IRAK4 Kinase Activity Research Articles

Emavusertib (CA-4948) in combination with ibrutinib in patients with relapsed/refractory primary central nervous system lymphoma (R/R PCNSL).

2087 Background: PCNSL is a rare and aggressive form of NHL in the central nervous system or vitreoretinal space. Despite high initial rates of responses to HD-MTX-based induction chemotherapies, most patients relapse in two years. R/R PCNSL is associated with poor prognosis, representing a clear unmet medical need, as there are no currently approved treatments for this disease. IRAK4 kinase activity is required for TLR and IL-1R signaling in a variety of myeloid and lymphoid cell types, including PCNSL. Recruitment of IRAK4 to these receptors and subsequent activation is facilitated by the MyD88 adaptor protein, which is mutated in ~70% of PCNSL. This leads to constitutive activation of the NF-κB signaling pathway, increased inflammation, and tumor growth. Emavusertib is a novel and potent oral inhibitor of IRAK4 and FLT3, which has demonstrated the ability to cross the blood-brain barrier in PCNSL xenografts. In preclinical studies, the combination of emavusertib with ibrutinib overcomes resistance to BTK inhibitors. This combination has an acceptable safety profile across a broad population of R/R NHL patients, including PCNSL. Here we present updated safety and efficacy data for emavusertib in combination with ibrutinib in patients with R/R PCNSL. Methods: The safety, clinical activity, and potential biomarkers of emavusertib in R/R PCNSL are being investigated in the ongoing open-label, Phase 1/2 TakeAim Lymphoma trial (NCT03328078). The eligible patients with R/R PCNSL received emavusertib (100-300 mg BID) in combination with ibrutinib (560 mg QD) in a 21-day cycle until tumor progression or unacceptable toxicity. Results: Here, we present updated data from R/R PCNSL patients with prior ibrutinib exposure who were treated with emavusertib in combination with ibrutinib. The median number of prior lines of anti-cancer therapies was 3 (range: 2-5), all patients were R/R to both frontline therapy and subsequent ibrutinib regimens. Prior responses to ibrutinib based regimens included only 1 CR. Here, we report a higher CR rate when these patients are subsequently treated with emavusertib in combination with ibrutinib.Results revealed no dose-limiting toxicities. Conclusions: Emavusertib in combination with ibrutinib was well tolerated with an acceptable safety profile and promising efficacy in R/R PCNSL patients with previous exposure to ibrutinib (BTKi). Enrollment in this trial is ongoing (NCT03328078). Clinical trial information: NCT03328078 .

A noncanonical IRAK4-IRAK1 pathway counters DNA damage-induced apoptosis independently of TLR/IL-1R signaling.

Interleukin-1 receptor (IL-1R)-associated kinases (IRAKs) are core effectors of Toll-like receptors (TLRs) and IL-1R in innate immunity. Here, we found that IRAK4 and IRAK1 together inhibited DNA damage-induced cell death independently of TLR or IL-1R signaling. In human cancer cells, IRAK4 was activated downstream of ATR kinase in response to double-strand breaks (DSBs) induced by ionizing radiation (IR). Activated IRAK4 then formed a complex with and activated IRAK1. The formation of this complex required the E3 ubiquitin ligase Pellino1, acting structurally but not catalytically, and the activation of IRAK1 occurred independently of extracellular signaling, intracellular TLRs, and the TLR/IL-1R signaling adaptor MyD88. Activated IRAK1 translocated to the nucleus in a Pellino2-dependent manner. In the nucleus, IRAK1 bound to the PIDD1 subunit of the proapoptotic PIDDosome and interfered with platform assembly, thus supporting cell survival. This noncanonical IRAK signaling pathway was also activated in response to other DSB-inducing agents. The loss of IRAK4, of IRAK4 kinase activity, of either Pellino protein, or of the nuclear localization sequence in IRAK1 sensitized p53-mutant zebrafish to radiation. Thus, the findings may lead to strategies for overcoming tumor resistance to conventional cancer treatments.

The IRAK4 scaffold integrates TLR4-driven TRIF and MYD88 signaling pathways.

SUMMARYInterleukin-1 receptor-associated kinases (IRAKs) −4, −2, and −1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-β (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 signaling.

