IRAK1 Signaling Research Articles

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

The IRAK-M death domain: a tale of three surfaces.

The anti-inflammatory interleukin-1 receptor associated kinase-M (IRAK-M) is a negative regulator of MyD88/IRAK-4/IRAK-1 signaling. However, IRAK-M has also been reported to activate NF-κB through the MyD88/IRAK-4/IRAK-M myddosome in a MEKK-3 dependent manner. Here we provide support that IRAK-M uses three surfaces of its Death Domain (DD) to activate NF-κB downstream of MyD88/IRAK-4/IRAK-M. Surface 1, with central residue Trp74, binds to MyD88/IRAK-4. Surface 2, with central Lys60, associates with other IRAK-M DDs to form an IRAK-M homotetramer under the MyD88/IRAK-4 scaffold. Surface 3; with central residue Arg97 is located on the opposite side of Trp74 in the IRAK-M DD tetramer, lacks any interaction points with the MyD88/IRAK-4 complex. Although the IRAK-M DD residue Arg97 is not directly involved in the association with MyD88/IRAK-4, Arg97 was responsible for 50% of the NF-κB activation though the MyD88/IRAK-4/IRAK-M myddosome. Arg97 was also found to be pivotal for IRAK-M's interaction with IRAK-1, and important for IRAK-M's interaction with TRAF6. Residue Arg97 was responsible for 50% of the NF-κB generated by MyD88/IRAK-4/IRAK-M myddosome in IRAK-1/MEKK3 double knockout cells. By structural modeling we found that the IRAK-M tetramer surface around Arg97 has excellent properties that allow formation of an IRAK-M homo-octamer. This model explains why mutation of Arg97 results in an IRAK-M molecule with increased inhibitory properties: it still binds to myddosome, competing with myddosome IRAK-1 binding, while resulting in less NF-κB formation. The findings further identify the structure-function properties of IRAK-M, which is a potential therapeutic target in inflammatory disease.

EXTH-93. THE IRAK-4 INHIBITOR EMAVUSERTIB (CA-4948) FOR THE TREATMENT OF PRIMARY CNS LYMPHOMA

Abstract BACKGROUND PCNSL is an aggressive brain tumor accounting for 3% of all CNS malignancies and is associated with poor prognosis. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients cannot tolerate this intensive therapeutic intervention. Novel treatments are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK-4 that demonstrates clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. HYPOTHESIS: Emavusertib has anti-tumor activity against preclinical PCNSL. METHODS Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Survival responses in preclinical PCNSL models were measured in response emavusertib treatment, along with in vivo tumor proliferation and downstream biomarker expression by multi-parameter IHC. RESULTS Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We show that emavusertib can achieve therapeutically relevant concentrations in the brain and CSF. Emavusertib impairs tumor cell proliferation in vitro and in vivo, and shows dose-dependent single-agent activity in preclinical PCNSL models. We confirm decreased ERK1/2, MAPK and NF-κB activation, indicative of downstream IRAK-4 inhibition. CONCLUSIONS Our data supports that emavusertib demonstrates anti-tumor activity in the CNS space, warranting further preclinical and clinical evaluation of this agent for the treatment of PCNSL.

Lead exposure promotes the inflammation via the circRNA-05280/miR-146a/IRAK1 axis in mammary gland

Lead, the most widely used heavy metal in industry, is detrimental to human health if exposed to living and occupational environment. Although several studies have been conducted on lead exposure, little has been reported on its harm to mammary gland and its mechanisms. In view of this, our study is the first to verify that lead exposure could promote apoptosis and inflammation in mouse mammary tissue (in vivo) and cow mammary epithelial cells (in vitro). After establishing a lead exposed mouse model, the expression profile of mammary gland tissue was constructed by high-throughput sequencing technology. In the profile, 917 differentially expressed genes were screened, of which IRAK1 was up-regulated by 4.33 times. Then, from qRT-PCR, Western blot and Luciferase report, IRAK1 was found to promote the release of inflammatory factors and tissue apoptosis and could be a specific target of miR-146a.On the other hand, double luciferase reporter system and qRT-PCR predicted the existence of a binding site between circRNA-05280 and miR-146a sequence. Experiments such as immunohistochemistry, apoptosis and EdU demonstrated that circRNA-05280 could promote not only cell apoptosis but also the expression level of inflammatory genes. Nevertheless, the function of miR-146a is opposite to that of circRNA-05280. Specifically, circRNA-05280 can regulate the level of apoptosis and inflammation of mammary gland by binding miR-146a and releasing the expression of miR-146a on target gene IRAK1. This study concludes that circRNA-05280/ miR-146a/ IRAK1 signaling pathway could mediate the mammary gland damage resulting from lead exposure. Accordingly, it sheds new light on further exploration of molecular mechanisms of mammary gland tissue damage caused by lead exposure, the risk assessment of lead, and the mechanism of lead mammary gland toxicity.

