Inhibitor BX795 Research Articles

Duck STING mediates antiviral autophagy directing the interferon signaling pathway to inhibit duck plague virus infection

Migratory birds are important vectors for virus transmission, how migratory birds recognize viruses and viruses are sustained in birds is still enigmatic. As an animal model for waterfowl among migratory birds, studying and dissecting the antiviral immunity and viral evasion in duck cells may pave a path to deciphering these puzzles. Here, we studied the mechanism of antiviral autophagy mediated by duck STING in DEF cells. The results collaborated that duck STING could significantly enhance LC3B-II/I turnover, LC3B-EGFP puncta formation, and mCherry/EGFP ratio, indicating that duck STING could induce autophagy. The autophagy induced by duck STING is not affected by shRNA knockdown of ATG5 expression, deletion of the C-terminal tail of STING, or TBK1 inhibitor BX795 treatment, indicating that duck STING activated non-classical selective autophagy is independent of interaction with TBK1, TBK1 phosphorylation, and interferon (IFN) signaling. The STING R235A mutant and Sar1A/B kinase mutant abolished duck STING induced autophagy, suggesting binding with cGAMP and COPII complex mediated transport are the critical prerequisite. Duck STING interacted with LC3B through LIR motifs to induce autophagy, the LIR 4/7 motif mutants of duck STING abolished the interaction with LC3B, and neither activated autophagy nor IFN expression, indicating that duck STING associates with LC3B directed autophagy and dictated innate immunity activation. Finally, we found that duck STING mediated autophagy significantly inhibited duck plague virus (DPV) infection via ubiquitously degraded viral proteins. Our study may shed light on one scenario about the control and evasion of diseases transmitted by migratory birds.

TBK1 pharmacological inhibition mitigates osteoarthritis through attenuating inflammation and cellular senescence in chondrocytes

ObjectivesTANK-binding kinase 1 (TBK1) is pivotal in autoimmune and inflammatory diseases, yet its role in osteoarthritis (OA) remains elusive. This study sought to elucidate the effect of the TBK1 inhibitor BX795 on OA and to delineate the underlying mechanism by which it mitigates OA. MethodsInterleukin-1 Beta (IL-1β) was utilized to simulate inflammatory responses and extracellular matrix degradation in vitro. In vivo, OA was induced in 8-week-old mice through destabilization of the medial meniscus surgery. The impact of BX795 on OA was evaluated using histological analysis, X-ray, micro-CT, and the von Frey test. Additionally, Western blot, RT-qPCR, and immunofluorescence assays were conducted to investigate the underlying mechanisms of BX795. ResultsPhosphorylated TBK1 (P-TBK1) levels were found to be elevated in OA knee cartilage of both human and mice. Furthermore, intra-articular injection of BX795 ameliorated cartilage degeneration and alleviated OA-associated pain. BX795 also counteracted the suppression of anabolic processes and the augmentation of catabolic activity, inflammation, and senescence observed in the OA mice. In vitro studies revealed that BX795 reduced P-TBK1 levels and reversed the effects of anabolism inhibition, catabolism promotion, and senescence induction triggered by IL-1β. Mechanistically, BX795 inhibited the IL-1β-induced activation of the cGAS–STING and TLR3–TRIF signaling pathways in chondrocytes. ConclusionsPharmacological inhibition of TBK1 with BX795 protects articular cartilage by inhibiting the activation of the cGAS–STING and TLR3–TRIF signaling pathways. This action attenuates inflammatory responses and cellular senescence, positioning BX795 as a promising therapeutic candidate for OA treatment. The translational potential of this articleThis study furnishes experimental evidence and offers a potential mechanistic explanation supporting the efficacy of BX795 as a promising candidate for OA treatment.

