TANK-binding Kinase 1 Expression Research Articles

CDK12 loss inhibits cell proliferation by regulating TBK1 in non-small cell lung cancer cells

Lung cancer is one of the most common malignant tumors and has a poor prognosis and a low survival rate. Traditional treatments, such as radiotherapy and chemotherapy, still face some challenges because of high drug resistance and toxicity. Therefore, it is necessary to discover a new kind of targeted drug with low toxicity and high efficiency. CDK12 is a cell cycle-dependent kinase whose main function is to activate RNA polymerase II (RNAPII) and promote the transcriptional extension of RNA. However, the role and molecular mechanism of CDK12 in lung cancer are still unclear.In this study, the mutation and RNA-Seq data of CDK12 in lung adenocarcinoma and squamous cell carcinoma were downloaded from The Cancer Genome Atlas (TCGA) database and analyzed with the custom scripts. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and cell colony formation assays. A subcutaneous tumor experiment in nude mice was used to examine the effects of CDK12 knockdown on the in vivo tumor growth of NSCLC cells. The cell cycle distribution and the apoptosis rate of lung cancer cells were assessed by flow cytometry. Regulation of TANK-binding kinase 1 (TBK1) by CDK12 was evaluated by quantitative PCR, immunoprecipitation and Western blot analysis.In this study we have analyzed the mutation and expression data of The Cancer Genome Atlas (TCGA) database and found that CDK12 is highly expressed in lung cancer tissues. Clinical correlation analysis showed that high expression of CDK12 in NSCLC reduces patient survival, but its high expression is only related to early tumor progression and has no significant correlation with late tumor progression and metastasis. Furthermore, we present evidence that CDK12 depletion in lung cancer cell lines not only leads to the inhibition of cell growth and induces apoptosis but also inhibits tumor growth of NSCLC cells in vivo. CDK12 positively regulates the expression of the oncogene TBK1 in lung cancer cells. These results revealed that CDK12 affects the progression of non-small cell lung cancer through positive regulation of TBK1 expression, suggesting that CDK12 might be a potential molecular target for the treatment of non-small cell lung cancer.

Serine/threonine kinase TBK1 promotes cholangiocarcinoma progression via direct regulation of β-catenin

Cholangiocarcinoma (CCA) is a highly heterogeneous and metastatic malignancy with a poor prognosis even after curative hepatectomy. Studies exploring its pathogenesis and identifying effective therapeutic targets are urgently needed. In this study, we found that TANK-binding kinase 1 (TBK1), a serine/threonine-protein kinase, showed a dynamic increase during the different stages of murine spontaneous CCA carcinogenesis (hyperplasia, dysplasia, and CCA). TBK1 was upregulated in human tissues, including intrahepatic (n = 182) and extrahepatic (n = 40) CCA tissues, compared with nontumor tissues, and the elevated expression of TBK1 was positively correlated with larger tumour diameter, lymph node metastasis, and advanced TNM stage. Functional studies indicated that TBK1 promoted CCA growth and metastasis both in vitro and in vivo. TBK1 directly interacts with β-catenin, promoting its phosphorylation at the S552 site and its nuclear translocation, which further activates EMT-related transcriptional reprogramming. GSK-8612, a TBK1 inhibitor or a kinase-inactivating mutation, effectively suppresses the above processes. In addition, we found that low-density lipoprotein receptor (LDLR), which mediates the endocytosis of cholesterol, was upregulated in CCA. Therefore, we designed a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting TBK1 (Cho-TBK1-HDO), which could accumulate in CCA cells via LDLR, reduce the TBK1 mRNA level and inhibit intrahepatic metastasis of CCA. Besides, in the experimental group of 182 ICC patients, high TBK1 expression combined with high nuclear β-catenin expression predicted a worse prognosis. In summary, TBK1 might serve as a potential prognostic biomarker and therapeutic target for patients with CCA.

