TLR and IRAK

Toll-Like Receptors and Interleukin-1 Receptor Kinases Technical Bulletin

Mediators of Chronic Inflammation in Autoimmunity and Cancer Source: Allan Mah, PhD. Product Manager - SignalChem

Toll-like receptors (TLRs) are membrane bound receptor proteins that are essential for activating innate and active immune responses. TLRs are members of the pattern recognition receptor (PRR) superfamily, which are conserved from plants to animals, thus representing evolutionarily ancient alert and response system against invading organisms [1]. PRRs identify sets of distinct and conserved molecules known as pathogen- associated molecular patterns (PAMPs). Many of these recognized molecules are pathogen-derived or released as a consequence of cellular stress. The extracellular domains of TLRs contain multiple leucine-rich repeats (LRRs); these are required to form the binding surface for PAMP interaction. Upon pathogen-recognition, TLRs dimerize and their cytosolic Toll/IL- receptor (TIR) domains recruit adaptor proteins, such as myeloid differentiation primary response gene 88 (MyD88) or TIR-domain- Figure 1: TLR/IRAK Signaling is a pro-inflammatory pathway linked to autoimmune disease and cancers. containing adapter-inducing interferon-β (TRIF). These adaptors are essential, as TLRs themselves lack enzymatic activity. well established roles as protein kinases. In contrast, IRAK2 and Nine out of ten of the human TLRs signal through the MyD88- IRAK3 appear to function mainly as regulatory proteins in protein dependent pathway. TLR3 stands out as the exception, because complexes. Following TLR stimulation, MyD88 recruits IRAK2 it relies exclusively on the TRIF-dependent pathway. TLR4 is and IRAK4 to the cell membrane. IRAK4 then phosphorylates also unique because it is able to signal through both pathways. IRAK1, which enables IRAK1 to autophosphorylate and join the Overall, MyD88 and TRIF stimulate independent downstream membrane-bound complex. IRAK1 then targets tumor necrosis pathways, which lead to the activation of partially overlapping factor receptor-associated factor 6 (TRAF6), an adaptor with sets of transcription factors (TFs), including activator protein 1 E3 ubiquitin ligase activity. Phosphorylated TRAF6 joins the (AP-1), nuclear factor (NF)-kB and interferon response factors membrane complex where it oligomerizes; in conjunction with (IRFs). These TFs are responsible for the expression of various the E2 ubiquitin ligase Ubc13/Uev1A, TRAF6 polyubiquitinates inflammatory cytokines, thus playing important roles in many several targets including itself and the negative regulatory subunit biological processes including innate and adaptive immunity [2]. of the IκB kinase (IKK) complex, IKK gamma/NEMO. Ubiquitinated TRAF6 then leaves the receptor complex to a new enzyme The interleukin-1 receptor kinases (IRAKs) are the primary complex with TAK1-TAB1 (or TAB2). This TRAF6/TAK1-TAB1 downstream effectors of the MyD88-dependent pathway [3]. complex is responsible for phosphorylating IKK beta, resulting in Humans have four IRAK proteins: IRAK1, IRAK2, IRAK3 (IRAK-M) full activation of the IKK complex, an essential regulator of NF-κB and IRAK4. IRAKs are pro-inflammatory factors, with the activity [5]. NF-κB is normally inactive in the cytoplasm where it exception of IRAK3, which participates in a negative feedback loop is bound to its inhibitory subunit. The IKK complex regulates the to regulate innate immune responses [4]. IRAK4 and IRAK1 have removal of IκBα, enabling the TF to translocate to the nucleus

www.signalchem.com Tel: 1.866.954.6273 [email protected] where it activates genes responsible for inflammatory responses cancers, such as: melanoma, non-squamous cell lung cancer and cell survival. TLR signaling is sensitive to perturbation (NSCLC), leukemia and lymphoma [12]. Likewise, dysregulated and therefore, it is frequently implicated in disease such as IRAK4 is also implicated in the development of leukemia and autoimmune disorders and cancers. lymphoma [12].

IRAKs in Autoimmune Diseases Interestingly, mutations in IRAK genes are rarely found in cancers, with only a couple that are presently identified. One of these is IRAKs are indispensable for pro-inflammatory signal IRAK2 (R214G), a mutant that prevents NF-κB activation, thus transduction, because they are the first group of kinases that contributing to the survival of colorectal cancer (CRC) patients are activated following TLR stimulation. Due to their pivotal roles [14]. The R214G mutation falls within the kinase domain of IRAK2 in activating downstream signaling components, deficiencies raising questions as to whether the protein functions as a in IRAK1 or IRAK4 lead to reduced responses to PAMPs or a kinase in CRC. Despite the need for further investigation into its complete attenuation of TLR signaling [6]. For instance, loss of mechanism, the IRAK2 (R214G) mutant represents a promising IRAK4 causes rheumatoid arthritis RA-sensitive mice to become biomarker in CRC patients, which would enable physicians to resistant to various RA-stimulating conditions [7]. This protection better weigh out therapeutic options. IRAK1 (F196S) is another from developing RA has prompted scientists to create IRAK4- mutation that is implicated in cancer. This amino acid substitution targeted inhibitors for the treatment of autoimmune diseases constitutively activates the kinase, which plays a vital role in such as collagen-induce arthritis (CIA), imiquimod-induced the survival of primary effusion lymphoma (PEL) cells. PEL is psoriasis and mono-sodium urate (MSU) gout [8]. In addition, a devastating form of lymphoma that threatens patients who IRAK1 is emerging as an important marker for systematic lupus already suffer from underlying immunodeficiencies, such as erythematosus (SLE), a disease that largely affects females. In HIV [15, 16]. Moreover, PEL is resistant to chemotherapy drugs the past, the gender bias of SLE was largely attributed to the traditionally used against other types of lymphomas, making role of sex hormones. However, recent studies have identified IRAK1 (F196S) a promising targets pharmaceutical development X-linked genetic polymorphisms in the IRAK1 gene, suggesting [16]. With TLR signaling as a causal link between inflammation that defective TLR signaling could play a larger role in the and cancer, there is a strong motivation to develop therapeutics development of SLE [9, 10, 11]. that modulate this pathway. IRAK1 and IRAK4 are clear targets for drug design because of their pivotal roles for transducing pro- IRAKs in Cancer inflammatory signals. Several chemical inhibitors of IRAKs are already in pre-clinical stages, including: imidazo[1,2-a]pyridino- Chronic inflammation is implicated in cancer initiation, tumor pyridines, benzimidazole-pyridine derivatives, ND-346, ND- progression and drug resistance. Due to its role in regulating 2110, and ND-2158 and the ginseng-derived Ginsenoside Rb1 pro-inflammatory responses, TLR/IRAK signaling is an active [17]. These efforts and emerging drug development programs area of study in oncology research. For instance, IRAK1 function will lead to effective options to treat various diseases, including is implicated in Helicobacter pylori-stimulated gastric cancer; the underserved and aggressive cancers such as PEL. strongest known risk factor for developing this disease. H. pylori is found in approximately a half of the world’s population, and it SignalChem Pharmaceuticals manufactures products to has been suggested that this bacteria is a normal component of support the development of new cancer therapies through the gut flora. Interestingly, H. pylori does not cause symptoms the manufacturing production of high quality and consistent in most individuals, but the infection is the leading cause of biological reagents. Along with proteins involved directly with the peptic ulcers; a chronic inflammatory condition that triggers the TLR/IRAK signaling, our offerings include a wide range of active overexpression of TLR2 and TLR5 in the gut resulting in amplified wild-type and mutant kinases, cell signaling proteins, enzyme signaling. The sustained IRAK1 phosphorylation the promotes inhibitors, siRNAs, antibodies, growth factors and other active the expression of tumor promoting cytokines and cell survival enzymes. genes [12, 13]. Elevated IRAK1 activity is also observed in other

www.signalchem.com Tel: 1.866.954.6273 [email protected] References: 1. Roach JM et al., Phylogeny of Toll-like receptor signaling: adapting 10. Zhai Y et al., Association of interleukin-1 receptor-associated kinase the innate response. PLoS One. 2013;8(1):e54156. doi: 10.1371/journal. (IRAK1) gene polymorphisms (rs3027898, rs1059702) with systemic pone.0054156. lupus erythematosus in a Chinese Han population. Inflamm Res. 2013 2. Piras V and Selvarajoo K., Beyond MyD88 and TRIF Pathways in Toll- Jun;62(6):555-60. Like Receptor Signaling. Front Immunol. 2014 Feb 24;5:70. 11. Jacob CO et al., Identification of IRAK1 as a risk gene with critical 3. Chen JQ et al., The critical role of Toll-like receptor signaling pathways role in the pathogenesis of systemic lupus erythematosus. Proc Natl in the induction and progression of autoimmune diseases. Curr Mol Med. Acad Sci U S A. 2009 Apr 14;106(15):6256-61. 2009 Apr;9(3):365-74. 12. Kumar P et al., Induction of TLR-2 and TLR-5 expression by 4. Kobayashi K et al., IRAK-M is a negative regulator of Toll-like receptor Helicobacter pylori switches cagPAI-dependent signalling leading to signaling. Cell. 2002 Jul 26;110(2):191-202. the secretion of IL-8 and TNF-α. PLoS One. 2011 May 9; 6(5):e19614. 5. Chen ZJ., Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol. 13. Jain A et al., IL-1 Receptor-Associated Kinase Signaling and Its Role 2005 Aug;7(8):758-65. in Inflammation, Cancer Progression, and Therapy Resistance. Front 6. Suzuki N et al., Severe impairment of interleukin-1 and Toll-like receptor Immunol. 2014 Nov 17;5:553. signalling in mice lacking IRAK-4. Nature. 2002 Apr 18;416(6882):750-6. 14. Wang H et al., A coding IRAK2 protein variant compromises Toll- 7. Koziczak-Holbro M et al., The critical role of kinase activity of like receptor (TLR) signaling and is associated with colorectal cancer interleukin-1 receptor-associated kinase 4 in animal models of joint survival. J Biol Chem. 2014 Aug 15;289(33):23123-31. inflammation. Arthritis Rheum. 2009 Jun;60(6):1661-71. 15. Yang D et al., Interleukin 1 receptor-associated kinase 1 (IRAK1) 8. Bahia MS et al., Interleukin-1 receptor associated kinase inhibitors: mutation is a common, essential driver for Kaposi sarcoma herpesvirus potential therapeutic agents for inflammatory- and immune-related lymphoma. Proc Natl Acad Sci U S A. 2014 Nov 4;111(44):E4762-8. disorders. Cell Signal. 2015 Jun;27(6):1039-55. 16. Chen YB et al., Primary effusion lymphoma. Oncologist. 2007 9. Zhang Y et al., Meta-analysis of GWAS on two Chinese populations May;12(5):569-76. followed by replication identifies novel genetic variants on the X 17. Rhyasen GW and Starczynowski DT., IRAK signalling in cancer. Br J chromosome associated with systemic lupus erythematosus. Hum Mol Cancer. 2015 Jan 20;112(2):232-7. Genet. 2015 Jan 1;24(1):274-84..