BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a typical neurodegenerative disorder typically characterized by inflammation activation. However, the relationship between non-canonical NF-κB (ncNF-κB) pathway activation and ALS progression is not clear.
METHODS: We tested the ncNF-κB pathway in the ALS animal model including hSOD1-G93A transgenic mice and TBK1 deletion mice.We treated age-matched SOD1-G93A mice with B022 (a NIK inhibitor) to investigate the role of NIK in the ALS animal model. We also established a new mice model by crossing SOD1-G93A mice with NIK+/- mice to further evaluate the interrelationship between the NIK and the disease progression in ALS animal model.
RESULTS: In this study, we found the ncNF-κB pathway was activated in SOD1-G93A animal model and TBK1 deletion model. Inhibition of NIK activity by small molecule B022 significantly improved the motor performance of the ALS animal model. However, NIK deletion enhanced the mutant SOD1 toxicity by inflammatory infiltration.
CONCLUSIONS: TBK1 deletion and mutant SOD1 shared the common pathological feature possibly via effects on NIK activation and inhibitor of NIK could be a novel strategy for treating ALS.

The NFκB pathway, known as the central regulator of inflammation, has a well-established role in colorectal cancer (CRC) initiation, progression, and therapy resistance. Due to the pathway\'s overarching roles in CRC, there have been efforts to characterise NFκB family members and target the pathway for therapeutic intervention. Initial research illustrated that the canonical NFκB pathway, driven by central kinase IKKβ, was a promising target for drug intervention. However, dose limiting toxicities and specificity concerns have resulted in failure of IKKβ inhibitors in clinical trials. The field has turned to look at targeting the less dominant kinase, IKKα, which along with NFκB inducing kinase (NIK), drives the lesser researched non-canonical NFκB pathway. However prognostic studies of the non-canonical pathway have produced conflicting results. There is emerging evidence that IKKα is involved in other signalling pathways, which lie outside of canonical and non-canonical NFκB signalling. Evidence suggests that some of these alternative pathways involve a truncated form of IKKα, and this may drive poor cancer-specific survival in CRC. This review aims to explore the multiple components of NFκB signalling, highlighting that NIK may be the central kinase for non-canonical NFκB signalling, and that IKKα is involved in novel pathways which promote CRC.

NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKβ has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKβ, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKβ. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKβ was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKβ up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKβ coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.

Inflammation resolution is an essential process for preventing the development of chronic inflammatory diseases. However, the mechanisms that regulate inflammation resolution in psoriasis are not well understood. Here, we report that ANKRD22 is an endogenous negative orchestrator of psoriasiform inflammation because ANKRD22-deficient mice are more susceptible to IMQ-induced psoriasiform inflammation. Mechanistically, ANKRD22 deficiency leads to excessive activation of the TNFRII-NIK-mediated noncanonical NF-κB signaling pathway, resulting in the hyperproduction of IL-23 in DCs. This is due to ANKRD22 being a negative feedback regulator for NIK because it physically binds to and assists in the degradation of accumulated NIK. Clinically, ANKRD22 is negatively associated with IL-23A expression and psoriasis severity. Of greater significance, subcutaneous administration of an AAV carrying ANKRD22-overexpression vector effectively hastens the resolution of psoriasiform skin inflammation. Our findings suggest ANKRD22, an endogenous supervisor of NIK, is responsible for inflammation resolution in psoriasis, and may be explored in the context of psoriasis therapy.

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

The critical roles of NF-κB Inducing Kinase (NIK) in tumor progression have been elucidated in various tumors; however, its effects on hepatocellular carcinoma (HCC) progression are still confusing. Here, we found that NIK level was upregulated in HCC tissues compared to that of normal tissues, and positively correlated with the levels of cancer stem cell (CSC) markers. Then we established HCC cells with NIK-stable knockdown and found that NIK knockdown suppressed the CSC-like traits of HCC cells through in vivo and in vitro experiments. Mechanistically, we revealed that SIX2 protein level, but not its mRNA level, was significantly reduced in HCC cells with NIK knockdown, which was rescued by MG132 treatment. Furthermore, NIK knockdown promoted the ubiquitination level of SIX2 and decreased its protein stability. Moreover, Six2 overexpression partially reversed the inhibition of NIK knockdown on the CSC-like traits of HCC cells. This study identified a novel NIK/SIX2 axis conferring HCC stemness.

