Interleukin-1 Receptor-associated Kinase 4 Kinase Research Articles

Discovery of LC-MI-3: A Potent and Orally Bioavailable Degrader of Interleukin-1 Receptor-Associated Kinase 4 for the Treatment of Inflammatory Diseases.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a promising therapeutic target in inflammation-related diseases. However, the inhibition of IRAK4 kinase activity may lead to moderate anti-inflammatory efficacy owing to the dual role of IRAK4 as an active kinase and a scaffolding protein. Herein, we report the design, synthesis, and biological evaluation of an efficient and selective IRAK4 proteolysis-targeting chimeric molecule that eliminates IRAK4 scaffolding functions. The most potent compound, LC-MI-3, effectively degraded cellular IRAK4, with a half-maximal degradation concentration of 47.3 nM. LC-MI-3 effectively inhibited the activation of downstream nuclear factor-κB signaling and exerted more potent pharmacological effects than traditional kinase inhibitors. Furthermore, LC-MI-3 exerted significant therapeutic effects in lipopolysaccharide- and Escherichia coli-induced acute and chronic inflammatory skin models compared with kinase inhibitors in vivo. Therefore, LC-MI-3 is a candidate IRAK4 degrader in alternative targeting strategies and advanced drug development.

Pharmacological inhibition of IRAK4 kinase activity does not prevent cachexia in mice with pancreatic cancer

AbstractBackgroundInflammation is a hallmark of cachexia; however, effective anti‐inflammatory treatments have not yet been identified. Interleukin‐1 receptor‐associated kinase 4 (IRAK4) is a key signalling node linking interleukin‐1 receptor (IL‐1R) and toll‐like receptor (TLR) activation to the production of multiple proinflammatory cytokines that are elevated in cancer cachexia. The purpose of this work is to evaluate whether pharmacological inhibition of IRAK4 kinase activity with PF‐06426779 could prevent cachexia using a model of pancreatic cancer. The effect of appetite stimulation via the ghrelin receptor agonist anamorelin was also examined as a benchmark of clinically validated mechanisms.MethodsFemale C57Bl/6J mice were given an intraperitoneal injection of KrasG12D; p53R172H; Pdx1‐Cre (KPC) pancreatic tumour cells. PF‐06426779 or anamorelin treatment was initiated at the onset of anorexia. Body weight and food intake were measured throughout the study. Body composition, muscle function (force), and physical activity (treadmill running endurance) were assessed at the end of the study.ResultsChronic treatment with PF‐06426779, at doses covering in vitro IC50 and IC90 at Cmin, did not increase body weight, food intake, and muscle function in the KPC tumour model. In contrast, anamorelin (vs. vehicle) increased food intake (P < 0.01), hindlimb skeletal muscle mass (P < 0.01), and muscle strength (P < 0.05); however, treadmill running endurance was not increased.ConclusionsThese data suggest that inhibition of IRAK4 kinase activity is not sufficient to treat cachexia, at least in pancreatic cancer, and exploration of alternative anti‐inflammatory strategies that increase appetite is required.

Deciphering the intrinsic dynamics of unphosphorylated IRAK4 kinase bound to type I and type II inhibitors

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a vital protein involved in Toll-like and interleukin-1 receptor signal transduction. Several studies have reported regarding the crystal structure, dynamic properties, and interactions with inhibitors of the phosphorylated form of IRAK4. However, no dynamic properties of inhibitor-bound unphosphorylated IRAK4 have been previously studied. Herein, we report the intrinsic dynamics of unphosphorylated IRAK4 (uIRAK4) bound to type I and type II inhibitors. The corresponding apo and inhibitor-bound forms of uIRAK4 were subjected to three independent simulations of 500 ns (total 1.5 μs) each, and their trajectories were analyzed. The results indicated that all three systems were relatively stable, except for the type II inhibitor-bound form of uIRAK4, which exhibited less compact folding and higher solvent surface area. The intra-hydrogen bonds corroborated the structural deformation of the type-II inhibitor-bound complex, which could be attributed to the long molecular structure of the type-II inhibitor. Moreover, the type II inhibitor bound to uIRAK4 showed higher binding free energy with uIRAK4 than the type I inhibitor. The free energy landscape analysis showed a reorientation of Phe330 side chain from the DFG motif at different metastable states for all the systems. The intra-residual distance between residues Lys213, Glu233, Tyr262, and Phe330 suggests a functional interplay when the inhibitors are bound to uIRAK4, thereby hinting at their crucial role in the inhibition mechanism. Ultimately, the intrinsic dynamics study observed between type I/II inhibitor-bound forms of uIRAK4 may assist in better understanding the enzyme and designing therapeutic compounds.