IRF5 knockdown reverses TDP-related phenotypes partially by increasing TBK1 expression

Interferon-regulatory factor 5 (IRF5) participates in the regulation of apoptosis, affects the phenotype of inflammatory macrophages and plays an essential role in the inflammatory response. However, the role of IRF5 in the progression of amyotrophic lateral sclerosis (ALS) remains largely unknown. Here, we show that IRF5 mainly accumulated in the nucleus in cells expressing the truncated 25 kD C-terminal fragments of TDP-43 (TDP-25, named TDP-25 cells hereafter). IRF5 knockdown using a lentivirus carrying an shRNA in TDP-25 cells exerted a protective effect and reduced the level of the apoptosis-related protein cleaved caspase-9 and the cell cycle arrest protein p21, while increasing the expression of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its target molecule glutamate-cysteine ligase modulatory subunit (GCLM). Furthermore, IRF5-knockdown cells showed improved mitochondrial swelling and cristae dilation. In addition, we found that IRF5 mediated neuronal injury partly through the negative regulation of TANK-binding kinase 1 (TBK1). These data indicate that the loss of IRF5 in TDP-25 cells exerts a protective effect mainly by inhibiting apoptosis, regulating cell cycle arrest and alleviating oxidative stress.

LINC00483 promotes proliferation and metastasis through the miR-19a-3p/TBK1/MAPK axis in pancreatic ductal adenocarcinoma (PDAC).

BackgroundLong noncoding RNAs (lncRNAs) have been found to promote tumor progression. However, the role of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) requires more investigation.MethodsIn this study, microarray was used to measure lncRNA levels in 3 pairs of PDAC tissues. As the highest upregulated lncRNA, LINC00483 was selected for further investigation to determine its functions in PDAC. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to confirm LINC00483 level in PDAC. PDAC cell lines were transfected with short hairpin RNA (shRNA) or microRNA (miRNA). 5-ethynyl-2’-deoxyuridine (EdU) assay, colony formation assay, wound healing assay, transwell assay, and xenograft mouse models were used to evaluate LINC00483 inhibition in vitro and in vivo. Luciferase reporter assay was performed to confirm binding sites of LINC00483 with miR-19a-3p, and miR-19a-3p with TANK-binding kinase 1 (TBK1). Immunohistochemistry (IHC) was performed to evaluate TBK1 and c-myc expression in PDAC tissues. Western blot was used to elucidate the LINC00483/miR-19a-3p/TBK1/mitogen-activated protein kinase (MAPK) axis.ResultsOur data showed that LINC00483 was significantly upregulated in PDAC compared to normal tissue. High level of LINC00483 was correlated with advanced clinical stage, tumor invasion and metastasis, and adverse prognosis in PDAC patients. LINC00483 suppression inhibited proliferation and invasion in vitro and tumor development in vivo via modulation of miR-19a-3p expression. Subsequently, we found that miR-19a-3p binds to TBK1 in PDAC and LINC00483 could regulate PDAC cell progression by regulating miR-19a-3p via the TBK1/MAPK pathway.ConclusionsThe results of our study suggested that the LINC00483/miR-19a-3p/TBK1/MAPK axis contributed to PDAC progression, which provides a potential therapeutic target for PDAC treatment.

Abstract P5-08-10: TBK1 inhibition potentiates the efficacy of AXL-targeted therapy in aggressive breast cancer preclinical models

