canonical NF-kB signaling in LTb-stimulated MEF cells, transcription of specific canonical NF-kB dependent genes such as MCP-1 and IkBa is NIKdependent [31]. So, NIK also regulates transactivation, possibly through activation of the nuclear functions of IKKa. Akt (also known as protein kinase B (PKB)) is a serine-threonine kinase that acts downstream of phosphatidylinositol 3-kinase (PI3K) [38] and has also been implicated in NF-kB signaling. Akt phosphorylates IKKa on Thr23 in response to TNF, in parallel to its phosphorylation by NIK on Ser176, and dominant-negative mutants of Akt and NIK inhibit each other’s ability to activate NF-kB [39,40]. Thus, Akt and NIK are both necessary for NF-kB activation. However, the role of Akt in TNF-induced NF-kB activation is cell-type dependent and reflects the ratio of IKKa to IKKb in the cell [41]. More specifically, the requirement for Akt is associated with a higher IKKa to IKKb ratio. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a serine/threonine protein kinase essential for the function of the PI3K/Akt signaling pathway. PDK1 binds IKKb and directly phosphorylates Ser181 in its activation loop, leading to NF-kB nuclear translocation and NF-kB-dependent anti-apoptotic gene expression [42]. IKKb Ser177 phosphorylation and Akt-mediated IKKa activation did not participate in PDK1-dependent IKKb and NF-kB activation. Constitutive PDK1 activity in resting cells was proposed to induce low levels of NF-kB activity, leading to the expression of anti-apoptotic genes and cell survival. A role for PDK1-induced IKK phosphorylation in response to receptor stimulation has not yet been reported. In fact, TNF-induced NFkB activation was not affected by PDK1 silencing [42]. PDK1 was reported to play a critical role in T cell receptor-induced NF-kB signaling [43], but this report focused on PDK1-induced recruitment of IKK and MALT1/Bcl10 signaling complexes to the T cell receptor, and IKK kinase activity of PDK1 was not studied at all. PDK1 may contribute to NF-kB activation both to promote complex formation and to phosphorylate and activate IKKb.

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3.2. Therapeutic targeting of IKKKs Because of its role in NF-kB and MAP kinase signaling in response to proinflammatory cytokines, TAK1 might be an attractive therapeutic target in inflammatory disorders and in cancer. In this context, TAK1 deficiency reduces renal inflammation and fibrosis in progressive kidney disease, prevents cardiomyocyte hypertrophy, and blocks neuronal death in cerebral ischemia (reviewed in [18]). TAK1 deficiency in dendritic cells ameliorates contact hypersensitivity and 5Z-7-oxozeaenol, a TAK1 inhibitor of fungal origin, can reduce ear swelling caused by picryl chloride treatment [44,45]. Also, TAK1 activation promotes metastasis in different cancer models [18]. In addition, the orally active TAK1-selective small-molecule inhibitor LYTAK1 (Eli Lilly) reduces NF-kB activation in human pancreatic cancer and reverts chemoresistance in cell lines and nude mice with human pancreatic tumor xenografts [46]. In addition, the TAK1 inhibitor 5Z-7-oxozeaenol inhibits KRAS-dependent tumor growth [47], further underlining the potential of TAK1 inhibitors in anti-cancer therapy. Despite these beneficial effects in murine models, it should be noted that global ablation of TAK1 in mice is embryonically lethal and that several studies on mice deficient in TAK1 in specific cell types have shown that TAK1 is essential for homeostasis in immune and non-immune tissues in the steady state [18]. Moreover, TAK1 has also been shown to mediate tumor suppression, illustrating that the outcome of TAK1 inhibition may depend on cell type and context. It is also important to note that TAK1 was discovered as a mediator of transforming growth factorb signaling. We are not aware of any clinical trials using TAK1 inhibitors, and it is unlikely that direct inhibition of TAK1 will be an acceptable therapy. Upstream modulators of TAK1 may be more suitable targets. Downregulation of NIK kinase expression is associated with tumor regression in several cancer types, such as multiple myeloma, diffuse large B-cell lymphoma (DLBCL), adult T-cell leukemia and melanoma [32,34,48,49]. In these tumors, high NIK levels are driven by NIK gene amplifications or by mutations in NIK or the ubiquitin ligase TRAF3, which regulates NIK stability. NIK downregulation in multiple myeloma cells expressing high levels of NIK led to cell death, but this was not the case in cells expressing low levels, indicating that the former cells were dependent on NIK for their survival [32]. Therefore, screening of NIK expression levels in multiple myeloma patients might indicate the therapeutic outcome of NIK inhibitors. Depletion of NIK inhibits both canonical and non-canonical NF-kB signaling in multiple myeloma cell lines [32,50], but the survival role of NIK in multiple myeloma was shown to be mainly due to its role in canonical NF-kB signaling. In pancreatic tumors, however, the NIK oncogenic potential was dependent solely on non-canonical NF-kB activation [51], whereas in non-small cell lung cancers both were necessary [52]. NIK expression is also enhanced in peripheral T-cell lymphoma, in which NIK activates both canonical and non-canonical NF-kB signaling [33]. In this case, knockdown of NIK induced cell death, whereas knockdown of the IKK kinases only had a minor effect, demonstrating the need for more specific targeting of NF-kB signaling depending on the cancer type. Intervention with the noncanonical NF-kB pathway via NIK inhibition may not only be beneficial in certain cancers, but also for some inflammatory disorders. For instance, mice deficient in NIK are protected from bone destruction in models of rheumatoid arthritis [53]. Because blockade of RANKL, which signals via NIK, has been approved for osteoporosis treatment and also prevents bone erosion in rheumatoid arthritis [54], inhibition of NIK activity may be of clinical benefit in human disease. The first NIK inhibitor that was reported is a substituted pyrazolo [4,3-c] isoquinoline [55]. However, it was later shown that this inhibitor does not inhibit NIK and