Selective IRAK4 degradation, not kinase inhibition, blocks TLR-activated NF-Kb and p38 Signaling leading to broad cytokine inhibition

Abstract IL-1R/TLR activation plays a central role in the pathophysiology of multiple autoimmune and inflammatory diseases via the Myddosome that is dependent on both the kinase and scaffolding functions of Interleukin-1 receptor associated kinase 4 (IRAK4). Therefore, hetero-bifunctional molecules that selectively target IRAK4 for degradation and elimination by the ubiquitin proteasome pathway have the greatest potential to block IL-1R/TLR signaling, including NF-kb activation and cytokine production. To interrogate downstream signaling, phosphorylation events were monitored in monocytes and B cells following TLR7/8 or TLR9 activation, respectively. IRAK4 degradation, but not kinase inhibition, inhibited NF-kb p65 activation and phosphorylation of p38 pathway members by TLR agonists in both cell types. PBMCs pretreated with an IRAK4 degrader and then stimulated with the TLR7/8 agonist, R848, exhibited significantly broader inhibition of cytokines (IL-6, TNFa, IL-8 and IL-1b) compared to those pretreated with a selective IRAK4 kinase inhibitor. Compound washout experiments demonstrated a sustained effect of IRAK4 degrader on both target pharmacodynamics and cytokine inhibition that was differentiated from IRAK4 kinase inhibition. Overall, through removal of both scaffolding and kinase functions, IRAK4 degradation demonstrated greater activity compared to kinase inhibition across multiple TLR stimuli and cell types. These data highlight the potential for IRAK4 degraders to block multiple TLR signaling pathways across different immune cell types and thereby impact TLR/IL-1R-driven inflammatory and autoimmune diseases.

Proteomic Analysis Reveals IRAK4 As a Therapeutic Target in Chronic Lymphocytic Leukemia

Dimethylfumarate (DMF) and other electrophilic compounds are highly toxic to CLL cells in vitro and in xenograft animal models [Wu et al., 2010; Wu et al., 2016]. To study the mechanism of action, competitive isotopic tandem Orthogonal Proteolysis Activity Based Protein Profiling (isoTOP ABPP) - a chemical proteomic platform, which utilizes broad-spectrum cysteine reactive probes, - was applied to quantitatively map DMF protein targets in whole proteomes. Cells from 10 CLL patients were treated with 30 μM DMF for 4h followed by an isoTOP ABPP sample preparation and analysis workflow. Based on reproducible high isoTOP ABPP ratios across multiple biological replicates, cysteine 13 of interleukin-1 receptor-associated kinase 4 (IRAK4) was identified as a prominent target of DMF in CLL patient cells.IRAK4 plays a critical role in Toll-like receptor (TLR) signal transduction. Engagement of IRAK4 with the signaling adaptor protein MyD88 in TLRs activates downstream NF-κB and type-I IFN pathways. Approximately 15-20% of CLL patients have dominant activating mutations of MyD88, and nearly all patients with the CLL-related disease Waldenstrom's Macroglobulinemia have such mutations. Hence, we sought to evaluate known inhibitors of IRAK4 kinase activity for their effect on CLL cells. Multiple IRAK4 inhibitors are currently in clinical development, primarily for the treatment of autoimmune conditions. We used PF06650833, a specific inhibitor of IRAK4, previously tested in phase 1 trials for patients with systemic lupus erythematosus, to evaluate its effect on CLL cells viability. In vitro , PF06650833 reduced CLL cell viability in a dose-dependent manner. Reduction in IRAK4 and IκBα protein levels were also observed in samples treated with at least 15 µM PF06650833 by Western blot analysis. In vivo studies provide a better opportunity to evaluate the effect of this drug in blocking microenvironment signaling. We utilized a murine xenograft model in which primary CLL cells are transplanted into the peritoneum of Rag2/gamma chain immunodeficient mice. Each mouse received a single intraperitoneal injection of 1-10 mg/kg PF06650833 one day after transplantation. Cells were recovered 6 days after the treatment and analyzed by flow cytometry, showing remarkable reduction in CLL survival in a dose-dependent manner.In conclusion, unbiased proteomics analysis of a non-specific drug, DMF, identified IRAK4 as a target in cells from CLL patients. While the exact mode of action for DMF and PF06650833 might differ (disruption of higher IRAK4 complexes, inhibition of kinase dependent signaling or other), our results suggest that IRAK4 represents a potential druggable target in the management of patients with CLL. [Display omitted] DisclosuresChoi:Gilead, Genentech, Abbvie, and PCYC: Speakers Bureau; AbbVie: Other: Institutional research funding; AbbVie, Genentech, and PCYC: Consultancy.

