Role Of TANK-binding Kinase 1 Research Articles

TBK1 is involved in M-CSF-induced macrophage polarization through mediating the IRF5/IRF4 axis.

TANK binding kinase 1 (TBK1) is an important kinase that is involved in innate immunity and tumor development. Macrophage colony-stimulating factor (M-CSF) regulates the differentiation and function of macrophages towards the immunosuppressive M2 phenotype in the glioblastoma multiforme microenvironment. The role of TBK1 in macrophages, especially in regulating macrophage polarization in response to M-CSF stimulation, remains unclear. Here, we found high TBK1 expression in human glioma-infiltrating myeloid cells and that phosphorylated TBK1 was highly expressed in M-CSF-stimulated macrophages but not in granulocyte-macrophage CSF-induced macrophages (granulocyte-macrophage-CSF is involved in the polarization of M1 macrophages). Conditional deletion of TBK1 in myeloid cells induced M-CSF-stimulated bone marrow-derived macrophages to exhibit a proinflammatory M1-like phenotype with increased protein expression of CD86, interleukin-1β and tumor necrosis factor-α, as well as decreased expression of arginase 1. Mechanistically, TBK1 deletion or inhibition by amlexanox or GSK8612 reduced the expression of the transcription factor interferon-regulatory factor (IRF)4 and increased the level of IRF5 activation in macrophages stimulated with M-CSF, leading to an M1-like profile with highly proinflammatory factors. IRF5 deletion reversed the effect of TBK1 inhibition on M-CSF-mediated macrophage polarization. Our findings suggest that TBK1 contributes to the regulation of macrophage polarization in response to M-CSF stimulation partly through the IRF5/IRF4 axis.

Crosstalk and Prospects of TBK1 in Inflammation.

TANK-binding kinase 1 (TBK1) is a pivotal mediator of innate immunity, activated by receptors such as mitochondrial antiviral signaling protein (MAVS), stimulator of interferon genes (STING), and TIR-domain-containing adaptor inducing interferon-β (TRIF). It modulates immune responses by exerting influence on the type I interferons (IFN-Is) signaling and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, Over the past few years, TBK1 multifaceted role in both immune and inflammatory responses is increasingly recognized. This review aims to scrutinize how TBK1 operates within the NF-κB pathway and the interferon regulatory transcription factor 3 (IRF3)-dependent IFN-I pathways, highlighting the kinases and other molecules involved in these processes. This analysis reveals the distinctive characteristics of TBK1's involvement in these pathways. Furthermore, it has been observed that the role of TBK1 in exerting anti-inflammatory or pro-inflammatory effects is contingent upon varying pathological conditions, indicating a multifaceted role in immune regulation. TBK1's evolving role in various diseases and the potential of TBK1 inhibitors as therapeutic agents are explored. Targeting TBK1 may provide new strategies for treating inflammatory disorders and autoimmune diseases associated with IFN-Is, warranting further investigation.

TBK1 promotes thyroid cancer progress by activating the PI3K/Akt/mTOR signaling pathway.

Thyroid cancer has received increasing attention; however, its detailed pathogenesis and pathological processes remain unclear. We investigated the role of TANK-binding kinase 1 (TBK1) in the progression of thyroid cancer. The expression of TBK1 in thyroid cancer and normal control tissues was analyzed using real-time quantitative polymerase chain reaction. The function of TBK1 on thyroid cancer cells was detected using MTT, colony formation, wound healing, and Transwell assays. The xenograft assay was carried out to check on the role of TBK1 in thyroid cancer. TBK1 was highly expressed in thyroid tumors. High expression of TBK1 raised viability, proliferation, migration, and invasion of thyroid cancer cells. Gene set enrichment analysis revealed that TBK1 activated the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway. In addition, Myc-associated zinc finger protein (MAZ) was overexpressed in thyroid cancer and transcriptionally activated BK1. MAZ silence reversed the effects of TBK1 overexpression on thyroid cancer progression. Cotransfection with MAZ small-interfering RNA(siRNA) and TBK1 siRNA did not strengthen the inhibitory effect of TBK1 silencing on the thyroid cancer cells. The xenograft tumor assay showed that TBK1 short hairpinRNA inhibited tumor growth. MAZ silencing inhibited tumor progress of thyroid cancer cells, whereas this inhibitory effect was reversed by TBK1 overexpression.

