NF-κB-inducing Kinase Research Articles

Tunable Signal Processing through a Kinase Control Cycle: the IKK Signaling Node

The transcription factor NFκB, a key component of the immune system, shows intricate stimulus-specific temporal dynamics. Those dynamics are thought to play a role in controlling the physiological response to cytokines and pathogens. Biochemical evidence suggests that the NFκB inducing kinase, IKK, a signaling hub onto which many signaling pathways converge, is regulated via a regulatory cycle comprising a poised, an active, and an inactive state. We hypothesize that it operates as a modulator of signal dynamics, actively reshaping the signals generated at the receptor proximal level. Here we show that a regulatory cycle can function in at least three dynamical regimes, tunable by regulating a single kinetic parameter. In particular, the simplest three-state regulatory cycle can generate signals with two well-defined phases, each with distinct coding capabilities in terms of the information they can carry about the stimulus. We also demonstrate that such a kinase cycle can function as a signal categorizer classifying diverse incoming signals into outputs with a limited set of temporal activity profiles. Finally, we discuss the extension of the results to other regulatory motifs that could be understood in terms of the regimes of the three-state cycle.

C-IAP1 and c-IAP2 Redundancy Differs between T and B Cells

Cellular Inhibitors of Apoptosis 1 and 2 (c-IAP1 and c-IAP2) are ubiquitin protein ligases (E3s) that constitutively ubiquitinate and induce proteasomal-mediated degradation of NF-κB Inducing Kinase (NIK) and repress non-canonical NF-κB activation. Mice expressing an E3-inactive c-IAP2 mutant (c-IAP2H570A) have constitutive activation of non-canonical NF-κB, resulting in B cell hyperplasia and T cell costimulation-independence. If, and if so to what extent, c-IAP1 and c-IAP2 are redundant in NF-κB regulation in these mice is not known. Here we have generated mice expressing a mutant c-IAP1 that lacks E3 activity (c-IAP1H582A). These mice were phenotypically normal and did not have constitutive NF-κB activation in B cells or MEFs. siRNA-mediated knockdown of c-IAP2 showed that accumulated c-IAP2, resulting from lack of c-IAP1-dependent degradation, compensated for absent c-IAP1 E3 activity. Surprisingly, c-IAP1H582A T cells had a lower p100/p52 ratio than wild type T cells, and in the absence of costimulation proliferated to a degree intermediate between wild type and c-IAP2H570A T cells. Therefore, although c-IAP1 and c-IAP2 both can repress constitutive NF-κB activation, the relative importance of each varies according to cell type.

THU0083 NF-KB-Inducing Kinase (NIK) Expression in Synovial Blood Vessels Correlates with Systemic Markers of Inflammation and Local Disease Activity in Early Arthritis Patients, but is not Disease Specific

BackgroundThe NF-κB family of transcription factors is strongly involved in synovial inflammation. We have previously shown that NF-κB-inducing kinase (NIK) is a key regulator of inflammation-induced angiogenesis in rheumatoid arthritis...

Inducible Tumor Necrosis Factor (TNF) Receptor-associated Factor-1 Expression Couples the Canonical to the Non-canonical NF-κB Pathway in TNF Stimulation

The NF-κB transcription factor mediates the inflammatory response through distinct (canonical and non-canonical) signaling pathways. The mechanisms controlling utilization of either of these pathways are largely unknown. Here we observe that TNF stimulation induces delayed NF-κB2/p100 processing and investigate the coupling mechanism. TNF stimulation induces TNF-associated factor-1 (TRAF-1) that directly binds NF-κB-inducing kinase (NIK) and stabilizes it from degradation by disrupting its interaction with TRAF2·cIAP2 ubiquitin ligase complex. We show that TRAF1 depletion prevents TNF-induced NIK stabilization and reduces p52 production. To further examine the interactions of TRAF1 and NIK with NF-κB2/p100 processing, we mathematically modeled TRAF1·NIK as a coupling signaling complex and validated computational inference by siRNA knockdown to show non-canonical pathway activation is dependent not only on TRAF1 induction but also NIK stabilization by forming TRAF1·NIK complex. Thus, these integrated computational-experimental studies of TNF-induced TRAF1 expression identified TRAF1·NIK as a central complex linking canonical and non-canonical pathways by disrupting the TRAF2-cIAP2 ubiquitin ligase complex. This feed-forward kinase pathway is essential for the activation of non-canonical pathway.