P1298: THE IRAK-4 INHIBITOR EMAVUSERTIB (CA-4948) FOR THE TREATMENT OF PRIMARY CNS LYMPHOMA

Background: Primary central nervous system lymphoma (PCNSL) is an aggressive brain tumor accounting for 3% of all CNS malignancies and is usually associated with poor prognosis. 5-year survival is less than 30% with current treatment options. Standard of care treatment includes induction with high-dose methotrexate based chemoimmunotherapy followed by consolidation with autologous stem cell transplant or whole brain radiation. However, more than half of PCNSL patients are not eligible for this type of intensive therapeutic intervention. Novel treatment strategies with better efficacy and toxicity profile are urgently needed. Toll-like receptor signaling pathway via MyD88/IRAK-4 signalosome is constitutively active in PCNSL secondary to MYD88 L265P mutation and represents an excellent therapeutic target. Emavusertib (CA-4948) is an oral first-in-class small molecule inhibitor of IRAK4 that has demonstrated clinical activity in patients with systemic Non-Hodgkin’s Lymphoma, however it has not been evaluated in the CNS space. Aims: In this study we examine translational and clinical therapeutic applicability and efficacy of emavusertib for the treatment of PCNSL. Methods: Using multiparameter immunohistochemistry (IHC) and proteomics analysis, IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient PCNSL tissues as compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for emavusertib drug concentration following single oral high dose in a murine model. Using an aggressive preclinical A20 immunocompetent CNSL model, preclinical therapeutic activity of emavusertib was evaluated with assessment of downstream biomarker response to treatment including phospho-NF-κB, phospho-ERK1/2, and phospho-P38 in control and treated animals by multi-parameter IHC. We also report therapeutic response in a relapsed/refractory PCNSL patient enrolled in a Phase I/II clinical trial (TakeAim) assessing the combination of emavusertib and Ibrutinib. Results: Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient PCNSL. We also demonstrate that emavusertib can achieve therapeutically relevant concentrations in the brain parenchyma and shows single-agent activity in an aggressive preclinical model of PCNSL. We further confirm decreased ERK1/2, MAPK and NF-κB activation in response to preclinical emavusertib treatment, indicative of downstream effect of IRAK-4 inhibition. Finally, one patient who could not tolerate standard therapies and was resistant to Ibrutinib achieved complete response when treated with the combination of emavusertib and Ibrutinib, providing early clinical evidence of CNS penetration and anti-tumor activity of emavusertib. Summary/Conclusion: Herein we confirm for the first time that targeted inhibition of IRAK-4 with the oral small molecule inhibitor emavusertib demonstrates preclinical and clinical therapeutic activity against CNS lymphoma. Further evaluation of this agent for the treatment of PCNSL is warranted.

CA-4948 for the treatment of melanoma brain metastasis.