Oral IRAK4 inhibitor BAY-1834845 prevents acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a lethal clinical entity that has become an emergency event with the outbreak of COVID-19. However, to date, there are no well-proven pharmacotherapies except dexamethasone. This study is aimed to evaluate IRAK4 inhibitors as a potential treatment for ARDS-cytokine release syndrome (CRS). We applied two IRAK4 inhibitors, BAY-1834845 and PF-06650833 to an inhaled lipopolysaccharide (LPS)-induced ARDS mouse model with control of high dose dexamethasone (10 mg/kg). Unexpectedly, although both compounds had excellent IC50 on IRAK4 kinase activity, only BAY-1834845 but not PF-06650833 or high dose dexamethasone could significantly prevent lung injury according to a blinded pathology scoring. Further, only BAY-1834845 and BAY-1834845 combined with dexamethasone could effectively improve the injury score of pre-existed ARDS. Compared with PF-06650833 and high dose dexamethasone, BAY-1834845 remarkably decreased inflammatory cells infiltrating lung tissue and neutrophil count in BALF. BAY-1834845, DEX, and the combination of the two agents could decrease BALF total T cells, monocyte, and macrophages. In further cell type enrichment analysis based on lung tissue RNA-seq, both BAY-1834845 and dexamethasone decreased signatures of inflammatory cells and effector lymphocytes. Interestingly, unlike the dexamethasone group, BAY-1834845 largely preserved the signatures of naïve lymphocytes and stromal cells such as endothelial cells, chondrocytes, and smooth muscle cells. Differential gene enrichment suggested that BAY-1834845 downregulated genes more efficiently than dexamethasone, especially TNF, IL-17, interferon, and Toll-like receptor signaling.

Combination of IRAK4 Inhibitor CA-4948 with BCL2 Inhibitor Venetoclax Induces Tumor Regression in an ABC-DLBCL Xenograft Model

IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein, which is mutated in ~22% of diffuse large B-cell lymphoma (DLBCL) cases. The MYD88- L265P activating mutation is found in ~30% of the activated B-cell (ABC) and ~6% of germinal center B-cell (GCB) subtypes of DLBCL and leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. In Waldenstrom macroglobulinemia (WM), the MYD88- L265P activating mutation is present in >90% of cases. Thus, the development of small molecule inhibitors targeting IRAK4 is an attractive anticancer strategy for MYD88 mutation-containing cancers such as DLBCL and WM.We are developing a novel IRAK4 inhibitor, CA-4948, as a therapeutic agent for non-Hodgkin lymphoma (NHL) and hematological cancers with dysregulated TLR/MYD88/IRAK4 signaling. CA-4948 is a reversible kinase inhibitor that modulates IRAK4 function in both the toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling cascades, and demonstrates pharmacodynamic and antitumor activity in in vitro and in vivo nonclinical models. CA-4948 exhibits favorable DMPK properties, oral bioavailability, and is well tolerated in mice. Furthermore, CA-4948 was previously shown to exhibit dose-dependent efficacy in ABC-DLBCL MYD88 -L265P xenograft tumor models using cell lines OCI-LY3 and OCI-Ly10 and patient-derived tumors.Activating mutations in the B-cell receptor (BCR) signaling pathway are frequently present in various NHL subtypes, leading to activation of NF-kB signaling, and growth and survival pathways. Dysregulation of both TLR and BCR pathways suggest that targeting both pathways will be required for improved therapeutic responses in NHL. Here, we report efficacy results from combined treatment of CA-4948 and the BCL2 inhibitor venetoclax in an ABC-DLBCL xenograft tumor model.We performed an in vivo study in female SCID Beige mice implanted with the ABC-DLBCL cell line OCI-Ly10, which harbors TLR (MYD88 -L265P) and BCR (CD79A ITAM) pathway activating mutations. OCI-Ly10 tumor-bearing mice were treated with CA-4948 (50 mg/kg, qd, po), venetoclax (75 mg/kg, qd, po) or the combination for 21 continuous days. Single agent treatment of CA-4948 and venetoclax exhibited moderate tumor growth inhibition (TGI) of 63% and 71%, respectively, and the combination demonstrated tumor regression. The combination was tolerated at the end of treatment without body weight loss relative to predose weight. After the 21-day drug treatment period, the mice were given a 19-day dosing holiday. Within 5 days of the holiday rapid tumor growth occurred in the previously treated single drug-treated mice and this rapid growth continued for the remainder of the dosing holiday. In contrast, tumors from the combination drug-treated mice did not commence detectable regrowth until the 10th day of the drug holiday and proceeded with a slow growth rate for the subsequent 9 days.After the 19-day drug holiday, dosing of the respective drug treatments was reinstated to each cohort. After 7 days of re-dosing, the tumors in each cohort exhibited antitumor responses to respective drug treatments with the CA-4948 plus venetoclax combination again driving the tumors into regression. In summary, CA-4948 plus venetoclax was a well-tolerated drug combination that exhibited a much greater in vivo antitumor response as compared to the single agent treatments. These results underscore the therapeutic potential of targeted IRAK4 kinase inhibition by CA-4948 in combination with other targeted agents for the treatment of NHL. DisclosuresBooher:Curis, Inc: Employment, Equity Ownership. Dellarocca:Curis, Inc: Employment, Equity Ownership. Atoyan:Curis, Inc: Employment, Equity Ownership. Borek:Curis, Inc: Employment, Equity Ownership. Samson:Curis, Inc: Employment, Equity Ownership. Tuck:Curis, Inc: Employment, Equity Ownership.