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the non-canonical pathway, but does inhibit TAK1. Staurosporine, a pan-kinase inhibitor, also inhibits NIK, but is not so interesting for therapeutic use. By using virtual screening, Mortier and colleagues recently identified two new NIK inhibitors that work in the 50–100 mM range. The first is a 1,4-naphtoquinone substituted in the positions 2 and 5 by a methyl and a hydroxyl moiety, respectively. However, such compounds are DNA-alkylating agents that can lead to the formation of reactive oxygen species and are therefore not suitable in the clinic. The second is a 4Hisoquinoline-1,3-dione, but cellular studies have not been reported. Amgen recently described a number of additional NIK inhibitors identified by high-throughput screening and homology modeling [56], laying the foundation for further developing of potent and selective NIK inhibitors. The IKKKs Akt and PDK1 have also been targeted, mainly in the context of cancer. Several Akt and PDK1 inhibitors have been described, and some of them showed promise in early clinical research [57]. However, phase III studies for colorectal cancer and multiple myeloma with the Akt inhibitor perifosine (KRX-0401, Aeterna Zentaris/Keryx) were recently discontinued and further development was abandoned. The allosteric Akt inhibitor MK2206 (Merck) is currently in a phase II clinical trial for endometrial cancer [38]. Celecoxib, a cyclooxygenase 2 inhibitor that also inhibits PDK1, was shown to induce apoptosis in various cancer cells in vitro and to reduce the formation of polyps in familial adenomatous polyposis patients [58,59]. AR-12 (Arno Therapeutics), an oral celecoxib analogue lacking cyclooxygenase 2 inhibitory activity, inhibits PDK1 and induces the endoplasmic reticulum stress pathway. Preclinical studies in various models indicated that AR-12 has antitumor activity. The first clinical trial on its safety and tolerability, which was recently completed, demonstrated limited drug absorption and PK variability [60]. Besides IKK, Akt and PDK1 also activate several other kinases (e.g. mTOR) that are important promoters of cancer progression, which may be indirectly inhibited by Akt and PDK1 inhibitors. This off-target activity may be advantageous from a therapeutic standpoint but obviously complicates the interpretation of the role of NF-kB inhibition. 4. Receptor-proximal kinases in NF-kB signaling 4.1. Multiple roads lead to IKK and IKKKs The family of IL-1 receptor associated kinases (IRAK) consists of four members, IRAK-1, IRAK-2, IRAK-M (IRAK-3) and IRAK-4, which have mainly been studied as scaffolds and kinases in IL-1 and TLR signaling [61]. Human IRAK-1, IRAK-2 and IRAK-4 are ubiquitously expressed, whereas human IRAK-M is induced only in monocytes and macrophages. All four kinases have an N-terminal death domain, a proST domain, a central serine/threonine kinase domain, and a C-terminal domain (except for IRAK-4, which lacks a C-terminal domain). The C-terminal domain is important for interaction with TRAF6, which then recruits the TAB/TAK1 complex to activate NF-kB. IRAKs are recruited to the receptor complex via their N-terminal death domain, which interacts with the death domain of the adaptor protein MyD88 or the death domain of other IRAK family members. IRAK-4 (and most likely also IRAK-1) is activated upon recruitment to MyD88, forming the oligomeric Myddosome, which leads to trans-autophosphorylation, as well as the recruitment and phosphorylation of other signaling molecules such as IRAK-2 and IRAK-M [62–64]. The relative contributions of the IRAK family members in IKK signaling and the role of their catalytic activity are still under debate. IRAKs can also contribute to the production of proinflammatory cytokines by mechanisms that are independent of NF-kB but involve MAPKs and changes in mRNA stability [61]. Moreover, IRAK-mediated IKK signaling also contributes to the production of