Abstract Background: Novel mechanism-oriented targeted therapies are needed for aggressive breast cancers such as triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC). We previously showed the oncogenic receptor tyrosine kinase AXL crucially contributes to the aggressiveness of TNBC and TN-IBC, which suggests that AXL is a potential therapeutic target. However, AXL-targeted therapy is insufficient to control the progress of aggressive breast cancer in preclinical models. Therefore, to further enhance the efficacy of AXL-targeted therapy in aggressive breast cancer, we conducted a synthetic lethality screen of pooled kinome siRNA and identified TANK-binding kinase 1 (TBK1) as a potential target. TBK1 is a necessary serine/threonine protein kinase that regulates innate and adaptive immune responses, T cell migration, and anti-tumor immune responses. In the present work, we validated that AXL and TBK1 inhibition have a synergistic anti-tumor effect by modulating the tumor microenvironment (TME). Methods: To determine the synergistic effect of AXL and TBK1 inhibition on cell proliferation and colony formation in vitro, we first used siRNA-mediated knockdown (KD) or CRISPR/Cas9 knockout (KO) to independently silence AXL and TBK1 expression. We also used pharmacological inhibitors to inactivate AXL and TBK1 signaling and conducted similar studies. To determine the effect of targeting TBK1 and AXL pathways in vivo, we injected control or TBK1 KO murine 4T1.2 TNBC cells into BALB/c mice and assessed the efficacy of the AXL inhibitor TP-0903 in reducing the tumor growth. We used multicolor flow cytometry and multiplexed immunostaining to examine the population of TME components in control and TBK1 KO 4T1.2 tumors treated with vehicle or TP-0903. We also used an in vitro migration assay to test the effect of targeting AXL and TBK1 on the recruitment of CD8+ T cells. We performed a mouse chemokine array to identify intratumoral chemokines involved in regulating infiltrating immune cells and used RT-PCR to validate these findings. Results: Compared with AXL or TBK1 suppression alone, the genomic or pharmacological suppression of both AXL and TBK1 resulted in a significantly greater reduction of TNBC and TN-IBC cell growth and colony-forming ability (P < 0.01). In our syngeneic 4T1.2 TNBC mouse tumor model, TP-0903 was more effective at controlling tumor growth from TBK1-KO cells than from control cells (P < 0.0005). TBK1-KO 4T1.2 tumors treated with TP-0903 had a significantly higher population of functional cytotoxic T cells (GranzymeB+CD3+CD8+) than control KO tumors treated with vehicle or TP-0903. Furthermore, our migration assay showed that TBK1-KO/AXL-KD SUM149 cell-conditioned media attracted more CD8+ T cells than control cell-conditioned media, suggesting the inhibition of AXL/TBK1 signaling enhances the recruitment of CD8+ T cells through a paracrine effect. Our chemokine array revealed increased CXCL16 levels in TP-0903-treated TBK1-KO tumors. Conclusion: Targeting TBK1 could enhance the sensitivity of aggressive breast cancer cells to AXL-targeted therapy in vitro and in vivo. Our preliminary TME data suggest that combined AXL and TBK1 inhibition enhances the anti-tumor immune environment by upregulating secretion of CXCL16 by tumor cells, resulting in the increased recruitment of cytotoxic CD8+ T cells. In future studies, we will further elucidate the role that CXCL16 upregulation plays in this novel synergy. Significance of this findings: This study will enable us to develop hypothesis-driven novel combination therapies for patients with aggressive breast cancer that can be rapidly tested as a clinical trial, leading to an improvement of treatment and patients’ outcome. Citation Format: Lan Phi, Takashi Semba, Jason Foulks, Steven Warner, David Bearss, Savitri Krishnamurthy, James Long, James Reuben, Debu Tripathy, Naoto Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy in aggressive breast cancer preclinical models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-10.

TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression.

Intestinal inflammation is a vital precipitating factor of colorectal cancer (CRC), but the underlying mechanisms are still elusive. TANK-binding kinase 1 (TBK1) is a core enzyme downstream of several inflammatory signals. Recent studies brought the impacts of TBK1 in malignant disease to the forefront, we found aberrant TBK1 expression in CRC is correlated with CRC progression. TBK1 inhibition impaired CRC cell proliferation, migration, drug resistance and tumor growth. Bioinformatic analysis and experiments in vitro showed overexpressed TBK1 inhibited mTORC1 signaling activation in CRC along with elevated GLUT1 expression without inducing GLUT1 translation. TBK1 mediated mTORC1 inhibition induces intracellular autophagy, which in turn decreasing GLUT1 degradation. As a rescue, blocking of autophagosome and retromer respectively via autophagy-related gene 7 (ATG7) or TBC1 Domain Family Member 5 (TBC1D5) silence diminished the regulation of TBK1 to GLUT1. GLUT1 staining presented that TBK1 facilitated GLUT1 membrane translocation which subsequently enhanced glucose consumption. Inhibitor of TBK1 also decreased GLUT1 expression which potentiated drug-sensitivity of CRC cell. Collectively, TBK1 facilitates glucose consumption for supporting CRC progression via initiating mTORC1 inhibition induced autophagy which decreases GLUT1 degradation and increases GLUT1 membrane location. The adaptive signaling cascade between TBK1 and GLUT1 proposes a new strategy for CRC therapy.

TANK-Binding Kinase 1 (TBK1) Serves as a Potential Target for Hepatocellular Carcinoma by Enhancing Tumor Immune Infiltration.

BackgroundNumerous cancer types present the aberrant TANK-binding kinase 1 (TBK1) expression, which plays an important role in driving inflammation and innate immunity. However, the prognostic role of TBK1 and its relationship with immune cell infiltration in hepatocellular carcinoma (HCC) remain unclear.MethodsThe expression and prognostic value of TBK1 was analyzed by Tumor Immune Estimation Resource (TIMER), Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA), Clinical Proteomic Tumor Analysis Consortium (CPTAC) and further confirmed in the present cohort of patients with HCC. The association between TBK1 and HCC immune infiltrates, and its potential mechanism were investigated via analyses of the Tumor Immune Estimation Resource, tumor-immune system interactions database (TISIDB), CIBERSORT, STRING, and Metascape. The effect of TBK1 on immune infiltrates and the therapeutic value of targeting TBK1 were further investigated in a HCC mouse model by treatment with a TBK1 antagonist.ResultsThe level of TBK1 expression in HCC was higher than that measured in normal tissues, and associated with poorer overall survival (GEPIA: hazard ratio [HR]=1.80, P=0.038; Kaplan–Meier plotter: HR=1.87, P<0.001; CPTAC: HR=2.23, P=0.007; Our cohort: HR=2.92, P=0.002). In addition, high TBK1 expression was found in HCC with advanced TNM stage and identified as an independent poor prognostic factor for overall survival among patients with HCC. In terms of immune infiltration, tumor tissues from HCC patients with high TBK1 expression had a low proportion of CD8+ T cells, and TBK1 expression did not show prognostic value in HCC patients with enriched CD8+ T cells. Furthermore, TBK1 expression was positively correlated with the markers of T cell exhaustion and immunosuppressive cells in the HCC microenvironment. Mechanistically, the promotion of HCC immunosuppression by TBK1 was involved in the regulation of inflammatory cytokines. In vivo experiments revealed that treatment with a TBK1 antagonist delayed HCC growth by increasing the number of tumor-infiltrating CD8+ T cells.ConclusionsThe up-regulated expression of TBK1 may be useful in predicting poor prognosis of patients with HCC. In addition, TBK1, which promotes the HCC immunosuppressive microenvironment, may be a potential immunotherapeutic target for patients with HCC.

TBK1 regulates regeneration of pancreatic \u03b2-cells

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate β-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in β-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat β-cells. Conversely, TBK1 overexpression decreased sensitivity of β-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of β-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of β-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived β-cells and human islets. TBK1 expression was increased in β-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and β-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional β-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a β-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional β-cells.

Analysis of Sars-Cov-2-Associated Proteins Identify Tank-Binding Kinase-1 As an Immunotherapeutic Target in Multiple Myeloma

Background and Rationale Patients with multiple myeloma (MM) are at a higher risk of viral infection due to immune deficiency. Importantly, recent studies highlighted the severity of COVID-19 in MM. To date, however, the mechanism(s) underlying the lack of anti-viral immune response in MM are unclear. Plasmacytoid dendritic cells (pDCs) play a key role in both recognition of viral nucleic acid and initiating anti-viral activity via type 1 interferon (IFN) response signaling. We showed that interactions of dysfunctional pDCs with MM cells, T cells, and NK cells confer immune suppression in the host-MM bone marrow (BM) microenvironment (Chauhan et al, Cancer Cell 2009; 16: p309; Ray et al, Leukemia 2015; 29: p1441). Here, we analyzed the immune-pathway proteins implicated in Covid-19-host interactions [Gordon et al, Nature 2020; 583, p459] to assess whether pDC-MM interactions modulate these pathways to confer immune suppression and susceptibility to COVID-19. We identified the TLR pathway serine/threonine kinase TBK1 (TANK-binding kinase 1), involved in type I IFN induction, as a potential immunotherapeutic target in MM. Moreover, Covid-19 relies on host-Ubiquitin-proteasome-system for propagation, and we found that targeting ubiquitin receptor Rpn13 with a specific inhibitor RA190 restores TBK1 expression in MM.Methods For our studies, we examined SARS-CoV-2-human protein-protein interactions (PPIs) maps (Gordon et al, 2020). pDC-MM co-cultures, and RNAseq using next- generation sequencing (NGS): Purified MM patient pDCs were cocultured with autologous MM cells (1pDC/5MM) for 48h, followed by separation of MM cells from pDCs using FACS. Total RNA from MM cells was subjected to RNAseq analysis using Illumina NGS. Raw sequence data were analyzed to generate differential expression (DEseq2). Linear model (Limma) and its GUI (Glimma) were also used. Statistical significance: log2FC (fold change) values in coculture vs control, with a False Discovery Rate value of <0.05, was considered significant (CI > 95). The heatmap analysis was done using Morpheus software (Broad Institute, MIT). MM patient and pDCs data used for bioinformatics were from the NCBI GEO.Results A total of 41 genes involved in the Covid-19 host-pathogen immune interactions are also differentially expressed in MM after coculture with pDCs. (log2Fold change: ± 3.5-fold; p < 0.00001; pDC-MM vs MM alone). The gene expression widely varies in pDC-MM vs MM [Median (log10): -0.13 to 4.5; adj p < 0.001]. We identified 3 specific innate immunity-linked genes TBK1, IRF3 (Interferon regulatory factor 3), and RAE1 (interferon-inducible mRNA nuclear export factor) which are essential for IFN production in MM. Importantly, pDCs decrease TBK1 (Log2FC: -0.5) and RAE1 (Log2FC: -0.3) as well as induce IRF3 (Log2FC: 1.5) in MM (p < 0.0001). Analysis of publicly available gene profiling datasets on relapsed MM patient showed low levels of TBK1 and RAE1 and higher IRF3 (n = 50) [Log2FC: TBK1: -0.208; RAE1: -0.286; IRF3: 0.273; vs normal; p < 0.05). Of note, low TBK1 expression correlates with poor survival in MM patients (n = 350) and elevated TBK1 correlates with a better prognosis (p = 0.026). Similar analysis showed that most of 41 genes involved in the Covid-19 host-pathogen immune interactions are expressed in pDCs. In unstimulated pDCs, TBK1 expression is significantly lower than IRF3 and RAE1 (2-3-fold, p < 0.05). In functional studies, treatment of pDCs with IFN-α activating CpG-ODN type-A increases both TBK1 (adj P = 0.004) and RAE1 (adj P = 0.043), without significantly altering IRF3 expression. Taken together, we show that TBK1 is downregulated in pDCs, and pDCs-MM interactions further decreases TBK1 in MM, thereby attenuating IFN-mediated anti-viral immune response signaling in MM. Finally, we found that blockade of proteasome-mediated protein degradation via inhibition of Ubiquitin receptor Rpn13 upregulates TBK-1 expression, indicating potential clinical use of targeting Rpn13 in restoring anti-viral immune responses in MM.Conclusion We found that pDC-MM interactions downregulate TBK1, which in turn reduces IFN response signaling essential to generate an effective anti-viral immune response in the MM BM microenvironment. Our findings suggest that: 1) low levels of TBK1 may confer increased susceptibility of MM patients to COVID-19; and 2) targeting TBK1 may restore anti-viral immune response and reduce COVID-19 severity in MM.DisclosuresChauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Janssen: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees.

The therapeutic effect of TBK1 in intervertebral disc degeneration via coordinating selective autophagy and autophagic functions

IntroductionWhile its innate immune function has been known, recent works of literature have focused on the role of Tank binding kinase 1 (TBK1) in regulating autophagy and it is unknown whether TBK1 protects against intervertebral disc degeneration (IVDD) through affecting autophagy. ObjectivesHere, we aim to explore whether TBK1 is implicated in the pathogenesis of IVDD, and investigated the potential mechanism. MethodsWestern blotting and immunohistochemistry were used to detect the TBK1 expression in human and rat NP tissue. After TBK1 overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. Si-RNA , a utophagy inhibitors and protein phosphatase inhibitors were applied to study the mechanism of autophagy regulation. In vivo study, we further evaluated the therapeutic action of lentivirus-TBK1(Lv-TBK1)injection in a rodent IVDD model. ResultsThe TBK1 level was reduced in rat and human NP tissue. TBK1 overexpression protected against apoptosis and premature senescence. These functions of TBK1 were abolished by chloroquine-medicated autophagy inhibition.P-TBK1, an activation form of TBK, is involved in selective autophagy through directly phosphorylating P62 at Ser 403, and the activation of TBK1 is also dependent on Parkin manner. TBK1 also activated NPCs autophagy to relieve puncture injury in vivo. ConclusionWe demonstrated that TBK1 overexpression attenuated senescence and apoptosis and promoted NPCs survival via upregulating autophagy. TBK1 represents a promising avenue for IVDD treatment.

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.

Phosphorylation of the N‐terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK‐binding kinase 1 (TBK1), that efficiently phosphorylates full‐length and N‐terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1‐mediated neuroprotective effects are due to phosphorylation‐dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation.

The Specific IKKε/TBK1 Inhibitor Amlexanox Suppresses Human Melanoma by the Inhibition of Autophagy, NF-κB and MAP Kinase Pathways.

Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types. Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this “difficult-to-treat” cancer type has not been investigated so far. We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients. Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes. The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion. Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells. The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects. In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth. In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.

Investigating the TANK-binding kinase expression in multiple sclerosis patients in comparison with control group in the Iranian population

Introduction: Multiple sclerosis (MS) is one of the most common inflammatory autoimmune diseases in the central nervous system, which affects individuals ranged between 20 and 40 years of age. There is still no gene or genes set that is unique to the disease. On the other hand, TANK-binding kinase 1 (TBK1) gene is one of the most important interferon signalling molecules and has been shown to affect many autoimmune diseases, and it is obvious that it is involved in CNS inflammation. In this study, we investigated the expression of TBK1 gene as a major factor in the production and activity of interferons in patients with MS. Materials and Methods: Ninety patients with MS and 30 healthy individuals referring to Kashani Hospital in Shahrekord which matched for age and sex were selected. According to the received treatments, MS patients were divided into three groups as follows: the newly diagnosed group not receiving treatment, interferon β1α (IFNβ1α) treatment group and IFNβ1β treatment group. After filling the questionnaire, blood was sampled, total RNA was extracted and finally, cDNA was synthesised from the individuals. The TBK1 kinase gene expression was investigated using the TaqMan real-time with ΔΔCt method. Obtained data were analysed using GRAPHPAD 5 and SPSS version 21 software. Results: In this study, the two studied groups did not show any significant differences in age and sex parameters. Our results showed that TBK1 expression in newly diagnosed patients was significantly higher than the control group (P

Expression and prognostic role of IKBKE and TBK1 in stage I non-small cell lung cancer.

BackgroundThe inhibitors of nuclear factor kappa-B kinase subunit epsilon (IKBKE) and TANK-binding kinase 1 (TBK1) are important members of the nonclassical IKK family that share the kinase domain. They are important oncogenes for activation of several signaling pathways in several tumors. This study aims to explore the expression of IKBKE and TBK1 and their prognostic role in stage I non-small cell lung cancer (NSCLC).Patients and methodsA total of 142 surgically resected stage I NSCLC patients were enrolled and immunohistochemistry of IKBKE and TBK1 was performed.ResultsIKBKE and TBK1 were expressed in 121 (85.2%) and 114 (80.3%) of stage I NSCLC patients respectively. IKBKE expression was significantly associated with TBK1 expression (P=0.004). Furthermore, multivariate regression analyses showed there was a significant relationship between patients with risk factors, the recurrence pattern of metastasis and IKBKE+/TBK1+ co-expression (P=0.032 and P=0.022, respectively). In Kaplan–Meier survival curve analyses, the IKBKE+/TBK1+ co-expression subgroup was significantly associated with poor overall survival (P=0.014).ConclusionsThis is the first study to investigate the relationship between IKBKE and TBK1 expression and clinicopathologic characteristics in stage I NSCLC patients. IKBKE+/TBK1+ co-expression was significantly obvious in patients with risk factors and with recurrence pattern of distant metastasis. Furthermore, IKBKE+/TBK1+ is also an effective prognostic predictor for poor overall survival.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase, RIG-I, and MDA-5. However, its precise role is incompletely defined. By screening a human cDNA library, we showed that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes TBK1 from access to Hsp90 and interferon (IFN) promoter activation. Consistently, the Us11 protein, upon HSV infection, suppresses the expression of beta interferon (IFN-β), RANTES, and interferon-stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome-dependent pathway. Accordingly, Us11 expression facilitates HSV growth. In contrast, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrated that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.

TANK-Binding Kinase 1 (TBK1) Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation.

TANK-binding kinase 1 (TBK1) is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs) in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I) and mitochondria antiviral-signaling protein (MAVS). However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s) exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.

USP1-UAF1 deubiquitinase complex stabilizes TBK1 and enhances antiviral responses.

Optimal activation of TANK-binding kinase 1 (TBK1) is crucial for initiation of innate antiviral immunity and maintenance of immune homeostasis. Although several E3 ubiquitin ligases have been reported to regulate TBK1 activation by mediating its polyubiquitination, the functions of deubiquitinase on TBK1 activity remain largely unclear. Here, we identified a deubiquitinase complex, which is formed by ubiquitin specific peptidase 1 (USP1) and USP1-associated factor 1 (UAF1), as a viral infection-induced physiological enhancer of TBK1 expression. USP1-UAF1 complex enhanced TLR3/4 and RIG-I-induced IFN regulatory factor 3 (IRF3) activation and subsequent IFN-β secretion. Mechanistically, USP1 and UAF1 bound to TBK1, removed its K48-linked polyubiquitination, and then reversed the degradation process of TBK1. Furthermore, we found that ML323, a specific USP1-UAF1 inhibitor, attenuated IFN-β expression and enhanced viral replication both in vitro and in vivo. Therefore, our results outline a novel mechanism for the control of TBK1 activity and suggest USP1-UAF1 complex as a potential target for the prevention of viral diseases.