Design, Synthesis, and Biological Evaluation of IRAK4-Targeting PROTACs.

Interleukin-1 receptor associated kinase 4 (IRAK4) is a promising therapeutic target for diffuse large B-cell lymphoma driven by MYD88 L265P mutant, acting both as a kinase and a scaffolding protein for downstream signaling molecules. While previous efforts to modulate IRAK4 activity with kinase inhibitors alone displayed moderate efficacy, protein degradation may offer a solution to blocking both IRAK4 kinase activity and scaffolding capabilities. To this end, the potent IRAK4 degrader 9 was discovered, and it effectively inhibited the activation of downstream NF-κB signaling and outperformed the parent compound 1. In addition, compound 9 displayed a substantial advantage in reduction of the viability of OCI-LY10 and TMD8 cells over the parent compound 1. These results underline the potential that eliminating both the kinase and scaffolding functions of IRAK4 may result in superior and broader efficacy than inhibiting the kinase activity alone.

From the Editor's Desk…

From the Editor's Desk…

A Phase 1, Open Label Dose Escalation Trial Evaluating the Safety, Pharmacokinetics, Pharmacodynamics, and Clinical Activity of Orally Administered CA-4948 in Patients with Acute Myelogenous Leukemia or Myelodysplastic Syndrome

Introduction: CA-4948 is a novel small molecule oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4). IRAK4 kinase plays an essential role in toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways These pathways are frequently dysregulated in Non-Hodgkin Lymphomas (NHL) and AML/MDS. [1] Oncogenic IRAK4-L, which is driven by spliceosome mutation (including SF3B1 and U2AF1), is preferentially expressed in >50% of AML/MDS patients [2,3]. Recently, activated IRAK4 has been identified as a driver of adaptive resistance in AML and other tumors [4]. CA-4948 not only strongly inhibits IRAK4, but also FLT3-ITD AML in vitro and in-vivo models. It has shown safety and activity in patients with relapsed or refractory NHL and will now be evaluated in patients with high-risk MDS and AML. Study Design and Methods: This is a multicenter, open-label, single arm Phase 1 dose escalation study of orally administered CA-4948 monotherapy in adult patients with AML or high risk MDS (NCT04278768). This study will be conducted in 2 parts: an initial dose escalation and dose expansion phase. The starting dose level is 200 mg BID which was determined to be safe, capable of achieving relevant levels of drug exposure and has demonstrated signs of biologic activity and clinical efficacy in an ongoing NHL study CA-4948-101. Three patients with AML or MDS will be enrolled at the starting dose. If none of the first 3 patients experience a DLT during the first cycle, patients may be enrolled into the next higher dose level of 300 mg BID. Dose escalation is expected to enroll approximately 18 patients to establish the initial RP2D. The safety population will include all patients in the study who received any dose of CA-4948, and the efficacy population will include patients who have a valid baseline and post-baseline disease assessment and received at least one dose of the study drug. Each treatment cycle of CA-4948 will be 28 days in length and repeated in the absence of unacceptable toxicity or disease progression. The major study inclusion and exclusion criteria are as follows: Relapsed or refractory AML (primary or secondary, including treatment-related) after at least one standard treatment (including chemotherapy, re-induction therapy or stem cell transplantation) based on the assessment of the investigator or high/very high risk relapsed/refractory MDS (IPSS-R criteria), following at least 6 cycles of hypomethylating agents [HMA] or evidence of early progression. Patients diagnosed with acute promyelocytic leukemia (APL, M3), blast phase of CML, allogeneic hematopoietic stem cell transplant (Allo-HSCT) within 60 days of the first dose of CA-4948, or clinically significant graft-versus-host disease (GVHD) requiring ongoing up-titration of immunosuppressive medications prior to start of CA-4948 are excluded. The primary objective is to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D) for CA-4948 based on the safety and tolerability, DLTs and PK/PD findings. Secondary objective is characterization of the pharmacokinetic (PK) parameters and preliminary efficacy. Exploratory objective (1)is to assess the potential association between target-related biomarkers (including IRAK4-L and downstream signaling parameters), selected genetic mutations (including spliceosome mutations), gene expression signatures, cell of origin, or other molecular classification subtypes and anti-leukemic activity (including central morphology review), and (2) to assess the pharmacodynamic effects of CA-4948 on selected biomarkers in peripheral blood and bone marrow. As of 03 August 2020, the trial has enrolled 3 patients in Cohort 1. Clinical updates will be later reported. After the RP2D has been determined, it may be subsequently amended including patient selection and combination therapy in a controlled design.