Establishing and Validating Cellular Functional Target Engagement Assay for Selective IRAK4 Inhibitor Discovery.

One of the main reasons for the lack of drug efficacy in late-stage clinical trials is the lack of specific and selective target engagement. To increase the likelihood of success of new therapeutics, one approach is to conduct proximal target engagement testing during the early phases of preclinical drug discovery. To identify and optimize selective IRAK4 inhibitors, a kinase that has been implicated in multiple inflammatory and autoimmune diseases, we established an electrochemiluminescence (ECL)-based cellular endogenous IRAK1 activation assay as the most proximal functional evaluation of IRAK4 engagement to support structure-activity relationship (SAR) studies. Since IRAK1 activation is dependent on both the IRAK4 scaffolding function in Myddosome formation and IRAK4 kinase activity for signal transduction, this assay potentially captures inhibitors with different mechanisms of action. Data from this IRAK1 assay with compounds representing different structural classes showed statistically significant correlations when compared with results from both IRAK4 biochemical kinase activity and functional peripheral blood mononuclear cell (PBMC)-derived tumor necrosis factor α (TNFα) secretion assays, validating the biological relevancy of the IRAK1 target engagement as a biomarker of the IRAK4 activity. Plate uniformity and potency reproducibility evaluations demonstrated that this assay is amenable to high throughput. Using Bland-Altman assay agreement analysis, we demonstrated that incorporating such proximal pharmacological assessment of cellular target engagement to an in vitro screening funnel for SAR studies can prevent compound optimization toward off-target activity.

Dipterocarpus tuberculatus Roxb. Ethanol Extract Has Anti-Inflammatory and Hepatoprotective Effects In Vitro and In Vivo by Targeting the IRAK1/AP-1 Pathway.

Dipterocarpus tuberculatus Roxb. has been used traditionally as a remedy for many diseases, especially inflammation. Therefore, we analyzed and explored the mechanism of the anti-inflammatory effect of a Dipterocarpus tuberculatus Roxb. ethanol extract (Dt-EE). Dt-EE clearly and dose-dependently inhibited the expression of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β in lipopolysaccharide (LPS)-treated RAW264.7 cells. Also, Dt-EE suppressed the activation of the MyD88/TRIF-mediated AP-1 pathway and the AP-1 pathway related proteins JNK2, MKK4/7, and TAK1, which occurred as a result of inhibiting the kinase activity of IRAK1 and IRAK4, the most upstream factors of the AP-1 pathway. Finally, Dt-EE displayed hepatoprotective activity in a mouse model of hepatitis induced with LPS/D-galactosamine (D-GalN) through decreasing the serum levels of alanine aminotransferase and suppressing the activation of JNK and IRAK1. Therefore, our results strongly suggest that Dt-EE could be a candidate anti-inflammatory herbal medicine with IRAK1/AP-1 inhibitory and hepatoprotective properties.