Abstract B171: BX-795 enhances the efficacy of crizotinib in colorectal cancer cells by altering the activity of aurora kinases

Abstract Background: Metastatic Colorectal cancer (mCRC) is driven by various genetic mutations that, in turn, present potentially actionable targets. Recent improvements in combining therapies have shown modest benefits in patients with BRAF mutated mCRC. Several efforts focusing on various KRAS inhibitors are ongoing in mCRC with outcomes pending. With no significant advances to improve outcomes in most patients with mCRC, it is important to identify novel therapies that have the potential to significantly improve the outcomes of these patients. Aims: To perform high-throughput screening (HTS) using clinically relevant drugs to identify novel and effective therapeutic combinations for patients with KRAS/BRAF mutated mCRC. Methods: We performed unbiased HTS with ~250 drugs that were either currently in clinical trials or approved by the FDA. 57 drugs that decreased cell proliferation in both 2D and 3D cultures and were effective in both HCT116 (KRAS mutated) and HT29 (BRAF mutated) cell lines, were termed as “pan-active” drugs. These drugs were divided into 9 classes based on targets they inhibit. Using the BLISS model, synergistic drug combinations were identified by HTS where the top three effective drugs from each class of “pan-active” drugs were paired with the top three drugs of all other classes. Effects of one such combination (BX-795 with crizotinib) were further validated in vitro by cell growth assays and measuring changes in apoptotic mediators by western blotting (WB) and flow cytometry (FACS). Mechanisms of synergy were examined by Reverse Phase Protein Array (RPPA) followed by WB and immunostaining of CRC cells to assess inhibition of downstream targets. Results: Pairwise HTS studies identified the PDK1/TBK1 inhibitor BX-795 to be synergistic with the MET/ALK inhibitor crizotinib in multiple CRC cell lines. This combination was further validated in vitro by MTT and colony formation assays in various KRAS and BRAF mutated CRC cell lines. Analyses of markers for apoptosis by WB and FACS using FITC-Annexin V and PI confirmed that the drug combination increased cell death in multiple CRC cell lines as compared to single agents. Unbiased RPPA indicated that the combination led to very strong and unexpected inhibition of the Aurora kinases along with decreases in the expected AKT/mTOR pathway in multiple CRC cell lines when compared to monotherapies. Immunostaining revealed significant mitotic defects in CRC cells treated with the drug combination but not with single agents suggesting that mitotic defects was a possible mechanism of cell death. Further, combining an Aurora kinase A inhibitor with crizotinib enhanced the efficacy of crizotinib validating the proposed mechanism. Conclusions: Our unbiased HTS and validation studies demonstrated that combining BX-795 with crizotinib is a potential novel effective therapeutic combination for patients with mCRC. Our in vitro studies serve as the basis for future preclinical and, if warranted, clinical studies to determine the efficacy of this drug combination in patients with mCRC. Citation Format: Susmita Ghosh, Fan Fan, Reid Powell, Yong Sung Park, Clifford Stephan, Jason Roszik, Lee M Ellis, Rajat Bhattacharya. BX-795 enhances the efficacy of crizotinib in colorectal cancer cells by altering the activity of aurora kinases [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B171.

Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways.

The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host's response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-β mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-β expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-β via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-β and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-β via the RIG-I and MDA5 signaling pathways, and the IFN-β produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-β. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-β induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

The small molecule inhibitor BX-795 uncouples IL-2 production from inhibition of Th2 inflammation and induces CD4+ T cells resembling iTreg.

Treg cells have been shown to be an important part of immune-homeostasis and IL-2 which is produced upon T cell receptor (TCR)-dependent activation of T lymphocytes has been demonstrated to critically participate in Treg development. To evaluate small molecule inhibitors (SMI) for the identification of novel IL-2/Treg enhancing compounds. We used TCR-dependent and allergen-specific cytokine secretion of human and mouse T cells, next generation messenger ribonucleic acid sequencing (RNA-Seq) and two different models of allergic airway inflammation to examine lead SMI-compounds. We show here that the reported 3-phosphoinositide dependent kinase-1 (PDK1) SMI BX-795 increased IL-2 in culture supernatants of Jurkat E6-1 T cells, human peripheral blood mononuclear cells (hPBMC) and allergen-specific mouse T cells upon TCR-dependent and allergen-specific stimulation while concomitantly inhibiting Th2 cytokine secretion. RNA-Seq revealed that the presence of BX-795 during allergen-specific activation of T cells induces a bona fide Treg cell type highly similar to iTreg but lacking Foxp3 expression. When applied in mugwort pollen and house dust mite extract-based models of airway inflammation, BX-795 significantly inhibited Th2 inflammation including expression of Th2 signature transcription factors and cytokines and influx into the lungs of type 2-associated inflammatory cells such as eosinophils. BX-795 potently uncouples IL-2 production from Th2 inflammation and induces Th-IL-2 cells, which highly resemble induced (i)Tregs. Thus, BX-795 may be a useful new compound for the treatment of allergic diseases.