Inhibition of IRAK4 kinase activity improves ethanol-induced liver injury in mice

Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Interleukin-1 receptor associated kinase 4 (IRAK4), the master kinase of Toll-like receptor (TLR)/IL-1R-mediated signalling activation, is considered a novel therapeutic target in inflammatory diseases, but has not been investigated in the context of ALD. IRAK4 phosphorylation and IRAK1 protein were analysed in liver from alcohol-related hepatitis patients and healthy controls. IRAK4 kinase activity-inactive knock-in (Irak4 KI) mice and bone marrow chimeric mice were exposed to chronic ethanol-induced liver injury. IL-1β-induced IRAK4-mediated signalling and acute phase response were investigated in cultured hepatocytes. IRAK1/4 inhibitor was used to test the therapeutic potential for ethanol-induced liver injury in mice. Increased IRAK4 phosphorylation and reduced IRAK1 protein were found in livers of patients with alcoholic hepatitis. In the chronic ethanol-induced liver injury mouse model, hepatic inflammation and hepatocellular damage were attenuated in Irak4 KI mice. IRAK4 kinase activity promotes expression of acute phase proteins in response to ethanol exposure, including C-reactive protein and serum amyloid A1 (SAA1). SAA1 and IL-1β synergistically exacerbate ethanol-induced cell death exvivo. Pharmacological blockage of IRAK4 kinase abrogated ethanol-induced liver injury, inflammation, steatosis, as well as acute phase gene expression and protein production in mice. Our data elucidate the critical role of IRAK4 kinase activity in the pathogenesis of ethanol-induced liver injury in mice and provide preclinical validation for use of an IRAK1/4 inhibitor as a new potential therapeutic strategy for the treatment of ALD. Herein, we have identified the role of IRAK4 kinase activity in the development of alcohol-induced liver injury in mice. Hepatocyte-specific IRAK4 is associated with an acute phase response and release of proinflammatory cytokines/chemokines, which synergistically exacerbate alcohol-induced hepatocyte cell death exvivo. Pharmacological inhibition of IRAK4 kinase activity effectively attenuates alcohol-induced liver injury in mice and could have therapeutic implications.

TLR7 Protein Expression in Mild and Severe Lupus-Prone Models Is Regulated in a Leukocyte, Genetic, and IRAK4 Dependent Manner.

The global increase in autoimmunity, together with the emerging autoimmune-related side effects of cancer immunotherapy, have furthered a need for understanding of immune tolerance and activation. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disease, affecting multiple organs, and tissues. Studying SLE creates knowledge relevant not just for autoimmunity, but the immune system in general. Murine models and patient studies have provided increasing evidence for the innate immune toll like receptor-7 (TLR7) in disease initiation and progression. Here, we demonstrated that the kinase activity of the TLR7-downstream signaling molecule, interleukin-1 receptor associated kinase 4 (IRAK4), is essential for mild and severe autoimmune traits of the Sle1 and Sle1-TLR7 transgenic (Sle1Tg7) murine models, respectively. Elimination of IRAK4 signaling prevented all pathological traits associated with murine lupus, including splenomegaly with leukocyte expansion, detectable circulating antinuclear antibodies and glomerulonephritis, in both Sle1 and Sle1Tg7 mice. The expansion of germinal center B cells and increased effector memory T cell phenotypes that are typical of lupus-prone strains, were also prevented with IRAK4 kinase elimination. Analysis of renal leukocyte infiltrates confirmed our earlier findings of an expanded conventional dendritic cell (cDC) within the kidneys of nephritic mice, and this was prevented with IRAK4 kinase elimination. Analysis of TLR7 at the protein level revealed that the expression in immune cells is dependent on the TLR7-transgene itself and/or autoimmune disease factors in a cell-specific manner. Increased TLR7 protein expression in renal macrophages and cDCs correlated with disease parameters such as blood urea nitrogen (BUN) levels and the frequency of leukocytes infiltrating the kidney. These findings suggest that controlling the level of TLR7 or downstream signaling within myeloid populations may prevent chronic inflammation and severe nephritis.

Targeting IRAK4 for Degradation with PROTACs.

Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) is a key mediator of innate immunity. IRAK4 overactivation is linked with several autoimmune diseases. To date, many IRAK4 inhibitors have been developed to block the protein's kinase activity with the most advanced reaching Phase II clinical trials. Nevertheless, several reports suggest kinase activity is not disease-relevant in certain cell types, so removing scaffolding signaling in addition to IRAK4 kinase activity may offer a better therapeutic outcome. Herein, we describe the design and synthesis of an IRAK4 Proteolysis Targeted Chimera (PROTAC). We show that IRAK4 degradation induced by compound 9 leads to the inhibition of multiple cytokines in PBMCs. However, in IL-1β stimulated human dermal fibroblasts, inhibition of IL-6 and TNF-α release was not observed despite IRAK4 degradation. Nonetheless, the possibility of targeting both IRAK4 kinase and scaffolding function could potentially lead to new therapeutic opportunities to treat autoimmune, inflammatory, and oncological diseases.

PS991 CA‐4948, AN IRAK4/FLT3 INHIBITOR, SHOWS ANTILEUKEMIC ACTIVITY IN MOUSE MODELS OF FLT3 WILD‐TYPE AND FLT3 MUTATED ACUTE MYELOID LEUKEMIA (AML)

Background:AML is an aggressive hematopoietic malignancy that arises from a population of aberrant hematopoietic stem cells in the bone marrow (BM). Advances in understanding the molecular basis of AML has led to the development of new targeted therapies. CA‐4948 is a novel, oral IRAK4 kinase inhibitor with additional inhibitory activity against wild‐type (wt) and mutated FLT3 kinase. IRAK4 (Interleukin‐1 Receptor Associated Kinase 4) is required for interleukin 1 receptor (IL‐1R) and toll‐like receptor (TLR) innate immune pathway signaling, pathways that are frequently over activated in AML and myelodysplastic syndromes (MDS). For example, AML patients have increased IL‐1R agonist (IL‐1ß) levels that promote the survival of AML cells and IL‐1R KO represses AML cell growth in vitro and in vivo (Carey et al 2017). Dysregulation of the FLT3 signaling pathway is a well validated driver of AML. Constitutively activating mutations in FLT3 that comprise the ITD or the tyrosine kinase domain (KD) are frequently acquired late in AML disease and are poor prognostic factors with high relapse rates. FLT3 kinase inhibitors targeting FLT3‐ITD or ITD/KD double mutations show high remission rates; however, multiple resistance mechanisms have been reported in both nonclinical models and AML patients. CA‐4948 has both IRAK4 and FLT3 inhibitory activity, which may impart benefit to FLT3‐wt and FLT3‐mutant AML patients.Aims:Evaluate the ability of a novel IRAK4/FLT3 inhibitor, CA‐4948, to block IRAK4 and FLT3 in FLT3‐wt and FLT3‐mutant AML in vitro and in vivo.Methods:CA‐4948's kinome profile was assessed against 378 kinases and 9 FLT3 mutant variants (DiscoverX). CA‐4948's ability to inhibit TLR/IL‐1R or FLT3 signaling pathways was evaluated using NF‐kB reporter, cytokine production, or western blot. The growth inhibitory and pro‐apoptotic activity of CA‐4948 was tested in vitro against AML cell lines by viability assay or flow cytometry. For AML FLT3‐wt in vivo efficacy, THP‐1 cells were tail‐vein injected into mice and animal survival and degree of AML cell engraftment in BM were monitored in CA‐4948, FLT3i, or vehicle treated mice. For AML FLT3 mutant in vivo efficacy, subcutaneous MV4–11 and MOLM‐14 FLT3‐ITD and MOLM‐14 double FLT3‐ITD/KD tumor models were treated with CA‐4948 or other FLT3i.Results:CA‐4948 exhibited 23 nM (Kd) binding affinity for IRAK4, >500‐fold selectivity against IRAK1, and high binding affinity for FLT3‐wt, ‐ITD, ‐ITD/D835 V, and ‐ITD/F691L at 8–31 nM (Kd). CA‐4948 blocked cellular TLR and IL‐1R‐stimulated signaling from 150–220 nM (IC50). Consistent with CA‐4948's additional FLT3 inhibitory activity, CA‐4948 induced apoptosis and exhibited cytotoxic activity against AML FLT3 mutant lines in vitro at 58–200 nM (IC50). In FLT3‐wt in vivo studies, CA‐4948 blocked THP‐1 bone marrow engraftment, while FLT3i quizartinib (Q) and midostaurin (M) had no inhibitory effect. In AML FLT3‐ITD tumors, CA‐4948 induced tumor regression equivalent to Q and M. CA‐4948 also induced tumor regression in FLT3‐ITD/D835Y tumors similar to M and showed greater efficacy in FLT3‐ITD/F691L tumors as compared to Q.Summary/Conclusion:These results demonstrate that targeting the IL‐1R/TLR signaling pathway with IRAK4 inhibitor CA‐4849 may be an effective therapeutic strategy in FLT3‐wt AML. Furthermore, CA‐4948's additional FLT3 inhibitory activity may repress the emergence of FLT3‐mutant AML clones. CA‐4948's dual IRAK4/FLT3 inhibitory activity merits further clinical investigation in FLT3‐wt and FLT3‐mutant AML/MDS.

Conformational flexibility and inhibitor binding to unphosphorylated interleukin-1 receptor–associated kinase 4 (IRAK4)

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a key player in innate immune and inflammatory responses, performing a critical role in signal transduction downstream of Toll-like receptors and interleukin-1 (IL-1) receptors. Upon ligand binding and via its N-terminal death domain, IRAK4 is recruited to an oligomeric receptor that is proximal to the Myddosome signaling complex, inducing IRAK4 kinase domain dimerization, autophosphorylation, and activation. To date, all known IRAK4 structures are in the active conformation, precluding a good understanding of IRAK4's conformational dynamics. To address this issue, here we first solved three crystal structures of the IRAK4 kinase domain (at ≤2.6 Å resolution), in its unphosphorylated, inactive state bound to either the ATP analog AMP-PNP or to one of the two small-molecule inhibitors JH-I-25 and JH-I-17. The structures disclosed that although the structure in complex with AMP-PNP is in an "αC-out" inactive conformation, those in complex with type I inhibitors assume an active "Asp-Phe-Gly (DFG)-in" and "αC-in" conformation. The ability of unphosphorylated IRAK4 to take on variable conformations prompted us to screen for small-molecule inhibitors that bind preferentially to unphosphorylated IRAK4, leading to the identification of ponatinib and HG-12-6. Solving the structures of unphosphorylated IRAK4 in complex with these two inhibitors, we found that they both bind as type II inhibitors with IRAK4 in a "DFG-out" conformation. Collectively, these structures reveal conformational flexibility of unphosphorylated IRAK4 and provide unexpected insights into the potential use of small molecules to modulate IRAK4 activity in cancer, autoimmunity, and inflammation.

Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-κB signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

Interleukin-1 receptor–associated kinase 4 (IRAK4) plays a dual role in myddosome formation and Toll-like receptor signaling

Toll-like receptors (TLRs) form part of the host innate immune system, in which they act as sensors of microbial and endogenous danger signals. Upon TLR activation, the intracellular Toll/interleukin-1 receptor domains of TLR dimers initiate oligomerization of a multiprotein signaling platform comprising myeloid differentiation primary response 88 (MyD88) and members of the interleukin-1 receptor–associated kinase (IRAK) family. Formation of this myddosome complex initiates signal transduction pathways, leading to the activation of transcription factors and the production of inflammatory cytokines. To date, little is known about the assembly and disassembly of the myddosome and about the mechanisms by which these complexes mediate multiple downstream signaling pathways. Here, we isolated myddosome complexes from whole-cell lysates of TLR-activated primary mouse macrophages and from IRAK reporter macrophages to examine the kinetics of myddosome assembly and disassembly. Using a selective inhibitor of IRAK4's kinase activity, we found that whereas TLR cytokine responses were ablated, myddosome formation was stabilized in the absence of IRAK4's kinase activity. Of note, IRAK4 inhibition had only a minimal effect on NF-κB and mitogen-activated protein kinase (MAPK) signaling. In summary, our results indicate that IRAK4 has a critical scaffold function in myddosome formation and that its kinase activity is dispensable for myddosome assembly and activation of the NF-κB and MAPK pathways but is essential for MyD88-dependent production of inflammatory cytokines. Our findings suggest that the scaffold function of IRAK4 may be an attractive target for treating inflammatory and autoimmune diseases.