Central IRAK-4 kinase inhibition for the treatment of pain following nerve injury in rats

There is ample evidence for the role of the immune system in developing chronic pain following peripheral nerve injury. Especially Toll-like receptors (TLRs) and their associated signaling components and pro-inflammatory cytokines such as IL-1β, induced after injury, are involved in nociceptive processes and believed to contribute to the manifestation of chronic neuropathic pain states. Whereas the inhibition of the kinase function of IRAK-4, a central kinase downstream of TLRs and IL-1 receptors (IL-1Rs), seems efficacious in various chronic inflammatory and autoimmune models, it’s role in regulating chronic neuropathic pain remained elusive to date. Here, we examined whether pharmacological inhibition of IRAK-4 kinase activity using PF-06650833 and BMS-986147, two clinical-stage kinase inhibitors, is effective for controlling persistent pain following nerve injury. Both inhibitors potently inhibited TLR-triggered cytokine release in human peripheral blood mononuclear cell (PBMC) as well as human and rat whole blood cultures. BMS-986147 showing favorable pharmacokinetic (PK) properties, significantly inhibited R848-triggered plasma TNF levels in a rat in vivo cytokine release model after single oral dosing. However, BMS-986147 dose dependently reversed cold allodynia in a rat chronic constriction injury (CCI) model following intrathecal administration only, supporting the notion that central neuro-immune modulation is beneficial for treating chronic neuropathic pain. Although both inhibitors were efficacious in inhibiting IL-1β- or TLR-triggered cytokine release in rat dorsal root ganglion cultures, only partial efficacy was reached in IL-1β-stimulated human glial cultures indicating that inhibiting IRAK-4́’s kinase function might be partially dispensable for human IL-1β driven neuroinflammation. Overall, our data demonstrate that IRAK-4 inhibitors could provide therapeutic benefit in chronic pain following nerve injury, and the central driver for efficacy in the neuropathic pain model as well as potential side effects of centrally available IRAK-4 inhibitors warrant further investigation to develop effective analgesia for patients in high unmet medical need.

The kinase activity of IRAK4 controls sterile autoinflammation and manifestations of Murine Lupus

Abstract Interleukin-1 receptor-associated kinase (IRAK) 4 is a serine/threonine kinase involved in TLR/IL-1R responses. Upon TLR/IL-1R activation, autophosphorylation of IRAK4 induces proinflammatory gene transcription. In this study we hypothesize that the kinase activity of IRAK4 may be a druggable target in the treatment of sterile autoinflammation and Lupus. C57BL/6 female mice injected with the hydrocarbon oil Pristane, exhibited a significant Pristane-dependent induction of splenomegaly; whereas treatment with the IRAK4 kinase inhibitor reduced this inflammatory symptom. Conversely, no significant difference was observed in males. Using mass spectrometry, we found that the urine of Pristane-injected mice contained increased levels of putative markers for several inflammatory diseases, which were reduced by IRAK4 kinase inhibition. We showed that Pristane-injected mice had increased cell free DNA in serum, which was not impacted by kinase inhibitor treatment. However, chemokine release was significantly reduced in IRAK4 kinase inhibitor-treated mice. We further analyzed the kinase activity of IRAK4 in MRL-lpr mice, which were compared with MRL-lpr/IRAK4 kinase-inactive (KDKI) mice (where IRAK4 is expressed but its kinase activity is lost). Although MRL-lpr/IRAK4KDKI and MRL-lpr mice did not show differences in CD4+ and CD8+ cells activation, we found a significantly reduced splenomegaly in MRL-lpr/IRAK4KDKI mice compared to controls, also exhibiting a gender bias. Thus, IRAK4 kinase activity might represent a new therapeutic and druggable target in the recruitment of immune cells during sterile inflammatory diseases in humans, including Systemic Lupus Erythematous (SLE).

Discovery of Potent Benzolactam IRAK4 Inhibitors with Robust in Vivo Activity.

IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.

Preclinical Activity of IRAK4 Kinase Inhibitor CA-4948 Alone or in Combination with Targeted Therapies and Preliminary Phase 1 Clinical Results in Non-Hodgkin Lymphoma

Preclinical Activity of IRAK4 Kinase Inhibitor CA-4948 Alone or in Combination with Targeted Therapies and Preliminary Phase 1 Clinical Results in Non-Hodgkin Lymphoma

Targeted Degradation of IRAK4 Protein Via Heterobifunctional Small Molecules for Treatment of MYD88 Mutant Lymphoma

Recurrent mutations in the scaffolding protein MYD88 are present in 30-40% of activated B cell diffuse lymphocytic B cell lymphoma (ABC-DLBCL)(Ngo et al. Nature 2011). MYD88 links activated interleukin 1 receptor (IL1R) and Toll-like receptors (TLRs) to downstream effectors by nucleating assembly of the Myddosome, a multi-protein complex containing MYD88, the protein kinases IRAK4 and IRAK1 and the pseudokinase IRAK2, via oligomerization of the N-terminal Death Domains in each of these proteins (Motshwene et al. JBC 2009; Lin, Lo and Wu. Nature 2010). The most prevalent MYD88 mutation, L265P, constitutively activates assembly of the Myddosome, causing IRAK4-dependent NFκB and MAP kinase signaling and leading to lymphoma survival and proliferation (Ngo et al. Nature 2011).Constitutive activation of the Myddosome has proven to be difficult to drug. MYD88 and IRAK2 lack enzymatic activity and cannot be targeted by conventional small molecule catalytic inhibitors. There are no IRAK1 inhibitors in clinical trials. Specific inhibitors of the kinase activity of IRAK4 have shown limited activity as single agents in preclinical models of MYD88 mutant lymphoma (Lim et al. Blood 2012; Booher et al. Blood 2014). This is consistent with the observations that there is kinase-independent activity of IRAK4 in mediating downstream signals, such as those leading to induction of NFκB (Qin et al. JBC 2004; Fraczek et al. JBC 2008; Sun et al. Sci Signal. 2016). IRAK4 kinase inhibitors thus might not provide optimal blockade of NFκB-dependent tumor survival signals driven by MYD88 mutations in ABC-DLBCL.Kymera Therapeutics is advancing a new class of drugs based on targeted protein degradation: heterobifunctional molecules that recruit disease-causing proteins to specific E3 ubiquitin ligases, resulting in their ubiquitination and subsequent degradation. We have developed a portfolio of potent and specific degraders of IRAK4 with drug-like propertiesOur lead molecules cause potent and specific degradation of IRAK4 in the ABC-DLBCL cell lines OCI-LY10 (MYD88 L265P) and U2932 (MYD88 WT) while showing selective cytotoxic effects on OCI-LY10 vs U2932. The apoptotic response is triggered within 24-48 h in OCI-LY10, demonstrated by appearance of cleaved caspase 3 and PARP. Quantitative proteomic studies show selective degradation of IRAK4 vs five to six thousand other proteins detected in OCI-LY10.IRAK4 degraders were tested in an OCI-LY10 xenograft model with daily dosing for 28 days, to determine the level of IRAK4 degradation and impact of that degradation on tumor growth in vivo. We observed that degraders mediated decrease in IRAK4 in vivo and produced a statistically significant decrease in tumor growth rate.Here we will present in vitro and in vivo activity and mechanism of action of our most advanced IRAK4 degraders. These IRAK4 degrader molecules represent a new therapeutic modality and provide an exciting opportunity to treat MYD88 mutation-driven ABC-DLBCL. DisclosuresKelleher:Kymera Therapeutics: Employment, Equity Ownership. Audoly:Kymera Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Campbell:Kymera Therapeutics: Employment, Equity Ownership. Chen:Kymera Therapeutics: Employment, Equity Ownership. Ji:Kymera Therapeutics: Employment, Equity Ownership. Kamadurai:Kymera Therapeutics: Employment, Equity Ownership. Li:Kymera Therapeutics: Employment, Equity Ownership. Loh:Kymera Therapeutics: Employment, Equity Ownership. Rong:Kymera Therapeutics: Employment, Equity Ownership. Vigil:Kymera Therapeutics: Employment, Equity Ownership. Weiss:Kymera Therapeutics: Employment, Equity Ownership. Yuan:Kymera Therapeutics: Employment, Equity Ownership. Zhang:Kymera Therapeutics: Employment, Equity Ownership. Mainolfi:Kymera Therapeutics: Employment, Equity Ownership.