NF-κB inducing kinase as a key player in regulating autoreactive B cell maturation (P4048)

Abstract The regulation of autoreactive B cells is crucial in immune systems to prevent developing autoimmune diseases in human and animal models. A critical peripheral B cell tolerance mechanism is exclusion of autoreactive B cells from the mature repertoire due to a failure to compete with non-autoreactive B cells. B cell activation factor (Baff) is one of the most important factors to discriminate autoreactive and non-autoreactive B cells in periphery. Therefore, the level of Baff expression in vivo is significant, since excess Baff can override exclusion and lead to autoimmunity, and may contribute to some human autoimmune diseases. We show here using B cells with different affinities for Sm, a self-antigen targeted in systemic lupus erythematosus (SLE), that the Baff-induced level of NF-κB inducing kinase (NIK) is reduced commensurate with the affinity for Sm and independently of Baffr levels. Reduced NIK differentially affect downstream signaling pathways, proportionally decreasing Akt activation and its repertoire inclusion function, but leaving intact NF-κB2 activation and its survival function. Thus, NIK functions as a key player to regulate entry into the mature B cell repertoire and preferentially excluding autoreactive B cells.

Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity

Fn14 is a therapeutic target in various diseases. Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.

NIK Controls Classical and Alternative NF-κB Activation and Is Necessary for the Survival of Human T-cell Lymphoma Cells

Peripheral T-cell lymphomas (PTCL) are a heterogeneous entity of neoplasms with poor prognosis, a lack of effective therapies, and a largely unknown molecular pathology. Deregulated NF-κB activity has been associated with several lymphoproliferative diseases, but its importance in T-cell lymphomagenesis is poorly understood. We investigated the function of the NF-κB-inducing kinase (NIK), in this pathway and its role as a potential molecular target in T-cell lymphomas. We used immunohistochemistry to analyze the expression of different NF-κB members in primary human PTCL samples and to study its clinical impact. With the aim of inhibiting the pathway, we used genetic silencing of NIK in several T-cell lymphoma cell lines and observed its effect on downstream targets and cell viability. We showed that the NF-κB pathway was activated in a subset of PTCLs associated with poor overall survival. NIK was overexpressed in a number of PTCL cell lines and primary samples, and a pivotal role for NIK in the survival of these tumor cells was unveiled. NIK depletion led to a dramatic induction of apoptosis in NIK-overexpressing cell lines and also showed a more pronounced effect on cell survival than inhibitor of kappa B kinase (IKK) knockdown. NIK silencing induced a blockage of both classical and alternative NF-κB activation and reduced expression of several prosurvival and antiapoptotic factors. The results of the present study indicate that NIK could be a promising therapeutic target in these aggressive malignancies.

A NIK-IKKα Module Expands ErbB2-Induced Tumor-Initiating Cells by Stimulating Nuclear Export of p27/Kip1

IκB kinase α (IKKα) activity is required for ErbB2-induced mammary tumorigenesis. Here, we show that IKKα and its activator, NF-κB-inducing kinase (NIK), support the expansion of tumor-initiating cells (TICs) that copurify with a CD24(med)CD49f(hi) population from premalignant ErbB2-expressing mammary glands. Upon activation, IKKα enters the nucleus, phosphorylates the cyclin-dependent kinase (CDK) inhibitor p27/Kip1, and stimulates its nuclear export or exclusion. Reduced p27 expression rescues mammary tumorigenesis in mice deficient in IKKα kinase activity and restores TIC self-renewal. IKKα is also likely to be involved in human breast cancer, where its expression shows an inverse correlation with metastasis-free survival, and its presence in the nucleus of invasive ductal carcinomas (IDCs) is associated with decreased nuclear p27 abundance.