2011 Background: Melanoma is a growing clinical health concern where the incidence has doubled over the last 30 years. A major driver of melanoma associated deaths is the development of brain metastases (MBM). It is estimated that 40-60% of patients with metastatic melanoma will develop MBM. Current treatment strategies have a great local control rate, but do not prevent recurrent disease, with a distant intracranial failure rate of 50% at 1 year. Novel treatments and an improved understanding of the MBM tumor microenvironment are needed. Toll-like receptor signaling is activated in cutaneous melanoma, triggering innate inflammatory activation downstream through the adapter proteins MyD88 and interleukin (IL)-1 receptor-associated kinase (IRAK-1 and -4). Increased expression of IRAK-4 stimulates transcription of multiple cellular kinases and transcription factors, and production of inflammatory chemokines and cytokines. This places IRAK-4 as a central driver of both intrinsic tumorigenicity and immune evasion in melanoma. In this study we examine IRAK-4 activation in MBM, as well as preclinical responsiveness to CA-4948, an oral first-in-class small molecule inhibitor of IRAK-4 that has demonstrated clinical activity and has an acceptable safety in cancer patients. Methods: Using multiparameter IHC and proteomics analysis IRAK-1, IRAK-4, and NF-κB pathway activation were examined in patient MBM tissues and compared to normal controls. Plasma, cerebrospinal fluid, and brain tissue were assessed via UPLC-MS/MS for CA-4948 drug concentration following single oral high dose in a murine model. Using an aggressive preclinical model of MBM, B16F10, downstream biomarker response to treatment including phospho-NF-κB, phospho-ERK1/2, and phospho-P38 was measured in control and CA-4948 treated animals using multi-parameter IHC. Survival response was also tested using the B16F10 model. Results: Our data confirm elevated IRAK-1, IRAK-4, and NF-κB signaling in patient MBM. We also demonstrate that CA-4948 is capable of achieving therapeutically relevant concentrations in the brain parenchyma, and shows single agent activity in an aggressive preclinical model of MBM. We further confirm decreased ERK1/2, MAPK and NF-κB activation in response to CA-4948 treatment. Conclusions: We present here for the first time that IRAK-4 is a strong candidate for targeted therapy in MBM. We further validate CNS penetrance of the oral IRAK-4 inhibitor CA-4948, where single agent markedly reduces the proliferative capacity of aggressive preclinical MBM, resulting in improved survival. These data warrant further investigation of CA-4948 for the treatment of melanoma brain metastases, with far reaching potential as an adjunct to standard of care.

Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming.

Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (Mϴ) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human Mϴs exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated Mϴs nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of Mϴs by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated Mϴs, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated Mϴs was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human Mϴs as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated Mϴ hyperactivation.Graphical abstract IRAK4i therapy counteracts Mϴ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human Mϴs. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-022-04329-8.

Interleukin Receptor Associated Kinase 1 Signaling and Its Association with Cardiovascular Diseases.

Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) directly interact with intracellular interleukin receptor associated kinase (IRAK) family members to initialize innate immune and inflammatory responses following activation by pathogen-associated or host-derived elements. Although four IRAK family members [IRAK1, 2, 3 (i.e., IRAK-M), and 4] are involved in TLR and IL-1R signaling pathways, IL-1R > IRAK1 signaling appears to be the most studied pathway, with sufficient evidence to support its central role linking the innate immune response to the pathogenesis of various diseases, including cancers, metabolic disorders, and non-infectious immune disorders. However, IRAK1's involvement in cardiovascular diseases was only recently revealed and the detailed mechanism underling the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and heart failure (all non-infectious disorders), remains largely unknown with very limited publications to date. This review aims to summarize the overall roles of the IRAK family, especially IRAK1, in mediating the development of cardiovascular diseases.

Regulatory roles of an atypical ubiquitin ligase UBE2O in orphans of multiprotein complexes for degradation.

UBE2O as an atypical ubiquitin-conjugating enzyme possesses an E2-E3 hybrid enzyme activity. It can regulate substrate levels or transcriptional activities by cooperating with other E3 ubiquitin ligases or forming homomeric complexes displaying intrinsic E2 and E3 activities. UBE2O controls the quality of cell proteome including protein degradation, modification, transport and location. Recent studies reveal that UBE2O plays a vital role in intracellular protein ubiquitination processes by regulating BMP/SMAD, TRAF/NF-κB, mTOR/HIF1a and IL-1β/IRAK4 signaling pathways, c-Maf stability and BAP1 subcellular location, which is proposed as a quality control supervisor of multiprotein complexes for degradation. Its abnormality leads to a variety of physical activity disorders and even occurrence of cancer. UBE2O is entirely distinct in molecular structure and functions from other E2 ubiquitin ligase. Exploring and elucidating regulatory mechanism of UBE2O may identify novel crucial molecular targets so as to pave therapeutic approaches for ubiquitination-associated metabolic disorders and diseases. Here, we particularly feature regulatory pathways of UBE2O in orphans of multiprotein complexes for degradation and its potential application.