Hypoxic postconditioning restores mitophagy against transient global cerebral ischemia via Parkin-induced posttranslational modification of TBK1

Hypoxic postconditioning (HPC) has been reported to enhance Parkin-catalyzed mitochondrial ubiquitination to restore mitophagy in hippocampal CA1 against transient global cerebral ischemia (tGCI). However, the molecular mechanism leading ubiquitinated mitochondria to final clearance during HPC-mediated mitophagy after tGCI is unclear. This study aims to investigate whether HPC restores mitophagy after tGCI through Parkin-induced K63-linked poly-ubiquitination (K63-Ub) to activate tumor necrosis factor associated factor family member associated nuclear factor κB activator -binding kinase 1 (TBK1) in CA1 of male rats. We found that HPC maintained TBK1 expression, promoted p62 and TBK1 phosphorylation in mitochondria, and enhanced their recruitments to mitochondria in CA1 after tGCI. However, these effects were partially abolished by TBK1 inhibitor BX795. K63-Ub of mitochondrial TBK1 was disturbed at 26 h of reperfusion after tGCI, which was reversed by HPC. The maintenance of K63-Ub of mitochondrial TBK1 induced by HPC was counteracted under Parkin knockdown with AAV-mediated Prkn small-interfering RNA, accompanied by the suppression on TBK1 activation and the reduction of mitochondrial p62 phosphorylation. This innovative study indicated that HPC maintained K63-Ub of TBK1 in a Parkin-dependent manner to promote TBK1 phosphorylation, and then phosphorylated TBK1 activated p62 to restore mitophagy, thereby alleviating neuronal damage in CA1 after tGCI.

High content screening and proteomic analysis identify a kinase inhibitor that rescues pathological phenotypes in a patient-derived model of Parkinson\u2019s disease

Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)-based disease modeling represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinson’s disease bearing the G209A (p.A53T) α-synuclein (αSyn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores disease-associated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the protective effects of BX795, comprising a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-αSyn in neuronal cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have identified a promising small molecule with neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.

BX-795 inhibits neuroblastoma growth and enhances sensitivity towards chemotherapy

High-risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology due to relapse of metastatic, drug-resistant disease, and treatment-related toxicities. An analysis of 1235 primary NB patient dataset revealed significant increase in AKT1 and AKT2 gene expression with cancer stage progression. Additionally, Both AKT1 and AKT2 expression inversely correlate with poor overall survival of NB patients. AKT1 and AKT2 genes code for AKT that drive a major oncogenic cell signaling pathway known in many cancers, including NB. To inhibit AKT pathway, we repurposed an antiviral inhibitor BX-795 that inhibits PDK1, an upstream activator of AKT. BX-795 potently inhibits NB cell proliferation and colony growth in a dose-dependent manner. BX-795 significantly enhances apoptosis and blocks cell cycle progression at mitosis phase in NB. Additionally, BX-795 potently inhibits tumor formation and growth in a NB spheroid tumor model. We further tested dual therapeutic approaches by combining BX-795 with either doxorubicin or crizotinib and found synergistic and significant inhibition of NB growth, in contrast to either drug alone. Overall, our data demonstrate that BX-795 inhibits AKT pathway to inhibit NB growth, and combining BX-795 with current therapies is an effective and clinically tractable therapeutic approach for NB.

PDK1 Inhibitor BX795 Improves Cisplatin and Radio-Efficacy in Oral Squamous Cell Carcinoma by Downregulating the PDK1/CD47/Akt-Mediated Glycolysis Signaling Pathway.

Background: Oral squamous cell carcinoma (OSCC) has a high prevalence and predicted global mortality rate of 67.1%, necessitating better therapeutic strategies. Moreover, the recurrence and resistance of OSCC after chemo/radioresistance remains a major bottleneck for its effective treatment. Molecular targeting is one of the new therapeutic approaches to target cancer. Among a plethora of targetable signaling molecules, PDK1 is currently rising as a potential target for cancer therapy. Its aberrant expression in many malignancies is observed associated with glycolytic re-programming and chemo/radioresistance. Methods: Furthermore, to better understand the role of PDK1 in OSCC, we analyzed tissue samples from 62 patients with OSCC for PDK1 expression. Combining in silico and in vitro analysis approaches, we determined the important association between PDK1/CD47/LDHA expression in OSCC. Next, we analyzed the effect of PDK1 expression and its connection with OSCC orosphere generation and maintenance, as well as the effect of the combination of the PDK1 inhibitor BX795, cisplatin and radiotherapy in targeting it. Results: Immunohistochemical analysis revealed that higher PDK1 expression is associated with a poor prognosis in OSCC. The immunoprecipitation assay indicated PDK1/CD47 binding. PDK1 ligation significantly impaired OSCC orosphere formation and downregulated Sox2, Oct4, and CD133 expression. The combination of BX795 and cisplatin markedly reduced in OSCC cell’s epithelial-mesenchymal transition, implying its synergistic effect. p-PDK1, CD47, Akt, PFKP, PDK3 and LDHA protein expression were significantly reduced, with the strongest inhibition in the combination group. Chemo/radiotherapy together with abrogation of PDK1 inhibits the oncogenic (Akt/CD47) and glycolytic (LDHA/PFKP/PDK3) signaling and, enhanced or sensitizes OSCC to the anticancer drug effect through inducing apoptosis and DNA damage together with metabolic reprogramming. Conclusions: Therefore, the results from our current study may serve as a basis for developing new therapeutic strategies against chemo/radioresistant OSCC.

TBK1 Inhibitor Exerts Antiproliferative Effect on Glioblastoma Multiforme Cells.

Glioma are common malignant brain tumors, among which glioblastoma multiforme (GBM) has the worst prognosis. Different studies of GBM revealed that targeting nuclear factor κB (NF-κB) induced an attenuation tumor proliferation and prolonged cell survival. TBK1 {TANK [TRAF (TNF (tumor-necrosis-factor) receptor-associated factor)-associated NF-κB activator]-binding kinase 1} is a serine/threonine protein kinase, and it is a member of the IκB kinase (IKK) family involved in NF-κB pathway activation. The aim of this study was to investigate the potential effect of BX795, an inhibitor of TBK1, in an in vitro and ex vivo model of GBM. GBM cell lines (U87 and U138) and primary GBM cells were treated with different concentrations of BX795 at different time points (24, 48, and 72h) to evaluate cell viability, autophagy, inflammation, and apoptosis. Our results demonstrated that BX795 10 μM was able to reduce cell viability, showing antiproliferative effect in U87, U138, and primary GBM cells. Moreover, treatment with BX795 10 μM increased the proapoptotic proteins Bax, p53, caspase 3, and caspase 9, whereas the antiapoptotic Bcl-2 expression was reduced. Additionally, our results showed a marked decrease in autophagy following BX795 treatment, reducing Atg 7, Atg 5/12, and AKT expression. The anti-inflammatory effect of BX795 was demonstrated by a significantly reduction in NIK, IKKα, and TNF-α expression, accompanied by a downregulation of angiogenesis. Furthermore, in primary GBM cell, BX795 10 μM was able to reduce TBK1 pathway activation and SOX3 expression. In conclusion, these findings showed that TBK1 is involved in GBM proliferation, demonstrating that the inhibitor BX795, thanks to its abilities, could improve therapeutic strategies for GBM treatment.

Quantitative Proteomics Reveals a Novel Role of the E3 Ubiquitin-Protein Ligase FANCL in the Activation of the Innate Immune Response through Regulation of TBK1 Phosphorylation during Peste des Petits Ruminants Virus Infection.

Peste des petits ruminants virus (PPRV) infection causes considerable innate immunosuppression in its host, which promotes viral replication. However, how the host rescues the innate immune response to counteract this immunosuppression during viral replication remains largely unknown. To explore the mechanisms of how a host counteracts PPRV-mediated innate immunosuppression, a high-throughput quantitation proteomic approach (isobaric tags for relative and absolute quantitation in conjunction with LC-MS/MS) was used to investigate the proteome landscape of goat fetal fibroblasts (GFFs) in response to PPRV infection. Eventually, 497 upregulated proteins and 358 downregulated proteins were identified. Many of the differentially expressed proteins were enriched in immune-related pathways. Blocking the activation of the innate immune response with a specific inhibitor BX795 in GFFs remarkably promoted PPRV replication, suggesting the significant antiviral role of the enriched immune-related pathways. The GO enrichment analysis showed that the host protein FANCL revealed a similar expression pattern to these innate immune-related proteins. In addition, the analysis of protein-protein interaction networks reveals a potential relationship between FANCL and the innate immune pathway. We determined that FANCL inhibited PPRV infection by enhancing type I interferon (IFN) and IFN-stimulated gene expression. Further investigation determined that FANCL induced type I IFN production by promoting TBK1 phosphorylation, thus impairing PPRV-mediated immunosuppression.

Human Metapneumovirus Induces IRF1 via TANK-Binding Kinase 1 and Type I IFN.

The innate immune and host-protective responses to viruses, such as the airway pathogen human metapneumovirus (HMPV), depend on interferons (IFNs) that is induced through TANK-binding kinase 1 (TBK1) and IFN regulatory factors (IRFs). The transcription factor IRF1 is important for host resistance against several viruses and has a key role in induction of IFN-λ at mucosal surfaces. In most cell types IRF1 is expressed at very low levels, but its mRNA is rapidly induced when the demand for IRF1 activity arises. Despite general recognition of the importance of IRF1 to antiviral responses, the molecular mechanisms by which IRF1 is regulated during viral infections are not well understood. Here we identify the serine/threonine kinase TBK1 and IFN-β as critical regulators of IRF1 mRNA and protein levels in human monocyte-derived macrophages. We find that inhibition of TBK1 activity either by the semi-selective TBK1/IKKε inhibitor BX795 or by siRNA-mediated knockdown abrogates HMPV-induced expression of IRF1. Moreover, we show that canonical NF-κB signaling is involved in IRF1 induction and that the TBK1/IKKε inhibitor BX795, but not siTBK1 treatment, impairs HMPV-induced phosphorylation of the NF-κB subunit p65. At later time-points of the infection, IRF1 expression depended heavily on IFN-β-mediated signaling via the IFNAR-STAT1 pathway. Hence, our results suggest that TBK1 activation and TBK1/IKKε-mediated phosphorylation of the NF-κB subunit p65 control transcription of IRF1. Our study identifies a novel mechanism for IRF1 induction in response to viral infection of human macrophages that could be relevant not only to defense against HMPV, but also to other viral, bacterial and fungal pathogens.

Systematic improvements in lentiviral transduction of primary human natural killer cells undergoing ex vivo expansion

Transduction of primary human natural killer (NK) cells with lentiviral vectors has historically been challenging. We sought to evaluate multiple parameters to optimize lentiviral transduction of human peripheral blood NK cells being expanded to large numbers using a good manufacturing practice (GMP)-compliant protocol that utilizes irradiated lymphoblastoid (LCL) feeder cells. Although prestimulation of NK cells with interleukin (IL)-2 for 2 or more days facilitated transduction with vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentivirus, there was a subsequent impairment in the capacity of transduced NK cells to proliferate when stimulated with LCL feeder cells. In contrast, incubation of human NK cells with LCL feeder cells plus IL-2 before transduction in the presence of the TBK1 inhibitor BX795 resulted in efficient lentiviral integration (mean of 23% transgene+ NK cells) and successful subsequent proliferation of the transduced cells. Investigation of multiple internal promoter sequences within the same lentiviral vector revealed differences in percentage and level of transgene expression per NK cell. Bicistronic lentiviral vectors encoding both GFP and proteins suitable for the isolation of transduced cells with magnetic beads led to efficient transgene expression in NK cells. The optimized approaches described herein provide a template for protocols that generate large numbers of fully functional and highly purified lentivirus-transduced NK cells for clinical trials.

Parkin overexpression alleviates cardiac aging through facilitating K63-polyubiquitination of TBK1 to facilitate mitophagy

Cumulative clinical and experimental evidence has revealed a cardinal role for mitochondrial integrity in cardiac aging. Parkin-mediated mitophagy is essential to ensure mitochondrial quality control in myocardium. This study was designed to examine the impact of Parkin overexpression on aging-induced myocardial anomalies and the underlying mechanisms with a focus on Parkin-regulated mitophagy. Cardiac function, myocardial apoptosis, mitochondrial ultrastructure and mitophagy were examined in young (3 mo) and old (24–26 mo) wild-type (WT) and Parkin transgenic mice. Our data revealed compromised myocardial function and mitochondrial morphology along with overtly apoptosis with advanced aging, the effects of which were attenuated by Parkin overexpression. Advanced aging dampened mitophagy as evidenced by decreased levels of Parkin, LC3II, phosphorylation of p62 and TBK1 in isolated mitochondria as well as reduced mitochondria autophagosomes, the effects of which were mitigated by restoration of mitophagy via Parkin overexpression. Using the low-dose doxorubicin (DOX) in vitro model of cell senescence, we noted that Parkin-offered beneficial effect against senescence was abolished by the TBK1 kinase inhibitor BX795. With TBK1 overexpression in cardiomyocytes, we uncovered the interaction of Parkin with TBK1 using a Co-immunoprecipitation (Co-IP) assay. The interaction of Parkin with TBK1 contributed to K63-linked polyubiquitination of TBK1. Our study also noted that DOX disturbed K63-linked polyubiquitination of TBK1 with downregulation of Parkin. Parkin overexpression promoted K63-linked polyubiquitination of TBK1 through Lys30 and Lys401 residues to foster TBK1 phosphorylation to facilitate efficient mitophagy. In summary, these findings suggested that Parkin effectively rescued cardiac aging through promoting K63-linked polyubiquitination of TBK1 to facilitate mitophagy.

Lentiviral delivery of combinatorial CAR/CRISPRi circuit into human primary T cells is enhanced by TBK1/IKK\u025b complex inhibitor BX795

BackgroundAdoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells.MethodsTo make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function.ResultsWe found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells.ConclusionsThese results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors.

Abstract 1011: Tank binding kinase (TBK1) inhibitor BX795 impairs anti-tumor efficacy of T cells by abrogating granzyme and perforin mobilization