Abstract 2099: Novel inhibitors of NFkB inducing kinase (NIK) are cytotoxic for myeloma cell lines with NIK-dependent activation of NFkB.

Abstract NFkB activity is critical for survival and proliferation of normal lymphoid cells and many kinds of B-cell tumors, including multiple myeloma (MM). NFkB activating mutations, which are apparent progression events, enable MM tumors to become less dependent on bone marrow signals that activate NFkB. Mutations that activate NFkB-inducing kinase (NIK) protein are the most prevalent among the many kinds of NFkB mutations in MM tumors. NIK is the main activating kinase of the alternative NFkB pathway, although over-expression of NIK also can activate the classical pathway. Three Amgen compounds (two NIK inhibitors and an isomeric control) were tested with human myeloma cell lines. These specific NIK inhibitors are selectively cytotoxic for cells with NIK-dependent activation of NFkB. The combination of NIK-inhibitors with IKKβ-inhibitors or dexamethasone show a synergistic effect that might more efficiently target some MM tumors. Citation Format: Yulia N. Demchenko, Walter Michael Kuehl. Novel inhibitors of NFkB inducing kinase (NIK) are cytotoxic for myeloma cell lines with NIK-dependent activation of NFkB. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2099. doi:10.1158/1538-7445.AM2013-2099 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

OTUB1 modulates c-IAP1 stability to regulate signalling pathways

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.

Delphinidin inhibits IL-1 -induced activation of NF- B by modulating the phosphorylation of IRAK-1Ser376 in human articular chondrocytes

In OA, there is enhanced expression of pro-inflammatory cytokines such as IL-1β in the affected joint. Delphinidin, an anthocyanidin found in pigmented fruits and vegetables, has been shown to possess anti-inflammatory and antioxidant properties. In the present study we determined whether delphinidin would inhibit the IL-1β-induced activation of NF-κB in human chondrocytes and determined the mechanism of its action. PGE2 levels and activation of NF-κB p65 in human OA chondrocytes were determined by ELISA-based assays. Protein expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of kinases was determined by western immunoblotting. Expression level of mRNAs was determined by TaqMan assays. Delphinidin inhibited IL-1β-induced expression of COX-2 and production of PGE2 in human chondrocytes. Delphinidin also inhibited IL-1β-mediated phosphorylation of IL-1 receptor-associated kinase-1(Ser376), phosphorylation of IKKα/β, expression of IKKβ, degradation of IκBα, and activation and nuclear translocation of NF-κB/p65. Phosphorylation of TGF-β-activated kinase 1 was not observed but NF-κB-inducing kinase (NIK) was phosphorylated and phosphorylation of NIK was blocked by delphinidin in IL-1β-treated human chondrocytes. These data identify delphinidin as a novel inhibitor of IL-1β-induced production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expression and provide new insight into the mechanism of its action. Our results also identify inhibition of IRAK1(Ser376) phosphorylation by delphinidin in IL-1β-induced activation of NF-κB in human chondrocytes. Given the important role played by IL-1β-induced NF-κB activation, COX-2 expression and PGE2 production in OA, our results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA.