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type I IFN by mechanisms that are partially NF-kB independent. We will discuss only their roles in NF-kB signaling. Deletion of IRAK-1 in mice attenuates, but does not eliminate, IL-1/TLR-induced NF-kB activation. Moreover, the activation of the canonical IKK complex and cytokine production were not significantly impaired in bone-marrow-derived macrophages from mice expressing a catalytically inactive IRAK1[D359A] mutant [65], which is in line with earlier data showing that overexpression of an IRAK-1 kinase inactive mutant can still activate NF-kB [66]. However, IRAK-1 catalytic activity is critical for IKK activation and IKK-driven type 1 IFN production by plasmacytoid dendritic cells [65], illustrating a cell-type specific role of IRAK-1 catalytic activity. Studies on macrophages and plasmacytoid dendritic cells from IRAK-2 knockout mice or knockin mice expressing an IRAK-2 mutant that does not interact with TRAF6 revealed that IRAK-2 functions redundantly with IRAK-1 in early signaling but is important for late and sustained NF-kB activation [65]. The role of IRAK-2 kinase activity is still unclear and awaits the generation of knock-in mice expressing catalytically inactive IRAK-2. IRAK-M is the only member of the IRAK kinase family to lack kinase activity. Studies on IRAK-M deficient mice showed that IRAK-M functions mainly as a negative regulator of TLR signaling [67]. However, it was recently shown that IRAK-M mediates TLR7-induced MEKK3dependent second wave NF-kB activation [68]. IRAK-4 is the most recently discovered member of the IRAK family and IL-1/TLRinduced NF-kB activation is defective in cells from IRAK-4 knockout mice. However, IL-1/TLR-induced NF-kB activation is not significantly affected in mice with knock-in of inactive IRAK-4 kinase, although IL-1/TLR-induced cytokine and chemokine production is strongly reduced, mainly reflecting an impaired activation of JNK activation [69]. Interestingly, the role of the IRAK family is not restricted to IL-1/ TLR signaling. IRAK-1 and IRAK-2 have been shown to participate in the RIG-I antiviral pathway, leading to type 1 IFN production in VSV-infected macrophages [70]. Also IRAK-4 seems to play a critical role in T cell receptor signaling and is required for Th17 differentiation [71]. Interestingly, T cells isolated from IRAK-4 deficient mice or from patients are impaired in NF-kB activation upon TCR stimulation. Moreover, IRAK-4 impairs PKCu phosphorylation by interacting with zeta-chain associated protein kinase (ZAP) 70 [72]. Several PKC family members can act as receptor proximal kinases in NF-kB signaling. For example, PKCu-deficient T cells and PKCb-deficient B cells are impaired in the activation of NF-kB [73,74]. Upon antigen receptor stimulation, PKCu and PKCb phosphorylate the adaptor protein CARD-containing MAGUK protein (CARMA) 1 on multiple serine residues, which triggers the recruitment of BCL10/MALT1 and IKK activation. Also, Ca2+/ calmodulin-dependent kinase II (CaMKII) leads to IKK activation upon TCR triggering [75]. In this case, CaMKII phosphorylates BCL10, enhancing its interaction with CARMA1 [76]. TCR-induced NF-kB activation has also been claimed to involve Tpl-2 kinase [77], which can trigger phosphorylation of IKKb in its activation loop upon overexpression [78]. However, studies on Tpl-2 deficient splenocytes did not support a role for Tpl-2 in TCR- or LPS-induced NF-kB activation [79]. The role of glycogen synthase kinase (GSK)-3 in NF-kB signaling is very controversial. GSK-3 has been proposed to control promoter-specific recruitment of NF-kB upon cytokine stimulation and to enhance NF-kB transactivation via NF-kB p65 phosphorylation, with no effect on IKK activation [80,81]. In contrast, others reported that GSK-3 deletion results in complete loss of TNFinduced IKK activation [82] and that treatment of pancreatic cancer cells with a GSK-3 inhibitor (AR-A014418) reduces IKK activity [83]. On the other hand, GSK-3 has also been described as a negative regulator of NF-kB signaling. For example, Akt-mediated