Regulatory roles of an atypical ubiquitin ligase UBE2O in orphans of multi-protein complexes for degradation

UBE2O as an atypical ubiquitin-conjugating enzyme possesses an E2-E3 hybrid enzyme activity. It can regulate substrate levels or transcriptional activities by cooperating with other E3 ubiquitin ligases or forming homomeric complexes displaying intrinsic E2 and E3 activities. UBE2O controls the quality of cell proteome including protein degradation, modification, transport and location. Recent studies reveal that UBE2O plays a vital role in intracellular protein ubiquitination processes by regulating BMP/SMAD, TRAF/ NF-κB, mTOR/HIF1a and IL-1ß/IRAK4 signaling pathways, c-Maf stability and BAP1 subcellular location, which is proposed as a quality control supervisor of multiprotein complexes for degradation. Its abnormality leads to a variety of physical activity disorders and even occurrence of cancer. UBE2O is entirely distinct in molecular structure and functions from other E2 ubiquitin ligase. Exploring and elucidating regulatory mechanism of UBE2O may identify novel crucial molecular targets so as to pave therapeutic approaches for ubiquitination-associated metabolic disorders and diseases. Here, we particularly feature regulatory pathways of UBE2O in orphans of multiprotein complexes for degradation and its potential application.

Tumor cell intrinsic RON signaling suppresses innate immune responses in breast cancer through inhibition of IRAK4 signaling.

Increasing evidence suggests that cancer cells require both alterations in intrinsic cellular processes and the tumor microenvironment for tumor establishment, growth, and progression to metastatic disease. Despite this, knowledge of tumor-cell intrinsic molecular mechanisms controlling both tumor cell processes as well as the tumor microenvironment is limited. In this study, we provide evidence demonstrating the novel role of RON signaling in regulating breast cancer initiation, progression, and metastasis through modulation of tumor cell intrinsic processes and the tumor microenvironment. Using clinically relevant models of breast cancer, we show that RON signaling in the mammary epithelial tumor cells promotes tumor cell survival and proliferation as well as an immunopermissive microenvironment associated with decreased M1 macrophage, natural killer (NK) cell, and CD8+ T cell recruitment. Moreover, we demonstrate that RON signaling supports these phenotypes through novel mechanisms involving suppression of IRAK4 signaling and inhibition of type I Interferons. Our studies indicate that activation of RON signaling within breast cancer cells promotes tumor cell intrinsic growth and immune evasion which support breast cancer progression and highlight the role of targeting RON signaling as a potential therapeutic strategy against breast cancer.

Discovery of a Selective, Covalent IRAK1 Inhibitor with Antiproliferative Activity in MYD88 Mutated B-Cell Lymphoma.

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström's macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development.

Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA.

BackgroundFibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play an important role in the pathogenesis of rheumatoid arthritis (RA). Dickkopf-1 (DKK-1) has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Here, potential impairation on the activity of FLSs derived from RA to small interfering RNAs (siRNAs) targeting DKK-1 was investigated.MethodssiRNAs targeting DKK-1 were transfected into FLSs of patients with RA. Interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-β1, TGF-β2 and monocyte chemoattractant protein (MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Invasion assay and 3H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-κB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK) and β-catenin in FLSs.ResultsDKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs (P < 0.01). siRNAs induced a significant reduction of the levels of IL-6, IL-8, MMP2, MMP3 and MMP9 in FLSs compared to the control group (P < 0.05). DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs (P < 0.05). Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, β-catenin, a pivotal downstream molecule of the Wnt signaling pathway was increased.ConclusionsBy inhibiting DKK-1, we were able to inhibit the proliferation, invasion and pro-inflammatory cytokine secretion of FLSs derived from RA, which was mediated by the ERK or the IRAK-1 signaling pathway. These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