The bulge region, a reservoir of multipotent stem cells, is possibly responsible for tumorigenesis. NF-κB-inducing kinase (NIK) is a kinase involved in the activation of the noncanonical NF-κB pathway and exhibits positive staining in tumor cells. However, whether high expression of NIK can result in tumorigenesis has not been reported in published papers. By establishing Nik-coe (Nik-stopF/F crossed with Chat-cre) and Nik-soe (Nik-stopF/F crossed with Sox9-cre) mice, we found that overexpression of Nik in the bulge region of hair follicles induced hair follicle loss and tumorigenesis. Furthermore, RNA sequencing, proteomic and phosphopeptide analyses revealed that multiple cancer pathways are involved in tumor formation. Taken together, these findings indicate that constitutive activation of Nik in the bulge region induces tumorigenesis.

Periodontal disease is an infectious disease that affects many people worldwide. Disease progression destroys the alveolar bone and causes tooth loss. We have previously shown that alymphoplasia (aly/aly) mice harboring a loss-of-function mutation in the map3k14 gene, which is involved in p100 to p52 processing of the alternative NF-κB pathway, exhibited mild osteopetrosis due to decreased number of osteoclasts, suggesting the alternative NF-κB pathway as a potential drug target for the amelioration of bone disease. In the present study, wild-type (WT) and aly/aly mice were subjected to silk ligation to establish a periodontitis model. Alveolar bone resorption was suppressed in aly/aly mice by decreased numbers of osteoclasts in the alveolar bone in comparison to WT mice. Furthermore, the expression of receptor activator of NF-κB ligand (RANKL) and TNFα (cytokines involved in osteoclast induction in periligative gingival tissue) was decreased. When primary osteoblasts (POBs) and bone marrow cells (BMCs) derived from WT and aly/aly mice were prepared and co-cultured, osteoclasts were induced from WT-derived BMCs, regardless of the origin of the POBs, but hardly formed from aly/aly mouse-derived BMCs. Furthermore, the local administration of an NIK inhibitor, Cpd33, inhibited osteoclast formation and thereby inhibited alveolar bone resorption in the periodontitis model. Therefore, the NIK-mediated NF-κB alternative pathway can be a therapeutic target for periodontal disease.

Inhibition of NF-κB inducing kinase (NIK) has been pursued as a promising therapeutic target for autoimmune disorders due to its highly regulated role in key steps of the NF-κB signaling pathway. Previously reported NIK inhibitors from our group were shown to be potent, selective, and efficacious, but had higher human dose projections than desirable for immunology indications. Herein we report the clearance-driven optimization of a NIK inhibitor guided by metabolite identification studies and structure-based drug design. This led to the identification of an azabicyclo[3.1.0]hexanone motif that attenuated in vitro and in vivo clearance while maintaining NIK potency and increasing selectivity over other kinases, resulting in a greater than ten-fold reduction in predicted human dose.

BACKGROUND: In vivo investigations with cancer cells have powerful tools to discover cancer progression mechanisms and preclinical candidate drugs. Among these in vivo experimental models, the establishment of highly malignancy cell lines with xenograft has been frequently used. However, few previous researches targeted malignancy-related genes whose protein levels translationally changed. Therefore, this study aimed to identify malignancy-related genes which contributed to cancer progression and changed at the protein level in the in vivo selected cancer cell lines.
METHODS: We established the high malignancy breast cancer cell line (LM05) by orthotopic xenograft as an in vivo selection method. To explore the altered genes by translational or post-translational regulation, we analyzed the protein production by western blotting in the highly malignant breast cancer cell line. Functional analyses of the altered genes were performed by in vitro and in vivo experiments. To reveal the molecular mechanisms of the regulation with protein level, we evaluated post-translational modification by immunoprecipitation. In addition, we evaluated translational production by click reaction-based purification of nascent protein.
RESULTS: As a result, NF-κB inducing kinase (NIK) increased at the protein level and promoted the nuclear localization of NF-κB2 (p52) and RelB in the highly malignant breast cancer cell line. The functional analyses indicated the NIK upregulation contributed to tumor malignancy via cancer-associated fibroblasts (CAFs) attraction and partially anti-apoptotic activities. Additionally, the immunoprecipitation experiment revealed that the ubiquitination of NIK decreased in LM05 cells. The decline in NIK ubiquitination was attributed to the translational downregulation of cIAP1.
CONCLUSIONS: Our study identified a dysregulated mechanism of NIK production by the suppression of NIK post-modification and cIAP1 translation. The aberrant NIK accumulation promoted tumor growth in the highly malignant breast cancer cell line.