Common and rare TBK1 variants in early-onset Alzheimer disease in a European cohort

TANK-binding kinase 1 (TBK1) loss-of-function (LoF) mutations are known to cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), often combined with memory deficits early in the disease course. We performed targeted resequencing of TBK1 in 1253 early onset Alzheimer's disease (EOAD) patients from 8 European countries to investigate whether pathogenic TBK1 mutations are enriched among patients with clinical diagnosis of EOAD. Variant frequencies were compared against 2117 origin-matched controls. We identified only 1 LoF mutation (p.Thr79del) in a patient clinically diagnosed with Alzheimer's disease and a positive family history of ALS. We did not observe enrichment of rare variants in EOAD patients compared to controls, nor of rare variants affecting NFκB induction. Of 3 common coding variants, rs7486100 showed evidence of association (OR 1.46 [95% CI 1.13–1.9]; p-value 0.01). Homozygous carriers of the risk allele showed reduced expression of TBK1 (p-value 0.03). Our findings are not indicative of a significant role for TBK1 mutations in EOAD. The association between common variants in TBK1, disease risk and reduced TBK1 expression warrants follow-up in FTD/ALS cohorts.

A novel function of TBK1 as a target of Cdon in oligodendrocyte differentiation and myelination.

During central nervous system development, oligodendrocyte progenitors elaborate multiple branched processes to contact axons and initiate myelination. Using cultured primary rat oligodendrocytes (OLGs), we have recently demonstrated that a cell surface protein belonging to the immunoglobulin superfamily, cell adhesion molecule-related, down-regulated by oncogenes (Cdon), is important in initiating OLG differentiation and axon myelination by promoting the formation of branched cellular processes; however, the molecular mechanism by which Cdon regulates OLG differentiation is not known. Here, using Cdon immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry analysis, we identified serine/threonine kinase TANK-binding kinase 1 (TBK1) as a candidate novel target of Cdon. We confirmed this interaction using co-IP and immunofluorescence with TBK1 antibodies, showing that TBK1 partly co-localizes with Cdon along cellular processes in puncta-like structures. We show that TBK1 is expressed throughout OLG differentiation, and surprisingly, that levels of phosphorylated TBK1 (ser172) increase during OLG maturation, while total levels of TBK1 protein decrease. To investigate function, TBK1 expression was knocked down using siRNA in OLG primary cultures, reducing protein levels by 69%. Two myelin-specific proteins, myelin basic protein and myelin-associated glycoprotein, were similarly reduced when examined at day 2 and day 4 of OLG differentiation. Reduced Cdon or TBK1 expression also decreased Akt phosphorylation at Threonine 308 in OLG. Our findings provide evidence that a Cdon-TBK1 complex is associated with Akt phosphorylation and early OLG differentiation.

IKKε and TBK1 expression in gastric cancer.

Inhibitor of kappa B kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IKKs. IKKε and TBK1 share the kinase domain and are similar in their ability to activate the nuclear factor-kappa B signaling pathway. IKKε and TBK1 are overexpressed through multiple mechanisms in various human cancers. However, the expression of IKKε and TBK1 in gastric cancer and their role in prognosis have not been studied.To investigate overexpression of the IKKε and TBK1 proteins in gastric cancer and their relationship with clinicopathologic factors, we performed immunohistochemical staining using a tissue microarray. Tissue microarray samples were obtained from 1,107 gastric cancer patients who underwent R0 gastrectomy with extensive lymph node dissection and adjuvant chemotherapy.We identified expression of IKKε in 150 (13.6%) and TBK1 in 38 (3.4%) gastric cancers. Furthermore, co-expression of IKKε and TBK1 was identified in 1.5% of cases. Co-expression of IKKε and TBK1 was associated with differentiated intestinal histology and earlier T stage. In a multivariate binary logistic regression model, intestinal histologic type by Lauren classification and early AJCC stage were significant predictors for expression of IKKε and TBK1 proteins in gastric cancer. Changes in IKKε and TBK1 expression may be involved in the development of intestinal-type gastric cancer. The overexpression of IKKε and TBK1 should be considered in selected patients with intestinal-type gastric cancer.In conclusion, this is the first large-scale study investigating the relationships between expression of IKKε and TBK1 and clinicopathologic features of gastric cancer. The role of IKKε and TBK1 in intestinal-type gastric cancer pathogenesis should be elucidated by further investigation.