IRAK4 activity controls immune responses to intracellular bacteria Listeria monocytogenes and Mycobacterium smegmatis.

IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4KI ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4KI macrophages expressed decreased TNF-α, IL-6, IL-1β, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4KI macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4KI mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO.

High throughput screening of IRAK4 small molecule inhibitors in TLR ligand stimulated whole blood

Abstract The IRAK4 kinase is a key regulator of TLR and IL1R signaling. Small molecule inhibitors of IRAK4 represent an important advance in targeting these pathways. We developed a high throughput whole blood assay to identify potent IRAK4 inhibitors through the inhibition of TLR ligand (R848, LPS, and Gardiquimod) induced inflammatory cytokine production (IL6, TNF, IFNa). Extensive optimization was carried out to ensure rigorous measurement of small molecule potencies, percent maximum inhibition, and cytokine endpoint correlations to clearly distinguish compounds from one another in the context of human whole blood variance. Whole blood treated with IRAK4 inhibitors resulted in partial inhibition of TLR induced cytokines. Whole blood is a complex mixture of cell types and serum proteins. Our analysis shows various dependency of TLR signaling on IRAK4 kinase activity varies among various immune cells allowing for residual inflammatory outputs. Ex vivo whole blood stimulation assays have proven to be an important tool in drug discovery to identify potent IRAK4 small molecule clinical candidates and further define the role of IRAK4 signaling in immune responses.

Deficiency in Interleukin-1 receptor-associated kinase (IRAK) 4 activity attenuates manifestations of murine Lupus

Abstract IL-1R-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of MyD88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumonia, but its role in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB male mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of IRAK1, mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced TNF-α and CCL5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein (Tollip) and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background had reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, decreased numbers of splenic macrophages, total and TNF-a+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared to lupus-prone mice with functional IRAK4. Furthermore, mice expressing kinase-inactive IRAK4 had decreased proteinuria and increased survival in the chemically-induced (pristine) lupus model. Thus, IRAK4 kinase activity significantly contributes to murine lupus and could represent a new target for therapeutic interventions.

Deficiency in IRAK4 activity attenuates manifestations of murine Lupus.

Interleukin-1 receptor-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of myeloid differentiation primary response protein (MyD) 88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumoniae, but its involvement in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced tumor necrosis factor (TNF)-α and C-C motif chemokine ligand (CCL) 5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background manifested blunted TLR signaling in macrophages and reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, numbers of splenic macrophages, total and TNF-α+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared with lupus-prone mice with functional IRAK4. Thus, IRAK4 kinase activity contributes to murine lupus and could represent a new therapeutic target.

Selective IRAK4 Inhibition Attenuates Disease in Murine Lupus Models and Demonstrates Steroid Sparing Activity.

The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use

Abstract Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)–based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor–associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and pro inflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.

Characterization of a small molecule IRAK4 kinase inhibitor for the treatment of autoimmune and inflammatory diseases

Abstract IRAK4 is a key protein kinase in immune cells. It is responsible for initiating MyD88-dependent signaling from most Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R), resulting in downstream production of pro-inflammatory cytokines. Hence, inhibitors of IRAK4 kinase activity represent valuable therapeutic tools to treat cytokine-driven autoimmune and inflammatory diseases. Through cell-based screening, we identified potent small molecule IRAK4 kinase inhibitors that block TLR4- and IL-1R-induced cytokine production with potencies less than 100nM. Our inhibitors exhibit good selectivity against a broad panel of kinases. In vivo, our lead compound decreases serum IL-6 in an acute mouse model of IL-1beta-induced cytokine release, demonstrating excellent pharmacokinetics properties. In addition, our IRAK4 inhibitor blocks monosodium urate crystals-induced peritonitis in mice, reduces paw swelling in the chronic rat model of collagen-induced arthritis, as well as the clinical score and overall inflammatory phenotype of mice in the imiquimod-induced psoriasis model. The in vitro and in vivo characterization of our lead candidate is promising and confirms IRAK4 as an attracting therapeutic target for the management of cytokine-driven diseases.

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use.

Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)-based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor-associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.