Jieduquyuziyin Prescription-Treated Rat Serum Suppresses Activation of Peritoneal Macrophages in MRL/Lpr Lupus Mice by Inhibiting IRAK1 Signaling Pathway.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, and Jieduquyuziyin prescription (JP) is a traditional Chinese medicine (TCM) formula that has been testified to be effective for SLE treatment as an approved hospital prescription for many years in China. However, its mechanism of action in the treatment of this disease is largely unknown. The purpose of this study was to determine whether JP-treated rat serum can inhibit the activation of peritoneal macrophages in MRL/lpr mice by downregulating the IRAK1 signaling pathway, thereby achieving the effect of improving SLE. The JP-treated rat serum was prepared, and the peritoneal macrophages of MRL/lpr lupus mice were isolated in vitro, and the effect of JP on cell viability was detected by the CCK8 method. After LPS induction and shRNA lentiviral transfection, the effect of JP on the expression of IRAK1 in cells was detected by immunofluorescence staining. The content of TNF-α and IL-6 in the cell supernatant was determined by ELISA. The expression of IRAK1, NF-κB, TNF-α, and IL-6 mRNA was detected by RT-PCR, and the expression levels of IRAK1, p-IRAK1, TRAF6, IKBα, p-IKBα, IKK + IKK, NF-κB, and p-NF-κB proteins was detected by western blot method. We investigated the role of JP in peritoneal macrophages of the MRL/lpr mouse and identified the possible mechanisms of action. The results showed that JP could reduce the phosphorylation of IRAK1 and its downstream proteins induced by LPS and inhibit the expression of inflammatory cytokines, including TNF-α and IL-6. In addition, after the transfection of cells with shRNA lentiviral, the results of JP tended to be consistent. In conclusion, JP may inhibit the activation of peritoneal macrophages in MRL/lpr mice by downregulating the IRAK1-NF-κB signaling pathway, and IRAK1 may be a potential target for JP treatment of SLE.

349 Hidradenitis Suppurativa Lesions are Characterized by Increased Kinase Signaling Activity

Hidradenitis Suppurativa (HS) is a chronic inflammatory skin disease that affects approximately 1 – 4% of the world population and is characterized by recurrent abscesses, nodules, and scars in the apocrine gland-bearing areas. The most frequently affected areas include the armpits, groin, genital area, abdomen, buttocks and under the breasts. Overall, HS is a painful and disfiguring disease and is associated with a significantly impaired quality of life. The underlying mechanisms which drive disease pathogenesis are not completely understood; therefore limited targeted therapies exist for these patients with surgical management being the primary treatment for severe HS. This study was conducted to improve our understanding of the downstream signaling pathways which are differentially expressed in HS patients and may drive disease pathogenesis. RNA was isolated from the skin of 16 healthy subjects and 34 patients with mild to severe HS. The expression of 15 genes was investigated using QuantiGene Plex assay with a focus on signaling molecules. Overall, a majority of the genes tested (12 of 15) showed a significant (p<0.0001) increase in gene expression in the HS patients compared to the healthy subjects. Specifically, HS patients had elevated expression of kinases such as IRAK1 (fold change = 7.3), IRAK2 (fold change = 52.4), SYK (fold change = 4.7) and JAK-STAT signaling molecules JAK1 (fold change = 2.3), JAK3 (fold change = 68.9), TYK2 (fold change = 3.8), STAT1 (fold change = 12.0), STAT2 (fold change = 23.3), STAT3 (fold change = 2.1), STAT5A (fold change = 2.2), STAT5B (fold change = 32.2), and STAT6 (fold change = 8.2), compared to healthy subjects. These data suggest a role for JAK signaling in the pathogenesis of HS and support the rationale for their subsequent antagonism in this population.

IRAK1 and IRAK4 signaling proteins are dispensable in the response of human neutrophils to Mycobacterium tuberculosis infection.

The involvement of neutrophils in the host response to Mycobacterium tuberculosis (Mtb) infection is not as well recognized as the involvement of macrophages and dendritic cells. Thus, this study gives more insight on the impact of the virulent Mtb H37Rv strain on proapoptotic and proinflammatory functions of human neutrophils in vitro. We found that neutrophils are not able to kill Mtb during the infection process, probably due to the lack of reactive oxygen species and nitric oxide production in response to bacteria. However, infected neutrophils effectively released cytokines, chemoattractant interleukin (IL) 8 and proinflammatory IL-1β. Moreover, Mtb enhanced the early apoptosis of neutrophils at 2 h postinfection. Additionally, this proapoptotic and proinflammatory response of neutrophils to Mtb infection occurred in an IRAK1- and IRAK4-independent manner. We also found that Mtb did not affect the surface expression of Toll-like receptor (TLR) 2 and slightly enhanced the surface expression of TLR4, but did not influence mRNA levels of both TLRs during the infection process. In conclusion, we show that the inhibition of signaling proteins activated by MyD88-dependent pathway did not participate in the biological activity of neutrophils against Mtb.