inactivation of GSK-3 was shown to enhance NF-kB activation [84]. Also, by interacting with IKKg, GSK-3 inhibits TNF-induced NF-kB activation in primary astrocytes, thereby inducing the release of IKKa/b [85]. Rho-associated kinase (ROCK) has also been implicated in NF-kB signaling in a cell-type and stimulus-specific manner. For example, ROCK inhibition was shown to prevent IKK activation in IL-1- and LPS-stimulated PBMC [86] and in thrombin-stimulated endothelial cells [87]. Other studies showed no effect of ROCK inhibition on TNFstimulated endothelial cells or IL-1-stimulated intestinal epithelial cells [88,89]. Moreover, ROCK inhibition was shown to enhance IL-1induced IKK activity in vascular smooth muscle cells [90]. However, it should be mentioned that evidence for a role of ROCK is mainly based on the use of Y27632 (N-(4-pyridyl)-4-(1-aminoethyl)cyclohexane-carboxamide dihydrochloride), a non-specific inhibitor of ROCK that competes with ATP for binding to ROCK catalytic sites. This inhibitor also inhibits Rho-dependent protein kinase C, complicating the interpretation of the results obtained with this inhibitor. 4.2. Therapeutic targeting of receptor proximal kinases: The place to be? Of the four IRAK proteins, IRAK-4 is considered the most interesting target in inflammatory disease. Mutations resulting in IRAK-4 deficiency have been linked only to infections by specific pyogenic bacteria in children, with susceptibility decreasing upon approaching adolescence, and immunity to other microbial infections was not affected [91,92]. Elucidation of the threedimensional structure of the IRAK-4 catalytic domain has facilitated the development of several classes of potent and selective small-molecule IRAK-4 inhibitors (aminobenzimidazoles, thiazole or pyridine amides, imidazo[1,2-a]pyridines, imidazo[1,2b]pyridazines and benzimidazole-indazoles) (for an overview see [93]). Many of them are currently being tested for their antiinflammatory effect by academic and industrial labs in preclinical studies, but experimental data are not publicly available. IRAK-4 inhibitors may also be useful in the treatment of certain types of lymphoma. More specifically, IRAK-4 kinase activity is absolutely required for the survival of ABC-type diffuse large B cell lymphoma cells (DLBCL), which are dependent on constitutive NF-kB activity for proliferation and survival [94]. Although most efforts for therapeutic targeting have focused on IRAK-4, IRAK-1 may also be an interesting target. IRAK-1 deficiency in mice was shown to be beneficial in a number of autoimmune disease models, such as experimental autoimmune encephalomyelitis [95] and lupusassociated phenotypes [96]. Moreover, polymorphisms in the IRAK-1 gene have been associated with systemic lupus erythematosus in humans [96,97]. IRAK-1 was shown to be strongly expressed and activated in bone marrow cells derived from patients with myelodysplastic syndromes, and treatment of these cells with a small-molecule inhibitor of IRAK-1/-4 (Amgen) led to a dose-dependent reduction in NF-kB activation, which impaired cell proliferation and increased apoptosis [98]. Finally, a study reported by Roche suggested that pharmacologic inhibition of both IRAK-4 and IRAK-1 might be necessary to block pro-inflammatory cytokine production [99]. Moreover, because IRAKs not only mediate inflammatory signaling and cytokine production via NFkB but also through other mechanisms, as mentioned in the previous section, the anti-inflammatory effects observed with IRAK-1/-4 inhibitors most likely reflect much more than NF-kB inhibition. PKCu and PKCb are important mediators of TCR- and BCRinduced NF-kB signaling, respectively. PKCu-deficient mice are protected in several murine inflammatory disease models, including OVA-induced asthma [100,101], antigen-induced

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arthritis [102] and experimentally induced encephalomyelitis [103,104]. Absence of PKCu in mice also delays rejection of a cardiac allograft [105]. Together, these results point to the potential of immunosuppressive therapy based on PKCu inhibition. Sotrastaurin (AEB071, Novartis) is a pan-PKC inhibitor that blocks T cell activation [106] and originally showed promising results as immunosuppressant in the prevention of solid organ transplant rejection [107]. However, clinical trials were recently halted because of insufficient effectiveness. Sotrastaurin is now in phase I/II clinical trials for DLBCL and uveal melanoma and has also shown promise in the treatment of psoriasis [108]. Promising results were also obtained with the PKCb inhibitor Enzastaurin in a phase II trial in patients with relapsed DLBCL [109]. Because of the role of PKCs in multiple signaling pathways, it is difficult to conclude how much of the beneficial effects are due to inhibition of NF-kB signaling. Pharmacological inhibition of GSK-3 is protective in a number of disease models, including LPS-induced shock and lung inflammation in mice (reviewed in [110]). However, long-term use of GSK-3 inhibitors is unwise because it has been linked to increased bone formation via activation of osteoblast development through the Wnt pathway. Also, in some cases it increases the risk of cancer and cardiac disease. Since the role of GSK-3 in NF-kB signaling is very controversial (see above) and GSK-3 has many other downstream effectors in addition to NF-kB, anti-inflammatory effects obtained with GSK-3 inhibitors most likely reflect changes in multiple pathways, which implies the risk of side-effects. CaMKII has been implicated in IKK and NF-kB activation upon H. pylori infection of gastric epithelial cells [111], leading to inflammation and an increased risk of the development of gastric cancer [112]. The CaMKII inhibitor KN-93 impaired IKK activation by H. pylori. Interestingly, CaMKII activates IKK independently of PKC and the CBM complex, but requires both MEKK3 and TAK1 [113]. The ROCK kinase inhibitor Y27632 is protective in the trinitrobenzene sulfonic acid-induced colitis model in rats [86]. Also, Y27632 impaired NF-kB and JNK activity in PBMCs and IECs obtained from Crohn’s disease patients [86,88], suggesting that ROCK inhibition might be useful in the treatment of inflammatory bowel disease. ROCK inhibition might also be useful for pulmonary hypertension, since Y27632 was shown to decrease pulmonary vasoconstriction in different mouse models [114]. However, as already mentioned, one should keep in mind that Y27632 is not a very selective ROCK kinase inhibitor. In contrast, the more selective ROCK inhibitor Fasudil was also shown to be more effective in prevention of pulmonary hypertension. Fasudil is also protective in several mouse models of neurodegeneration and is the first ROCK inhibitor to be approved for the clinical treatment of ischemiainduced brain damage.