Abstract TANK binding kinase 1 (TBK1) is a non-canonical IκB kinase that regulates the innate immune response and is highly expressed in lung, breast, pancreatic and colon cancers. It phosphorylates and activates downstream targets such as IRF3 and c-Rel and mediates NF-κB activation, facilitating the expression of pro-inflammatory genes and interferons. In addition to its role in regulating innate immunity, TBK1 also promotes oncogenesis downstream of K-Ras by phosphorylating Akt and promoting cell survival, proliferation, autophagy and mitophagy. Therefore, TBK1 inhibitors have been proposed to be potentially efficacious against inflammatory diseases and cancer. Unfortunately, TBK1 inhibitors have not been found to be highly efficacious anti-cancer agents and we made efforts to elucidate the underlying reasons. Earlier findings from our lab revealed a novel role for TBK1 in regulating mitosis. Specifically, we found that active phospho-TBK1 levels were increased during mitosis and TBK1 localized to the centrosomes, mitotic spindles and midbody; selective inhibition or silencing of TBK1 triggered defects in spindle assembly and prevented mitotic progression (Pillai et al., Nature Communications, 2015). TBK1 was found to facilitate the binding of the kinesis Kif2b to CEP170 and dynein to NuMA; depletion or inhibition of TBK1 significantly altered microtubule stability and dynamics, probably due to its regulation of dyenein-NuMA interaction. Since dynein function and appropriate microtubule dynamics is necessary for proper trafficking of vesicles for secretion, hypothesized that inhibition of TBK1 might adversely affect the tumor microenvironment by interfering with the mobilizing of cytotoxic molecules like granzyme and perforin by T cells. Hence we tested if inhibition of TBK1 would impair the anti-tumor activity of T-cells. Pilot immunofluorescence experiments conducted on JURKAT T-cells treated with the TBK1 inhibitor BX795 showed higher percent of cells retaining granzyme-b and perforin as compared to control cells. Further, we carried out in vitro cytotoxicity assay using normal human peripheral blood mononuclear cells (PBMC's) treated with BX795 (2.5uM for 24 hrs), activated with T cell mitogen concanavalin A (1.25ug/ml) and co-cultured with KRAS mutant A549 lung cancer cells for 72 hrs. Our results showed that BX795 treatment of PBMC's significantly inhibited the ability of the T cells to induce apoptosis in A549 cells, as measured by Annexin-V / PI +ve signal. These findings suggest that inhibiting TBK1 may be causing dysfunction of immune mediated T cell functions and interference with the anti-tumor immunity. Further experiments are in progress to study the regulatory role of TBK1 on different T cell populations and the associated cytokine signaling. Citation Format: Fatema Khambati, Jaya Padmanabhan, SriKumar Chellappan. Tank binding kinase (TBK1) inhibitor BX795 impairs anti-tumor efficacy of T cells by abrogating granzyme and perforin mobilization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1011.

The kinase inhibitor BX795 suppresses the inflammatory response via multiple kinases

BX795, a small molecule with an aminopyrimidine backbone, is a potent ATP-competitive inhibitor of phosphoinositide-dependent kinase 1 (PDK1) and TANK-binding kinase 1 (TBK1). BX795 has significant functions in various immune responses and cancer. Few reports on the anti-inflammatory effect of BX795 are available, and its molecular mechanisms have not been fully elucidated. In this study, lipopolysaccharide (LPS)-treated macrophages (RAW264.7 cells), luciferase reporter gene assay, knock-down and overexpression strategies, kinase assay, protein chip, immunoprecipitation, and immunoblotting analyses were employed to clarify the anti-inflammatory mechanism of BX795. BX795 was found to dose-dependently inhibit the production of pro-inflammatory mediators without exhibiting cytotoxicity. Luciferase assay and immunoblotting analysis with nuclear fractions showed that activator protein-1 (AP-1), signal transducer and activator of transcription 1 (STAT1), and interferon regulatory factor 3 (IRF3) are targeted by BX795 rather than nuclear factor (NF)-κB. Moreover, TBK1 and AKT, transforming growth factor activated kinase (TAK)-1/c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase kinase 4 (MKK4) for AP-1 activation, and Janus kinase 2 (JAK2)/STAT1 were inhibited by BX795. Consistent with these findings, BX795 strongly ameliorated inflammatory symptoms in colitis models. These results suggest that BX795 can suppress inflammatory responses triggered by Gram-positive bacteria by suppressing multiple pathways.

Chimeric antigen receptor expression in natural killer cell line NK-92 by transduction with lentiviral particles pseudotyped with the surface glycoproteins of the measles virus vaccine strain

Cancer immunotherapy with T-cells that carry chimeric antigen receptors is currently on cutting edge of modern oncology. Autotransplantation of T-lymphocytes with chimeric receptor specific for certain tumor antigen proves to be clinically effective, but costly. Linear carriers of chimeric antigen receptors based on natural killer NK-92 cell culture may be an affordable alternative, however, this culture is resistant to lentiviral transduction. Recently, lentiviral vectors, pseudotyped with surface glycoproteins of the measles virus vaccine strain, have recently been successfully applied for transduction of primary immune cells. The aim of the work was to assess the efficiency of transduction of NK-92 cells with lentivirus vectors, pseudotyped with measles F and H surface glycoproteins, as well as to establish optimal conditions for selection of NK-92 transduced with the chimeric receptor against CD20 and to evaluate the culture’s cytotoxic potential. The results showed that the maximum infectious titer is achieved using the H∆18 variant in combination with F∆30, and the use of the TBK1/IKKɛ inhibitor BX795 results in additional 3-fold increase in the infectious titer. CAR-expressing NK-92 were able to suppress the proliferation of CD20+ cell line Raji in lower effector-to-target ratios than unmodified NK-92.