Targeting TBK1 attenuates ocular inflammation in uveitis by antagonizing NF-κB signaling

Uveitis with complex pathogenesis is a kind of eye emergency involving refractory and blinding inflammation. Dysregulation of TANK binding kinase 1 (TBK1), which plays an important role in innate immunity, often leads to inflammatory diseases in various organs. However, the role of TBK1 in uveitis remains elusive. In this study, we identified that the mRNA expression level of TBK1 and its phosphorylation level were significantly increased in peripheral blood mononuclear cells (PBMCs) of patients with uveitis. Consistent with this, the expression of Tbk1 was elevated in the ocular tissues of uveitis rats and primary peritoneal macrophages while its phosphorylation levels, which present activation forms, were upregulated as well, accompanied by an increase in the level of nuclear factor-κB (NF-κB) and proinflammatory cytokines. In addition, inhibition of TBK1 may effectively reduce the inflammatory response of uveitis rats by blocking NF-κB entry into the nucleus and impeding the initiation of NLRP3 inflammasome- and caspase-1-mediated pyroptosis pathways.

Knockdown of TANK-Binding Kinase 1 Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Molecular-Targeted Drugs.

The protein kinase, TANK-binding kinase 1 (TBK1), not only regulates various biological processes but also functions as an important regulator of human oncogenesis. However, the detailed function and molecular mechanisms of TBK1 in hepatocellular carcinoma (HCC), especially the resistance of HCC cells to molecular-targeted drugs, are almost unknown. In the present work, the role of TBK1 in regulating the sensitivity of HCC cells to molecular-targeted drugs was measured by multiple assays. The high expression of TBK1 was identified in HCC clinical specimens compared with paired non-tumor tissues. The high level of TBK1 in advanced HCC was associated with a poor prognosis in patients with advanced HCC who received the molecular-targeted drug, sorafenib, compared to patients with advanced HCC patients and a low level of TBK1. Overexpression of TBK1 in HCC cells induced their resistance to molecular-targeted drugs, whereas knockdown of TBK1 enhanced the cells’ sensitivity to molecular-targeted dugs. Regarding the mechanism, although overexpression of TBK1 enhanced expression levels of drug-resistance and pro-survival-/anti-apoptosis-related factors, knockdown of TBK1 repressed the expression of these factors in HCC cells. Therefore, TBK1 is a promising therapeutic target for HCC treatment and knockdown of TBK1 enhanced sensitivity of HCC cells to molecular-targeted drugs.

TBK1 Mediates Innate Antiviral Immune Response against Duck Enteritis Virus.

Duck enteritis virus (DEV) can infect several types of waterfowl can cause high mortality and huge economic losses to the global waterfowl industry. Type I interferons (IFN) are important for host defense against virus infection through induction of antiviral effector molecules. TANK-binding kinase 1 (TBK1) is a key kinase required for the induction of type I IFNs; however, the role of TBK1 on DEV infection remains unclear. Here, we observed that the expression levels of TBK1 and IFN-β were upregulated during DEV infection in vivo and in vitro. Thus, the function of TBK1 on DEV infection was determined. The results showed that overexpression of TBK1 reduced DEV infection and knockdown of TBK1 resulted in the increased of DEV infection. Additionally, TBK1 overexpression upregulated the expression of IFN-β and a few interferon-stimulated genes (ISGs), which thus inhibited the synthesis of DEV glycoprotein B. On the other hand, the TBK1 inhibitor Amlexanox down-regulated the expression levels of IFN-β and IRF3. Interestingly, the expression levels of MAVS and GSK-3β were decreased in the cells treated with Amlexanox. Furthermore, overexpression of TBK1 activated the expression of upstream molecules MAVS and GSK-3β. Whereas, the expression of TBK1, IRF3 and IFN-β was inhibited by the GSK-3β inhibitor SB216763. Our findings suggest that DEV–stimulated TBK1 may be involved in defense against DEV infection.

Recent progress in small molecule TBK1 inhibitors: a patent review (2015– 2020)

ABSTRACT Introduction: TANK-binding kinase 1 (TBK1) is a key mediator of innate immunity processes and studies have reported on its role in inflammatory and autoimmune diseases. Moreover, several studies have also described the important role of TBK1 in cancer and metabolic disorders. Therefore, there is increasing interest in this noncanonical IKK serine/threonine kinase family member as a drug target in both the scientific community and the pharmaceutical industry as indicated by the growing number of patents reporting on these efforts. Areas covered: This review covers the patent literature from 2015 to 2020 issued by the World, US and European patent offices on novel TBK1 small molecule inhibitors as well as patents claiming new applications of TBK1 inhibitors. Expert opinion: The high complexity TBK1 biology greatly increases the challenge of pursuing it as a drug target. The recent discovery of several small molecule inhibitors, particularly those with high selectivity, will enable further exploration of TBK1s biological role and its validation as a drug target.