Therapeutic targeting of the NF-kB pathway may still have a future if one can find much more specific approaches. In this context, targeting of receptor proximal kinases that are unique to a subset of receptors and that activate IKKs can be successful. For instance, inhibition of IRAKs would block IL-1 and TLR signaling, while leaving TNF signaling intact. This approach might eventually lead to more disease specific treatments, as different autoimmune disorders are driven by different cytokines. This is also reflected for instance in the effectiveness of anti-TNF treatment in certain diseases, like Crohn’s disease, rheumatoid arthritis and psoriasis, while it is not effective for the treatment of systemic lupus erythematosus. As anti-TNF and other antibody-based therapies still have significant shortcomings such as high production costs, non-responsiveness of some patients, immunogenicity, and risks of infection and malignancy, inhibition of receptor proximal kinases through small-compound inhibitors might resolve at least some of these issues. The NF-kB pathway has served as a paradigm for inflammatory signaling and resulted in the discovery of multiple new kinases that directly or indirectly affect IKKs and that are interconnected with each other or with other kinase pathways, such as the MAP kinase pathway. Moreover, kinome-wide screens are revealing new kinases that affect NF-kB signaling. Therefore, more comprehensive knowledge on the role and mechanism of action of NF-kB regulatory kinases is needed for the development of novel therapeutics and to prevent unexpected and adverse effects of kinase inhibitors. Gene targeting in mice is important for elucidating the physiological role of a specific protein kinase, and the increasing availability of conditional knock-out mice is invaluable for validation of specific kinases as targets for drug development and for prediction of side effects. Furthermore, the use of knock-ins of catalytically inactive kinases will be essential to reveal whether the knockout phenotype is caused by ablation of the specific catalytic activity of a protein kinase or by loss or alteration of a scaffolding function. The availability of novel gene targeting techniques such as CRISPR/Cas makes such approaches feasible. Also the growing number of available three-dimensional structures of protein kinases will finally make it possible to carefully and iteratively design potent and specific protein kinase inhibitors by structure-based lead optimization. In addition to the use of inhibitors of catalytic activity, alternative strategies that affect specialized protein interaction domains that mediate the docking of kinases to their activators, inactivators and substrates are gaining interest. The rapidly mounting knowledge of NF-kB signaling combined with remarkable technological advances facilitate identification of novel small-molecule drug candidates. However, for treatments of chronic inflammation the way to the clinic is difficult because any new drug should be more effective and have fewer side effects and superior toxicity profile than available treatments.

5. Conclusions and future perspectives

Acknowledgements

Enthusiasm for therapeutic targeting of the NF-kB pathway has dropped substantially in recent years because of the lack of efficacy and the presence of adverse, non-tolerable side effects in clinical studies on existing IKK inhibitors. This is expected if one takes into account the role of NF-kB in many physiological processes. Moreover, since their original discovery as kinases responsible for IkB phosphorylation, IKKs have been found to exert many pleiotropic activities not related to NF-kB. In addition to these ontarget effects, several inhibitors may also suffer from off-target effects that contribute to toxicity and side effects. Although IKK inhibitors so far failed to reach the clinic, their use in cellular studies and animal models of inflammatory disease at least provided new insights into the regulation of inflammatory signaling networks.

L.V. holds a FWO postdoctoral fellowship. Research in the authors’ lab is supported by grants from the ‘‘Interuniversity Attraction Poles program’’ (IAP7/32), the FWO (G090914N, G027413N, G016413N, 1509712N, G046612N, G028712N), the IWT (SBO 110028), the ‘‘Belgian Foundation against Cancer’’, and the UGent (BOF13/GOA/005 and Group-ID MRP). References [1] Sun SC, Chang JH, Jin J. Regulation of nuclear factor-kappaB in autoimmunity. Trends Immunol 2013;34:282–9. [2] DiDonato JA, Mercurio F, Karin M. NF-kappaB and the link between inflammation and cancer. Immunol Rev 2012;246:379–400. [3] Sun SC. Non-canonical NF-kappaB signaling pathway. Cell Res 2011;21: 71–85.

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