Antagonism of Inhibitor of Apoptosis Proteins Increases Bone Metastasis via Unexpected Osteoclast Activation

Inhibitor of apoptosis (IAP) proteins play a central role in many types of cancer, and IAP antagonists are in development as anticancer agents. IAP antagonists cause apoptosis in many cells, but they also activate alternative NF-κB signaling through NF-κB-inducing kinase (NIK), which regulates osteoclasts. In bone metastasis, a positive feedback loop between tumors and osteoclasts promotes tumor growth and osteolysis. We therefore tested the effect of IAP antagonists on the bone microenvironment for metastasis. In both drug-sensitive and drug-resistant tumors, growth in bone was favored, as compared with other sites during IAP antagonist treatment. These drugs also caused osteoporosis and increased osteoclastogenesis, mediated by NIK, and enhanced tumor-associated osteolysis. Cotreatment with zoledronic acid, a potent osteoclast inhibitor, reduced IAP antagonist-enhanced tumor growth in bone and osteolysis. Thus, IAP antagonist-based cancer treatment may be compromised by osteoporosis and enhanced skeletal metastasis, which may be prevented by antiresorptive agents. Although IAP antagonists are a class of anticancer agents with proven efficacy in multiple cancers, we show that these agents can paradoxically increase tumor growth and metastasis in the bone by stabilizing NIK and activating the alternative NF-κB pathway in osteoclasts. Future clinical trials of IAP antagonist-based therapy may require detailed examination of this potential for enhanced bone metastasis and osteoporosis, as well as possible combination with antiresorptive agents.

Inhibiting NF-κB-inducing kinase (NIK): Discovery, structure-based design, synthesis, structure–activity relationship, and co-crystal structures

The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28. Compounds 25 and 31 were co-crystallized with NIK kinase domain to provide structural insights.

Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

BackgroundIncreased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated.Methodology/Principal FindingsHere we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells.Conclusions/SignificanceRapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer.

A Complex Relationship between TRAF3 and Non-Canonical NF-κB2 Activation in B Lymphocytes

The adaptor protein TRAF3 restrains B cell activating factor receptor (BAFFR) and CD40-mediated activation of the NF-κB2 pathway in B cells. Mice lacking TRAF3 specifically in B cells revealed the critical role of TRAF3 in restraining homeostatic B cell survival. Furthermore, loss-of-function mutations of the traf3 gene have been associated with human B cell malignancies, especially multiple myeloma (MM). It has been proposed that receptor-induced TRAF3 degradation leads to stabilization of the NF-κB inducing kinase (NIK), and subsequent NF-κB2 activation. However, it is unclear how receptor-mediated TRAF3 degradation or loss-of-function contributes to B cell-specific NF-κB2 activation. In the current study, we employed two complementary models to address this question. One utilized a mutant traf3 gene found in a human MM-derived cell line called LP1. The LP1 mutant TRAF3 protein lacks the TRAF-N and TRAF-C domains. Consistent with the paradigm described, expression of LP1 TRAF3 in B cells promoted higher basal levels of NF-κB2 activation compared to Wt TRAF3. However, LP1 did not associate with TRAF2, CD40, or BAFFR, and no LP1 degradation was observed following receptor engagement. Interestingly, LP1 showed enhanced NIK association. Thus, TRAF3 degradation becomes dispensable to activate NF-κB2 when it is unable to associate with TRAF2. In a second model, we examined several mutant forms of BAFFR that are unable to induce NF-κB2 activation in B cells. Signaling to B cells by each of these BAFFR mutants, however, induced levels of TRAF3 degradation similar to those induced by Wt BAFFR. Thus, in B cells, receptor-mediated TRAF3 degradation is not sufficient to promote NF-κB2 activation. We thus conclude that there is not a simple linear relationship in B lymphocytes between relative levels of cellular TRAF3, induced TRAF3 degradation, NIK activation, and NF-κB2 activation.

Carrageenan-Induced Colonic Inflammation Is Reduced in Bcl10 Null Mice and Increased in IL-10-Deficient Mice

The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μg/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (Bcl10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in Bcl10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBα in the colonic epithelial cells were significantly less (P < 0.001) in the carrageenan-treated Bcl10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-Bcl10, and exogenous IL-10 inhibited only the canonical pathway of NF-κB activation in cultured colonic cells. These findings demonstrate a Bcl10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.