IRAK4 kinase activity controls Toll-like receptor–induced inflammation through the transcription factor IRF5 in primary human monocytes

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate immune signaling by Toll-like receptors (TLRs), and loss of IRAK4 activity in mice and humans increases susceptibility to bacterial infections and causes defects in TLR and IL1 ligand sensing. However, the mechanism by which IRAK4 activity regulates the production of downstream inflammatory cytokines is unclear. Using transcriptomic and biochemical analyses of human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the activation of interferon regulatory factor 5 (IRF5), a transcription factor implicated in the pathogenesis of multiple autoimmune diseases. Following TLR7/8 stimulation by its agonist R848, chemical inhibition of IRAK4 abolished IRF5 translocation to the nucleus and thus prevented IRF5 binding to and activation of the promoters of inflammatory cytokines in human monocytes. We also found that IKKβ, an upstream IRF5 activator, is phosphorylated in response to the agonist-induced TLR signaling. Of note, IRAK4 inhibition blocked IKKβ phosphorylation but did not block the nuclear translocation of NFκB, which was surprising, given the canonical role of IKKβ in phosphorylating IκB to allow NFκB activation. Moreover, pharmacological inhibition of either IKKβ or the serine/threonine protein kinase TAK1 in monocytes blocked TLR-induced cytokine production and IRF5 translocation to the nucleus, but not nuclear translocation of NFκB. Taken together, our data suggest a mechanism by which IRAK4 activity regulates TAK1 and IKKβ activation, leading to the nuclear translocation of IRF5 and induction of inflammatory cytokines in human monocytes.

Increased IRAK-4 Kinase Activity in Alzheimer’s Disease; IRAK-1/4 Inhibitor I Prevents Pro-inflammatory Cytokine Secretion but not the Uptake of Amyloid Beta by Primary Human Glia

Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β (Aβ), which is associated with a neuroinflammatory response involving microglia and astrocytes. This neuroinflammatory response has detrimental effects on disease progression but also has a beneficial function on removal of excess Aβ. Microglia and astrocytes are involved in the clearance of Aβ from the brain, but neuroinflammation also promotes neurodegeneration. In order to identify signal transduction pathways critically involved in AD we analysed human brain tissue using protein kinase activity profiling. We identified increased activity of the Interleukin 1 Receptor Associated Kinase 4 (IRAK-4) in AD compared to control brain tissue. IRAK-4 is a component of the signal transduction pathway that functions downstream of the Toll-like receptors and the interleukin-1 receptor. Immunohistochemical analysis of human brain tissue revealed the presence of IRAK-4 in astrocytes and microglia. Quantification of IRAK-4 and the phosphorylated form of IRAK-1, a specific substrate for IRAK-4, revealed increased expression and activity of IRAK-4 in AD. Interestingly, IRAK-1/4 inhibitor I reduces the lipopolysaccharide-induced secretion of monocyte chemotactic protein-1 (MCP-1) by primary human microglia and the interleukin-1β-induced secretion of MCP-1 and interleukin 6 by primary human astrocytes. In contrast, the uptake of Aβ by astrocytes and microglia is not affected by IRAK-1/4 inhibition. Our data show that IRAK-4 protein kinase activity is increased in AD and selective inhibition of IRAK-1/4 inhibits a pro-inflammatory response without affecting the uptake of Aβ by glial cells, indicating that the IRAK signalling pathway is a potential target for modulating neuroinflammation in AD.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

The critical role of kinase activity of interleukin‐1 receptor–associated kinase 4 in animal models of joint inflammation

We have previously reported that the kinase activity of interleukin-1 receptor-associated kinase 4 (IRAK-4) is important for Toll-like receptor and interleukin-1 receptor signaling in vitro. Using mice devoid of IRAK-4 kinase activity (IRAK-4 KD mice), we undertook this study to determine the importance of IRAK-4 kinase function in complex disease models of joint inflammation. IRAK-4 KD mice were subjected to serum transfer-induced (K/BxN) arthritis, and migration of transferred spleen lymphocytes into joints and cartilage and bone degradation were assessed. T cell response in vivo was tested in antigen-induced arthritis (AIA) by measuring the T cell-dependent antigen-specific IgG production and frequency of antigen-specific T cells in the spleen and lymph nodes. T cell allogeneic response was tested in vitro by mixed lymphocyte reaction (MLR). Lipopolysaccharide-induced local neutrophil influx into subcutaneous air pouches was impaired in IRAK-4 KD mice. These mice were also protected from inflammation in the K/BxN and AIA models, as shown by reduced swelling of joints. Histologic analysis of joints of K/BxN serum-injected mice revealed that bone erosion, osteoclast formation, and cartilage matrix proteoglycan loss were reduced in IRAK-4 KD mice. Assessment of T cell response by MLR, by frequency of antigen-specific clones, and by production of antigen-specific IgG did not reveal substantial differences between IRAK-4 KD and wild-type mice. These results demonstrate that IRAK-4 is a key component for the development of proarthritis inflammation, but that it is not crucial for T cell activation. Therefore, the kinase function of IRAK-4 appears to be an attractive therapeutic target in chronic inflammation.