Development of Potent and Selective Pyrazolopyrimidine IRAK4 Inhibitors.

A series of pyrazolopyrimidine inhibitors of IRAK4 were developed from a high-throughput screen (HTS). Modification of an HTS hit led to a series of bicyclic heterocycles with improved potency and kinase selectivity but lacking sufficient solubility to progress in vivo. Structure-based drug design, informed by cocrystal structures with the protein and small-molecule crystal structures, yielded a series of dihydrobenzofurans. This semisaturated bicycle provided superior druglike properties while maintaining excellent potency and selectivity. Improved physicochemical properties allowed for progression into in vivo experiments, where lead molecules exhibited low clearance and showed target-based inhibition of IRAK4 signaling in an inflammation-mediated PK/PD mouse model.

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

Abstract 1131: IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that occurs disproportionately in the elderly population. First line therapy is a regimen of anthracycline and cytarabine treatment or, more recently, demethylating agents. However, elderly AML patients are unable to tolerate aggressive chemotherapy regimens and there is an urgent need for targeted, tolerable therapies. To identify candidate pathways for targeted treatments, a microarray on transcripts for inflammatory signaling was performed on 588 primary patient AML samples. Non-hierarchical clustering revealed that Toll-like receptor (TLR) signaling transcripts are overexpressed in myelomonocytic and monocytic AML subtypes (M4 and M5 AML subtypes, respectively). Myeloid differentiation primary response 88 (MYD88) and downstream IL-1 receptor-associated kinases (IRAKs) were found to be overexpressed in these AML subtypes. MYD88 is an adaptor molecule for IL-1 and most TLR mediated signaling. To determine whether MYD88-dependent signaling is required for leukemogenesis, we derived a CreERT2-LoxP inducible system to delete Myd88 in a murine leukemia model. Because M4 and M5 AML subtypes are enriched for mutations in the mixed lineage leukemia (MLL) gene and overexpressed TLRs, MYD88, and IRAK2, an MLL-AF9 expressing construct was utilized to transform murine progenitor cells into leukemia cells. To study whether Myd88-dependent signaling is required for in vitro colony formation of the leukemic cells, Myd88 knockout (Myd88-/-) MLL-AF9 leukemic cells were subjected to colony-forming assays. Compared to Myd88 wild-type (Myd88fl/fl and Myd88+/+) leukemia cells, reduced colony-forming capacity was observed in the Myd88-/- leukemia cells. Further, reduced proliferation was observed in Myd88-/- cells compared to Myd88+/+ cells. Cell surface staining indicates Myd88-/- cells exhibit a loss of CD117, a marker of myeloid stemness, and a gain of CD11b, a marker of myeloid differentiation, when compared to Myd88+/+ cells, indicating that Myd88 deletion correlates with MLL-AF9 leukemia cell partial differentiation. In vivo transplantation of Myd88-/- cells delays leukemia development compared to Myd88+/+ cells. To determine whether IRAK1, a downstream kinase of MYD88, is also required to prevent partial differentiation, an IRAK1 shRNA knockdown cell line was generated and assayed for partial differentiation. In IRAK1KD MLL-AF9 cells, no partial differentiation was detected. Similarly, application of an IRAK1/4 inhibitor to Myd88+/+ MLL-AF9 cells failed to induce partial differentiation, despite suppressing proliferation. This indicates that MYD88-dependent signaling has functions independent of IRAK1 signaling that prevent partial differentiation in MLL-AF9 leukemia cells. Thus, MYD88 and IRAK1 signaling appear to mediate divergent functions, and both MYD88 and IRAK1 function appear to be required for MLL-AF9 leukemia. Citation Format: Joseph M. Cannova, Wei Wei, Rafael Gutierrez, Peter Breslin, Jiwang Zhang. IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1131.

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.