Activation of stimulator of interferon genes (STING) induces ADAM17-mediated shedding of the immune semaphorin SEMA4D

Stimulator of interferon genes (STING) is an endoplasmic reticulum-resident membrane protein that mediates cytosolic pathogen DNA-induced innate immunity and inflammatory responses in host defenses. STING is activated by cyclic di-nucleotides and is then translocated to the Golgi apparatus, an event that triggers STING assembly with the downstream enzyme TANK-binding kinase 1 (TBK1). This assembly leads to the phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3), which in turn induces expression of type-I interferon (IFN) and chemokine genes. STING also mediates inflammatory responses independently of IRF3, but these molecular pathways are largely unexplored. Here, we analyzed the RAW264.7 macrophage secretome to comprehensively identify proinflammatory factors released into the extracellular medium upon STING activation. In total, we identified 1299 proteins in macrophage culture supernatants, of which 23 were significantly increased after STING activation. These proteins included IRF3-dependent cytokines, as well as previously unknown targets of STING, such as the immune semaphorin SEMA4D/CD100, which possesses proinflammatory cytokine-like activities. Unlike for canonical cytokines, the expression of the SEMA4D gene was not up-regulated. Instead, upon STING activation, membrane-bound SEMA4D was cleaved into a soluble form, suggesting the presence of a post-translational shedding machinery. Importantly, the SEMA4D shedding was blocked by TMI-1, an inhibitor of the sheddase ADAM metallopeptidase domain 17 (ADAM17) but not by the TBK1 inhibitor BX795. These results suggest that STING activates ADAM17 and that this activation produces soluble proinflammatory SEMA4D independently of the TBK1/IRF3-mediated transcriptional pathway.

TBK1: A key regulator and potential treatment target for interferon positive Sjögren's syndrome, systemic lupus erythematosus and systemic sclerosis

ObjectiveUpregulation of type I interferons (IFN-I) is a hallmark of systemic autoimmune diseases like primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). Expression of IFN-I is induced by three different receptor families: Toll-like receptors (TLRs), RIG-like receptors (RLRs) and DNA-sensing receptors (DSRs). TANK-binding kinase (TBK1) is an important signaling hub downstream of RLRs and DSRs. TBK1 activates IRF3 and IRF7, leading to IFN-I production and subsequent induction of interferon stimulated genes (ISGs). The objective of this study was to explore the potential of BX795, an inhibitor of TBK1, to downregulate IFN-I activation in pSS, SLE and SSc. MethodsTBK1, IRF3, IRF7 and STAT1 were determined by RT-PCR in PAXgene samples and phosphorylated-TBK1 (pTBK1) was analyzed by flowcytometry in plasmacytoid dendritic cells (pDCs) from IFN-I positive (IFNpos) patients. Peripheral blood mononuclear cells (PBMCs) of pSS, SLE and SSc patients and TLR7 stimulated PBMCs of healthy controls (HCs) were cultured with the TBK1 inhibitor BX795, followed by analysis of ISGs. ResultsIncreased gene expression of TBK1, IRF3, IRF7 and STAT1 in whole blood and pTBK1 in pDCs was observed in IFNpos pSS, SLE and SSc patients compared to HCs. Upon treatment with BX795, PBMCs from IFNpos pSS, SLE, SSc and TLR7-stimulated HCs downregulated the expression of the ISGs MxA, IFI44, IFI44L, IFIT1 and IFIT3. ConclusionsTBK1 inhibition reduced expression of ISGs in PBMCs from IFNpos patients with systemic autoimmune diseases indicating TBK1 as a potential treatment target.