Interleukin‐1 receptor–associated kinase 4 links innate immunity to the pathogenesis of rheumatoid arthritis

The innate immune response is linked to the adaptive immune response through several families of the pattern recognition receptors, among which Toll-like receptors (TLRs) are the most studied. Triggering of the pattern recognition receptors results in production of cytokines and chemokines and enhancement of both innate and adaptive immune responses. TLR signaling pathways share similar elements with interleukin-1 receptor (IL-1R)/IL-18R signaling pathways. Activation of the receptors after ligand binding leads to the recruitment of the Toll/IL-1R domain–containing adaptor molecule, myeloid differentiation factor 88, to the TLRs (1). Thereafter, several kinases such as IL-1R–associated kinase 4 (IRAK-4) and IRAK-1, as well as tumor necrosis factor receptor–associated factor 6, are recruited to the receptor complex (Figure 1). IRAK-4 is activated, and after hyperphosphorylation of IRAK-1, the receptor complex is further activated by transforming growth factor –activated kinase 1–dependent and –independent pathways, leading finally to activation of the downstream mediators NFB and activator protein 1 (2,3). An important factor in the activation of this inflammatory pathway is IRAK-4. IRAK-4 is a member of a family of protein kinases that also contains IRAK-1, IRAK-2, and IRAK-M. It has been demonstrated that IRAK-4 is crucial for the signaling pathway of the IL-1 and IL-18 receptors. In addition, IRAK-4 is needed for activation of nearly all of the TLRs, with the exception of TLR-3. Using IRAK-4 kinase-inactive knockin mice, it has been shown that IRAK-4 is important for IL-1–, lipopolysaccharide (LPS; TLR-4)–, or R848 (TLR-7)– induced cytokine and chemokine production (4–6). Interestingly, TLR-4 signaling is only partly disrupted in IRAK-4 kinase-inactive knockin mice, since cells from these mice respond to LPS exposure with subnormal cytokine production (7). The partial TLR-4 responsiveness is mediated by a secondary pathway involving the TRIF adaptor molecule (Figure 1). However, IRAK-4 kinase-inactive knockin mice are resistant to LPSor CpG-induced lethal shock (5). The role of active IRAK-4 has not been extensively studied in animal models of complex diseases. Very recently, it was shown that IRAK-4 is crucial in the development of vascular inflammation. IRAK-4 kinaseinactive knockin mice backcrossed in apolipoprotein E–knockout mice were almost completely protected against atherosclerosis (8). However, until now very little has been known about the effect of IRAK-4 in models of arthritis. In this issue of Arthritis & Rheumatism, KoziczakHolbro and colleagues demonstrate for the first time that IRAK-4 kinase-inactive knockin mice are completely protected against the development of antibodyinduced arthritis (K/BxN mouse model). Compared with wild-type (WT) mice, IRAK-4 kinase-inactive knockin mice did not have joint swelling, cartilage damage, or bone erosions (9). It has been previously shown that this particular animal model of arthritis is highly IL-1 dependent, since IL-1–deficient mice were extremely resistant to the K/BxN model of experimental arthritis (10). In line with these results, Koziczak-Holbro and colleagues confirmed the critical role of IRAK-4 in the IL-1R signaling pathway (9). Interestingly, however, the authors also showed that IRAK-4 kinase activity was not necessary for trafficking of T cells, B cells, and macrophages to the site of inflammation, whereas IRAK-4 was crucial for residential stromal cells to recruit inflammatory cells to the joint. These data identify IRAK-4 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in rheumatoid arthritis (RA). Furthermore, Koziczak-Holbro and colleagues Leo A. B. Joosten, PhD, Mihai G. Netea, MD, PhD: Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. Address correspondence and reprint requests to Leo A. B. Joosten, PhD, Department of Medicine (463), Radboud University Nijmegen Medical Centre, Geert Grooteplein zuid 8, 6525 GA Nijmegen, The Netherlands. E-mail: l.joosten@aig.umcn.nl. Submitted for publication February 3, 2009; accepted in revised form February 25, 2009.