Tank Binding Kinase 1 modulates spindle assembly checkpoint components to regulate mitosis in breast and lung cancer cells

Error-free progression through mitosis is critical for proper cell division and accurate distribution of the genetic material. The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase regulates the progression from metaphase to anaphase and its activation is controlled by the cofactors Cdc20 and Cdh1. Additionally, genome stability is maintained by the spindle assembly checkpoint (SAC), which monitors proper attachment of chromosomes to spindle microtubules prior to cell division. We had shown a role for Tank Binding Kinase 1 (TBK1) in microtubule dynamics and mitosis and here we describe a novel role of TBK1 in regulating SAC in breast and lung cancer cells. TBK1 interacts with and phosphorylates Cdc20 and Cdh1 and depletion of TBK1 elevates SAC components. TBK1 inhibition increases the association of Cdc20 with APC/C and BubR1 indicating inactivation of APC/C; similarly, interaction of Cdh1 with APC/C is also enhanced. TBK1 and TTK inhibition reduces cell viability and enhances centrosome amplification and micronucleation. These results indicate that alterations in TBK1 will impede mitotic progression and combining TBK1 inhibitors with other regulators of mitosis might be effective in eliminating cancer cells.

The E3 Ubiquitin Ligase TBK1 Mediates the Degradation of Multiple Picornavirus VP3 Proteins by Phosphorylation and Ubiquitination.

TANK-binding kinase 1 (TBK1) is essential for interferon beta (IFN-β) production and innate antiviral immunity. However, other, additional functions of TBK1 have remained elusive. Here, we showed that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. Further evidence showed that TBK1 could also be self-ubiquitylated in vivo Importantly, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Mechanistically, TBK1 phosphorylated multiple picornavirus VP3 proteins at serine residues and ubiquitinated them via K63-linked ubiquitination at lysine residues. In addition, the C426 and C605 residues of TBK1 were not essential for TBK1 innate immunity activity; however, these residues were required for degradation of multiple picornavirus VP3 proteins and for its E3 ubiquitin ligase activity. Hence, our findings identified a novel role of TBK1 in regulating the virus life cycle and provided new insights into the molecular mechanisms of TBK1-mediated antiviral response.IMPORTANCE TBK1 is an important adaptor protein required for innate immune response to viruses, but its other functions were unknown. In this study, we found that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. In addition, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Our report provides evidence that TBK1 plays a role in viral protein degradation.

Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo

choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine–threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.

Research progress on the role of TANK-binding kinase 1 in anti-virus innate immune response

The innate immune response against viral infection is mainly relies on type I interferon, the production of which is mediated by TANK-binding kinase 1 (TBK1). It is revealed that the downstream TBK1 is activated by viral nucleic acid sensors RIG-I, cGAS and TLR3. The activity of TBK1 is complexly and precisely regulated by different type of protein modifications, including phosphorylation, ubiquitination and Sumolylation. This article focuses on the role of TBK1 in anti-viral innate immunity and the regulatory mechanism for the TBK1 activation.

Abstract A40: Targeting TBK1 promotes apoptosis in MEK-inhibitor resistant mutant NRAS melanoma cells

Abstract Melanoma is a devastating form of skin cancer. Fifteen to twenty percent of melanoma patients have an activating mutation in the GTPase, NRAS. Despite advances in the targeted inhibitor treatment for mutant BRAF melanoma patients, the options for mutant NRAS patients remain poor. TANK-binding kinase 1 (TBK1) is an atypical IκB kinase family member that acts downstream of the RAS effector RalGEF but the role of TBK1 in melanoma is not known. We found that NRAS overexpression in wild-type NRAS melanoma cells increased TBK1 phosphorylation. In a panel of NRAS mutant melanoma cells, we characterized sensitivity to MEK inhibition in 2D as well as 3D cell cultures that mimic the dermal microenvironment. We show that there is a differential response to MEK inhibition in this panel. TBK1 inhibition, through either siRNA transfection or pharmacological inhibitors, cooperated with MEK inhibition to promote apoptosis. These effects were particularly noted in those NRAS mutant cell lines that are insensitive to MEK inhibition. By contrast, this effect was absent in melanoma cell lines wild type for NRAS. These results identify TBK1 as a downstream effector for NRAS mutant melanoma and as a potential target for use in combinatorial therapy in this subset of patients for whom current therapeutic options are limited. Citation Format: Ha Linh Vu, Andrew E. Aplin. Targeting TBK1 promotes apoptosis in MEK-inhibitor resistant mutant NRAS melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A40. doi: 10.1158/1557-3125.RASONC14-A40

TBK1 protects vacuolar integrity during intracellular bacterial infection.

TANK-binding kinase-1 (TBK1) is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella), more extensive bacterial proliferation was observed in tbk1−/− than tbk1+/+ cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1−/−cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity.