NF-κB-inducing Kinase Research Articles

Noncanonical NF-κB controls stem cell signatures in the colonic mucosa and affects susceptibility to inflammation-induced carcinogenesis

Abstract NF-κB signaling is divided into two distinct pathways, defined as either canonical or noncanonical. Canonical NF-κB signaling is well described and characterized. However, significantly less is known regarding mechanisms regulated by the noncanonical NF-κB pathway, especially outside of the lymphoid system. Recently, interest in the role of noncanonical NF-κB in the mucosal immune system has been increasing, with emphasis on NF-κB inducing kinase (NIK). NIK is an essential kinase in this cascade. Our data shows that lack of noncanonical signaling through the complete knockout of Nik in mice results in an aberrant large intestine phenotype characterized by reduced stem cell marker expression, altered regeneration and differentiation capacity under stress, changes in the microbiome, and predisposition to inflammation-associated tumorigenesis. Subsequent mechanistic studies utilizing novel mice with cell type specific deletions of either RelA/p65 or Nik revealed separate and distinct rolls for canonical and noncanonical NF-κB signaling in attenuating inflammation and tumor progression. RelA/p65 attenuates disease progression through the myeloid compartment in the gut, whereas Nik functions through the intestinal epithelial cell compartment. Dysregulation of noncanonical signaling is also observed in human colorectal cancer patients, which have marked suppression of this pathway in colonic biopsy samples. Together, these findings indicate that noncanonical NF-κB signaling is a critical regulator of immune system homeostasis in the gastrointestinal tract and plays a vital protective role against colorectal cancer.

Ubiquitin Ligases cIAP1 and cIAP2 Limit Cell Death to Prevent Inflammation

Cellular inhibitor of apoptosis proteins cIAP1 and cIAP2 ubiquitinate nuclear factor κB (NF-κB)-inducing kinase (NIK) to suppress non-canonical NF-κB signaling and substrates such as receptor interacting protein kinase 1 (RIPK1) to promote cellsurvival. We investigate how these functions contribute to homeostasis by eliminating cIap2 from adult cIap1-deficient mice. cIAP1 and cIAP2 (cIAP1/2) deficiency causes rapid weight loss and inflammation, with aberrant cell death, indicated by cleaved caspases-3 and -8, prevalent in intestine and liver. Deletion of Casp8 and Ripk3 prevents this aberrant cell death, reduces the inflammation, and prolongs mouse survival, whereas Ripk3 loss alone offers little benefit. Residual inflammation in mice lacking cIap1/2, Casp8, and Ripk3 is reduced by inhibition of NIK. Loss of Casp8 and Mlkl (mixed lineage kinase domain-like), but not Mlkl loss alone, also prevents cIAP1/2-deficient mice from dying around embryonic day 11. Therefore, a major function of cIAP1/2 invivo is to suppress caspase-8-dependent cell death.

Pectin oligosaccharide from hawthorn fruit ameliorates hepatic inflammation via NF-κB inactivation in high-fat diet fed mice

Abstract Effects of pectin oligosaccharide (POS) derived from hawthorn fruit on the nuclear factor kappa-B (NF-κB) pathway-related factors were investigated in the liver of mice fed a high-fat diet. The results showed that POS significantly reduced the total liver fat content and the levels of hepatic pro-inflammatory factors tumor necrosis factor (TNF)-α and interleukin-6 (IL-6), while increased the level of interleukin-10 (IL-10). Moreover, POS significantly down-regulated gene and protein expression of NF-κB, with concomitant decreased mRNA levels of receptor interacting protein kinase 1 (RIP1), NF-κB-inducing kinase (NIK), IκB kinase-α (IKKα), TNFα and its receptors 1 (TNFR1), receptor-associated factor 2 (TRAF2), compared to high fat control (HFC). However, expressions of AMP-activated protein kinase (AMPK) and silent information regulator T 1 (SIRT 1) were up-regulated by POS, compared to HFC. The results indicated that POS had significant inhibition effects on the liver inflammation in vivo, involving the inhibition on NF-kB activation.

Importance of Incorporating Protein Flexibility in Molecule Modeling: A Theoretical Study on Type I1/2 NIK Inhibitors.

NF-κB inducing kinase (NIK), which is considered as the central component of the non-canonical NF-κB pathway, has been proved to be an important target for the regulation of the immune system. In the past few years, NIK inhibitors with various scaffolds have been successively reported, among which type I1/2 inhibitors that can not only bind in the ATP-binding pocket at the DFG-in state but also extend into an additional back pocket, make up the largest proportion of the NIK inhibitors, and are worthy of more attention. In this study, an integration protocol that combines molecule docking, MD simulations, ensemble docking, MM/GB(PB)SA binding free energy calculations, and decomposition was employed to understand the binding mechanism of 21 tricyclic type I1/2 NIK inhibitors. It is found that the docking accuracy is largely dependent on the selection of docking protocols as well as the crystal structures. The predictions given by the ensemble docking based on multiple receptor conformations (MRCs) and the MM/GB(PB)SA calculations based on MD simulations showed higher linear correlations with the experimental data than those given by conventional rigid receptor docking (RRD) methods (Glide, GOLD, and Autodock Vina), highlighting the importance of incorporating protein flexibility in predicting protein–ligand interactions. Further analysis based on MM/GBSA demonstrates that the hydrophobic interactions play the most essential role in the ligand binding to NIK, and the polar interactions also make an important contribution to the NIK-ligand recognition. A deeper comparison of several pairs of representative derivatives reveals that the hydrophobic interactions are vitally important in the structural optimization of analogs as well. Besides, the H-bond interactions with some key residues and the large desolvation effect in the back pocket devote to the affinity distinction. It is expected that our study could provide valuable insights into the design of novel and potent type I1/2 NIK inhibitors.

Functional interplay between NF-κB-inducing kinase and c-Abl kinases limits response to Aurora inhibitors in multiple myeloma.

Considering that Aurora kinase inhibitors are currently under clinical investigation in hematologic cancers, the identification of molecular events that limit the response to such agents is essential for enhancing clinical outcomes. Here, we discover a NF-κB-inducing kinase (NIK)-c-Abl-STAT3 signaling-centered feedback loop that restrains the efficacy of Aurora inhibitors in multiple myeloma. Mechanistically, we demonstrate that Aurora inhibition promotes NIK protein stabilization via downregulation of its negative regulator TRAF2. Accumulated NIK converts c-Abl tyrosine kinase from a nuclear proapoptotic into a cytoplasmic antiapoptotic effector by inducing its phosphorylation at Thr735, Tyr245 and Tyr412 residues, and, by entering into a trimeric complex formation with c-Abl and STAT3, increases both the transcriptional activity of STAT3 and expression of the antiapoptotic STAT3 target genes PIM1 and PIM2. This consequently promotes cell survival and limits the response to Aurora inhibition. The functional disruption of any of the components of the trimer NIK-c-Abl-STAT3 or the PIM survival kinases consistently enhances the responsiveness of myeloma cells to Aurora inhibitors. Importantly, concurrent inhibition of NIK or c-Abl disrupts Aurora inhibitor-induced feedback activation of STAT3 and sensitizes myeloma cells to Aurora inhibitors, implicating a combined inhibition of Aurora and NIK or c-Abl kinases as potential therapies for multiple myeloma. Accordingly, pharmacological inhibition of c-Abl together with Aurora resulted in substantial cell death and tumor regression in vivo. The findings reveal an important functional interaction between NIK, Abl and Aurora kinases, and identify the NIK, c-Abl and PIM survival kinases as potential pharmacological targets for improving the efficacy of Aurora inhibitors in myeloma.

A Novel Regulatory Role of TRAPPC9 in Osteoarthritis

INTRODUCTIONTrafficking Protein Particle Complex 9 (TRAPPC9) is a major subunit of TRAPPII Complex. TRAPPC9 is implicated in endoplasmic to Golgi trafficking pathway and intra Golgi trafficking in yeast cells. TRAPPC9 has been reported to bind IKKb and NIK where it plays a role in NFkB signaling. Hence, plays a role in different cell types differentiation and function. In osteoarthritis, inflammation mediated by the NFkB signaling plays a role in the pathogenesis on the disease. There are two distinct pathways, which can activate the NF‐kB signaling cascades. The first one termed the canonical or the classical pathway, activation of IKKα/IKKβ/IKKβ‐NEMO complex will activate this pathway. On the other hand, the non‐canonical or the alternative pathway, activation relies on NF‐kB‐inducing kinase (NIK) signaling. Our data showed that TRAPPC9 binds IKKβ and NIK in chondrocytes. We believe such interaction could lead to a potential regulatory role of TRAPPC9 during NF‐kB signaling cascades given that the role of TRAPPC9 in OA has not been studied yet.METHODSIn this study, we used human chondrocytes and primary mouse chondrocyte cell culture, immunofluorescence, and retroviral transfection techniques.RESULTSTo explore the role of TRAPPC9 in OA, first, we assessed the expression of TRAPPC9 (mRNA and protein) levels in primary human chondrocytes treated with ILb to resemble chondrocytes within OA knee and found that TRAPPC9 expression is decreased in IL‐1β‐treated chondrocytes compared to controls. Next, we determined whether modulation of TRAPPC9 expression alters the inflammatory markers induced by IL1b that are regulated by NF‐kB signaling pathways. Human chondrocytes were transfected with either TRAPPC9 overexpression plasmid or control plasmid. Gene expression for metalloproteinases (what else a part of MMP) was measured and our data showed that TRAPPC9 overexpression induces inflammation by increasing the gene expression of MMPs‐1,3,9,13, and Interlukin‐6 (IL‐6).CONCLUSIONWe showed that TRAPPC9 binds/interacts with NIK and IKKb in chondrocytes. Also, we were able to show that TRAPPC9 expression is decreased in inflammatory conditions. In addition, our data showed that modulating TRAPPC9 expression prior to IL‐1β induction leads to elevate inflammatory conditions in chondrocytes. Studies are underway to determine the effects of modulating TRAPPC9 using genetic and pharmacologic approaches on osteoarthritis development and identifying the mechanism(s) by which inflammation in chondrocytes mediated by TRAPPC9, NIK, and IKKb interactions. Collectively, these studies are the first to report a novel role for TRAPPC9 in osteoarthritis.SIGNIFICANCE/CLINICAL RELEVANCEUnderstanding the role of TRAPPC9 in osteoarthritis are not reported previously. It is important to investigate whether TRAPPC9, NIK and IKKb interaction mediates inflammation in chondrocytes by modulating the NF‐kB signaling pathways thereby, suggesting new therapeutics for osteoarthritis disease.Support or Funding InformationKent State University, School of Biomedical Sciences. Ohio Physiological Society (Peter K Lauf Travel Award).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NIK-IKK complex interaction controls NF-κB-dependent inflammatory activation of endothelium in response to LTβR ligation.

NF-κB-inducing kinase (NIK; also known as MAP3K14) is a central regulator of non-canonical NF-κB signaling in response to stimulation of TNF receptor superfamily members, such as the lymphotoxin-β receptor (LTβR), and is implicated in pathological angiogenesis associated with chronic inflammation and cancer. Here, we identify a previously unrecognized role of the LTβR-NIK axis during inflammatory activation of human endothelial cells (ECs). Engagement of LTβR-triggered canonical and non-canonical NF-κB signaling promoted expression of inflammatory mediators and adhesion molecules, and increased immune cell adhesion to ECs. Sustained LTβR-induced inflammatory activation of ECs was NIK dependent, but independent of p100, indicating that the non-canonical arm of NF-κB is not involved. Instead, prolonged activation of canonical NF-κB signaling, through the interaction of NIK with IκB kinase α and β (also known as CHUK and IKBKB, respectively), was required for the inflammatory response. Endothelial inflammatory activation induced by synovial fluid from rheumatoid arthritis patients was significantly reduced by NIK knockdown, suggesting that NIK-mediated alternative activation of canonical NF-κB signaling is a key driver of pathological inflammatory activation of ECs. Targeting NIK could thus provide a novel approach for treating chronic inflammatory diseases.

A NIK-SIX signalling axis controls inflammation by targeted silencing of non-canonical NF-κB.

SUMMARYThe non-canonical NF-κB signaling cascade is essential for lymphoid organogenesis, B-cell maturation, osteoclast differentiation, and inflammation in mammals1,2, whereas dysfunction of this system is associated with human diseases, including immunological disorders and cancer3–6. While controlled expression of NF-κB Inducing Kinase (NIK) is the rate-limiting step in non-canonical NF-κB activation2,7, mechanisms of inhibition remain largely unknown. Here, we report the identification of the sine oculis homeobox homolog family transcription factors SIX1 and SIX2 as essential inhibitory components of the non-canonical NF-κB signaling pathway. The developmentally silenced SIX-proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit RelA and RelB trans-activation function in a negative feedback circuit. In support of a physiologically pivotal role for SIX-proteins in host immunity, human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/p53-driven lung adenocarcinoma cells from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents, small-molecule activators of the non-canonical NF-κB pathway. Collectively, our study reveals a NIK-SIX signaling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.

ZFYVE21 is a Novel Complement-Induced Rab5 Effector Protein Mediating Cardiac Allograft Vasculopathy

Purpose Complement promotes vascular inflammation in transplant organ rejection, a process leading to cardiac allograft vasculopathy. In this study, we identify a new Rab5 effector protein, ZFYVE21, that mediates this process. We elucidate a ZFYVE21-SMURF2-pAkt signaling axis which activates non-canonical NF-kB, and we define the clinical relevance of this sequence. Methods We developed FACS-assisted protocols enabling proteomic analysis of MAC+Rab5+ endosomes and semi-quantitative analyses of the phosphoinositide content of these structures. A humanized mouse model incorporating human coronary artery segments and human lymphoid cells was employed to define a role for ZFYVE21-associated signaling in cardiac allograft vasculopathy. Software-assisted analyses of human transplant biopsies analyzing ZFYVE21 colocalization was performed using a computer algorithm we helped to developed in-house. Results In response to deposition of membrane attack complexes (MAC) on endothelial cells (EC), ZFYVE21 is post-translationally stabilized via recruitment to MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of the phosphoinositide content of MAC+Rab5+ endosomes with miltefosine reduces ZFYVE21 induction, EC activation, and MAC-induced allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC- but not ligand-induced non-canonical NF-κB and is a biomarker for complement-mediated endothelial signaling in transplant biopsies with antibody-mediated rejection. Conclusion Our data identifies ZFYVE21 as a novel Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway as a drug target and biomarker for allograft vasculopathy.

Intestinal non-canonical NF\u03baB signaling shapes the local and systemic immune response

Microfold cells (M-cells) are specialized cells of the intestine that sample luminal microbiota and dietary antigens to educate the immune cells of the intestinal lymphoid follicles. The function of M-cells in systemic inflammatory responses are still unclear. Here we show that epithelial non-canonical NFkB signaling mediated by NFkB-inducing kinase (NIK) is highly active in intestinal lymphoid follicles, and is required for M-cell maintenance. Intestinal NIK signaling modulates M-cell differentiation and elicits both local and systemic IL-17A and IgA production. Importantly, intestinal NIK signaling is active in mouse models of colitis and patients with inflammatory bowel diseases; meanwhile, constitutive NIK signaling increases the susceptibility to inflammatory injury by inducing ectopic M-cell differentiation and a chronic increase of IL-17A. Our work thus defines an important function of non-canonical NFkB and M-cells in immune homeostasis, inflammation and polymicrobial sepsis.

The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate.

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).

Discovery of novel indoleaminopyrimidine NIK inhibitors based on molecular docking-based support vector regression (SVR) model

A set of NF-κB-inducing kinase (NIK) inhibitors was used to develop a molecular docking-based QSAR model by using nonlinear regression method. The accuracy of the QSAR model was remarkably improved by integrating the docking scores and key interaction profiles. Two indole-aminopyrimidine derivatives 32a and 32b predicted as NIK inhibitors were synthesized and biologically evaluated. The significant correlationship between experimental data and MD-SVR model-predicted results were observed. The binding mode of 32a and 32b with NIK were further investigated by dynamic simulations. Compound 32b was proposed as a promising lead for the findings of highly potent inhibitors.

Differential involvement of TAK1, RIPK1 and NF-κB signaling in Smac mimetic-induced cell death in breast cancer cells.

Smac mimetics (SMs) are considered promising cancer therapeutics. However, the mechanisms responsible for mediating cell death by SMs are still only partly understood. Therefore, in this study, we investigated signaling pathways upon treatment with the bivalent SM BV6 using two SM-sensitive breast cancer cell lines as models. Interestingly, genetic silencing of transforming growth factor (TGF)β activated kinase (TAK)1, an upstream activator of the nuclear factor-kappaB (NF-κB) subunit RelA (p65), increased BV6-induced cell death only in EVSA-T cells, although it reduced BV6-mediated upregulation of tumor necrosis factor (TNF)α in both EVSA-T and MDA-MB-231 cells. By comparison, genetic silencing of p65, a key component of canonical NF-κB signaling, blocked BV6-induced cell death in MDA-MB-231 but not in EVSA-T cells. Similarly, knockdown of NF-κB-inducing kinase (NIK) rescued MDA-MB-231 cells from BV6-induced cell death, while it failed to do so in EVSA-T cells. Consistently, silencing of p65 or NIK reduced BV6-stimulated upregulation of TNFα in MDA-MB-231 cells. In conclusion, TAK1, receptor-interacting kinase 1 (RIPK1) as well as canonical and non-canonical NF-κB signaling are differentially involved in SM-induced cell death in breast cancer cells. These findings contribute to a better understanding of SM-induced signaling pathways.

CRISPR/Cas9-Generated Models Uncover Therapeutic Vulnerabilities of Del(11q) Chronic Lymphocytic Leukemia Cells to Dual BCR and PARP Inhibition

Chromosome 11q22.3 deletion (del(11q)) is one of the most common cytogenetic alterations in CLL and usually involves both ATM and BIRC3 genes. Concomitant mutations in ATM and/or BIRC3 in the remaining allele have been associated with poor survival. Despite the encouraging efficacy of novel agents targeting BCR and BCL2 pathways, del(11q) patients still have an inferior outcome and the development of resistance to these drugs has been increasingly reported. We therefore investigated the functional impact of del(11q) together with loss-of-function mutations in ATM and/or BIRC3 and whether CLLs harboring these alterations could benefit from novel combinatorial therapies.To address these questions, we used the CRISPR/Cas9 system to generate an isogenic CLL cell line to model del(11q) derived from HG3 cells by introducing two guide RNAs targeting the 11q22.1 and 11q23.3 regions. The presence of a monoallelic deletion (size ~17 Mb) was confirmed in 100% of the cells by FISH. Truncating mutations in ATM and/or BIRC3 were introduced in the remaining allele, generating HG3 del(11q) ATMKO, del(11q) BIRC3KO and del(11q) ATMKOBIRC3KO (three clones per condition). In addition, single ATMKO and BIRC3KO mutations, or the combination of both, were introduced into both HG3 and MEC1 CLL-derived cells (three clones per condition).Functional in vitro studies revealed that del(11q) BIRC3KO cells had increased growth rates compared to del(11q) BIRC3WT clones (P<0.01). Similar results were observed in HG3 and MEC1 BIRC3KO cells (P<0.01; P<0.05). Moreover, biallelic inactivation of BIRC3 in del(11q) cells resulted in cytoplasmic stabilization of NF-kB-inducing kinase (NIK), leading to higher nuclear NF-kB2 (p52) activation (P<0.01) as measured by ELISA. In parallel, we analyzed the DNA damage response (DDR) of these cells, and showed that del(11q) ATMKO cells displayed reduced pH2AX levels (P<0.001) and an accumulation of unrepaired double strand breaks (DSB) (P<0.001) after irradiation, as determined by comet assays. Consistently, in vivo subcutaneous xenografts showed that HG3 ATMKOBIRC3KO tumors presented proliferative advantage, higher p52 levels and greater genomic and mitotic instability than HG3WT tumors, indicating a more aggressive phenotype.We next assessed the response of these CRISPR/Cas9-edited CLL cell lines to therapy. Of note, only TP53KO clones (also generated by CRISPR/Cas9), and not del(11q) BIRC3KO cells, showed resistance to fludarabine (mean IC50 16.9 uM vs. 4.1 uM; mean apoptotic cells (5 uM) 5.5% vs. 22.5%; P<0.05). Moreover, del(11q) cells were slightly more resistant to ibrutinib (IBRU) treatment compared to WT cells (mean IC50 10 uM vs. 3.7 uM; P<0.05). Interestingly, exploiting the DDR deficiencies underlying del(11q) by targeting the single strand break repair pathway with the PARP inhibitor olaparib (OLA), del(11q) ATMKO cells were not able to proliferate even 12 days after treatment (3 uM), independently of the mutational status of BIRC3 (P<0.01). In vivo intravenous HG3-derived xenografts (N=20) showed that OLA (100 mg/kg) reduced hCD45+ cells in the peripheral blood (P<0.01) and significantly improved survival of del(11q) ATMKOBIRC3KO xenografted mice (P<0.01).Moreover, IBRU potentiated the effects of OLA in cell viability (72h) in all the del(11q) clones (combination indexes 0.69-0.85), leading to an increased necrotic cell death, as shown by annexin V/PI staining (P<0.001) and HMGB1 release. Remarkably, we found that IBRU caused downregulation of the DNA repair protein RAD51, leading to impaired RAD51 foci formation in DSB lesions (P<0.01). Consistently, IBRU (1 uM) reduced the homologous recombination (HR) repair efficiency in HG3 cells (P=0.001), as determined by an HR-reporter construct. This IBRU-dependent impairment of HR repair could explain the synergistic effects with OLA by synthetic lethality.In conclusion, we demonstrate that del(11q) CLL cells with biallelic inactivation of BIRC3 and ATM show enhanced proliferation through activation of the non-canonical NF-kB pathway, and accumulation of DNA damage contributing to genomic instability. We show that these defects on the DDR can be therapeutically targeted by synthetic lethal approaches using PARP inhibitors either alone or in combination with BCR inhibitors, providing a rationale for the study of this combination in relapsed del(11q) CLL patients.PI15/01471 SA085U16 JCyL-MQ FEHH-MH DisclosuresGarcía-Tuñón:Novartis: Research Funding. Wu:Neon Therapeutics: Equity Ownership.

Lymphotoxin‐β receptor‐NIK signaling induces alternative RELB/NF‐κB2 activation to promote metastatic gene expression and cell migration in head and neck cancer

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-β (LTβ), receptor LTβR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTβR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTβ, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTβR, NIK, and RELB proteins. Recombinant LTβ, and siRNA depletion of endogenous LTβR and NIK, modulated expression of LTβR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTβ induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTβ-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTβ is expressed. Our findings show that LTβ/LTβR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

DDX3 directly facilitates IKKα activation and regulates downstream signalling pathways.

DDX3 is a DEAD-box RNA helicase that we and others have previously implicated in antiviral immune signalling pathways leading to type I interferon (IFN) induction. We previously demonstrated that it directly interacts with the kinase IKKε (IκB kinase ε), enhances it activation, and then facilitates phosphorylation of the transcription factor IRF3 by IKKε. However, the TLR7/9 (Toll-like receptor 7/9)-mediated pathway, one of the most physiologically relevant IFN induction pathways, proceeds independently of IKKε or the related kinase TBK1 (TANK-binding kinase 1). This pathway induces type I IFN production via the kinases NIK (NF-κB-inducing kinase) and IKKα and is activated when plasmacytoid dendritic cells sense viral nucleic acids. In the present study, we demonstrate that DDX3 also directly interacts with IKKα and enhances its autophosphorylation and -activation. Modulation of DDX3 expression consequently affected NIK/IKKα-mediated IRF7 phosphorylation and induction of type I interferons. In addition, alternative NF-κB (nuclear factor-κB) activation, another pathway regulated by NIK and IKKα, was also down-regulated in DDX3 knockdown cells. This substantially broadens the effects of DDX3 in innate immune signalling to pathways beyond TBK1/IKKε and IFN induction. Dysregulation of these pathways is involved in disease states, and thus, our research might implicate DDX3 as a potential target for their therapeutic manipulation.

Immunologic reconstitution following hematopoietic stem cell transplantation despite lymph node paucity in NF-κB–inducing kinase deficiency

This case demonstrates successful immune reconstitution following hematopoietic stem cell transplantation in NIK deficiency.

Inhibitory-κB Kinase (IKK) α and Nuclear Factor-κB (NFκB)-Inducing Kinase (NIK) as Anti-Cancer Drug Targets.

The cellular kinases inhibitory-κB kinase (IKK) α and Nuclear Factor-κB (NF-κB)-inducing kinase (NIK) are well recognised as key central regulators and drivers of the non-canonical NF-κB cascade and as such dictate the initiation and development of defined transcriptional responses associated with the liberation of p52-RelB and p52-p52 NF-κB dimer complexes. Whilst these kinases and downstream NF-κB complexes transduce pro-inflammatory and growth stimulating signals that contribute to major cellular processes, they also play a key role in the pathogenesis of a number of inflammatory-based conditions and diverse cancer types, which for the latter may be a result of background mutational status. IKKα and NIK, therefore, represent attractive targets for pharmacological intervention. Here, specifically in the cancer setting, we reflect on the potential pathophysiological role(s) of each of these kinases, their associated downstream signalling outcomes and the stimulatory and mutational mechanisms leading to their increased activation. We also consider the downstream coordination of transcriptional events and phenotypic outcomes illustrative of key cancer ‘Hallmarks’ that are now increasingly perceived to be due to the coordinated recruitment of both NF-κB-dependent as well as NF-κB–independent signalling. Furthermore, as these kinases regulate the transition from hormone-dependent to hormone-independent growth in defined tumour subsets, potential tumour reactivation and major cytokine and chemokine species that may have significant bearing upon tumour-stromal communication and tumour microenvironment it reiterates their potential to be drug targets. Therefore, with the emergence of small molecule kinase inhibitors targeting each of these kinases, we consider medicinal chemistry efforts to date and those evolving that may contribute to the development of viable pharmacological intervention strategies to target a variety of tumour types.

A proteasome-resistant fragment of NIK mediates oncogenic NF-κB signaling in schwannomas.

Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.

Manipulation of the Alternative NF-κB Pathway in Mice Has Sexually Dimorphic Effects on Bone.

ABSTRACTAlternative NF‐κB signaling promotes osteoclastogenesis and pathological bone loss, but the effect of sex on phenotype has not been explored. We disrupted alternative NF‐κB signaling by deletion of upstream kinase NF‐κB‐inducing kinase (NIK) or NF‐κB subunit RelB and found that both NIK‐deficient and RelB‐deficient female mice possessed more than twofold higher trabecular bone mass compared to controls, whereas no differences were observed in males. In vitro, RelB‐deficient precursors from female mice showed a more severe osteoclast (OC) differentiation defect than male, while WT had no sex bias. Next, we asked whether pharmacologic activation of alternative NF‐κB by inhibitor of apoptosis (IAP) antagonist BV6 has sex‐dependent effects on bone. Unlike male mice that lost bone, female mice on BV6 for 4 weeks showed no changes in either trabecular bone mass or OC number. Because estrogen generally suppresses NF‐κB, we hypothesized that estrogen protects bone from BV6 effects in vivo. Thus, we performed ovariectomy or sham surgery in female mice, then treated with BV6 or vehicle for 4 weeks. Although ovariectomy caused bone loss, BV6 did not have any additional impact, suggesting that direct estrogen effects do not cause resistance to BV6 in vivo. The osteopenic effects of IAP antagonists in males may have implications for their use in cancer therapy. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.