Regulation of the Alternative NF-κb Pathway and Its Role in Cancer

NF-κB is regulated by two distinct pathways namely the Classical NF-κB pathway and the recently discovered alternative NF-κB pathway. While the classical NF-κB pathway has been extensively studied for its role in cancer, our understanding of the regulation of the alternative NF-κB pathway and its role in cancer has been limiting. Nevertheless, significant progress has been made recently that revolves around the regulation of NF-κB inducing kinase and its role in cancer. These recent developments will be discussed in this editorial.

Glucocorticoid Receptor Signaling Contributes to Constitutive Activation of the Noncanonical NF-κB Pathway in Term Human Placenta

Our recent study demonstrated that constitutively activated RelB/NF-κB2 positively regulates the CRH in the human placenta. In the current study, we explored the role of the glucocorticoid receptor (GR) signaling in constitutive activation of the noncanonical NF-κB pathway. A glucocorticoid response element (GRE) motif search suggests that both NF-κB inducing kinase (NIK) and RelB genes, which are key regulators of the noncanonical NF-κB pathway, have a putative GRE within their promoter, approximately 1 kb upstream from the transcription start site. By using chromatin immunoprecipitation assay we identified that the GR and phosphorylated GR at Ser211 were associated with the GREs of both NIK and RelB. Dexamethasone stimulated expression of NIK, RelB, NF-κB2 as well as CRH and cyclooxygenase-2 (COX-2). Repression of GR by short interfering RNA resulted in inhibition of NIK, RelB, NF-κB2, CRH, and COX-2. In addition, depletion of GR attenuated glucocorticoid-mediated up-regulation of NIK, RelB, NF-κB2, CRH, and COX-2. Furthermore, siRNA specifically targeting NIK down-regulated CRH and COX-2. Taken together, these results suggest that constitutive activation of the noncanonical NF-κB pathway in term human placenta is driven by the GR signaling, which in turn up-regulates placental CRH and other NF-κB-responsive genes.

Apoptosis Repressor with Caspase Recruitment Domain Is Regulated by the cIAP1-NIK Axis and Confers Resistance to SMAC Mimetic Birinapant-Induced Cell Death in AML

Abstract 534The inhibitors of apoptosis (IAPs), including cIAP1, cIAP2, and XIAP are a family of anti-apoptotic proteins that play important roles in regulating cell survival. SMAC, a mitochondrial protein, is a natural cellular inhibitor of IAPs. SMAC mimetics, mimicking the IAP-binding site in the N-terminal AVPI peptide sequence of SMAC, are a new class of anticancer agents that degrade cIAPs and suppress XIAP activity. ARC (Apoptosis repressor with caspase recruitment domain) is an anti-apoptotic protein that inhibits the activation of caspase-8. We previously reported that the SMAC mimetic birinapant (TL32711; Tetralogic Pharmaceuticals, Malvern, PA) degrades cIAP1 and promotes apoptosis via the death receptor/caspase-8-mediated extrinsic pathway in primary AML cells and in AML cell lines in the presence of death receptor ligands (Carter BZ et al., ASH 2011). High ARC levels also predict adverse outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report that birinapant-induced reduction in cIAP1 is accompanied by increased ARC levels.cIAPs are known E3 ligases for NF-κB-inducing kinase (NIK), an upstream kinase of non-canonical NF-κB. SMAC mimetics, including birinapant cleave cIAPs, leading to stabilization of NIK and activation of non-canonical NF-κB signaling and its downstream targets. To determine whether ARC is regulated via the cIAP1-NIK axis, we knocked down NIK in OCI-AML3 and Molm13 cells by siRNAs and found that inhibition of NIK decreased ARC RNA and protein levels in these cells and suppressed birinapant-induced increases of ARC, suggesting that ARC is regulated via the cIAP1/NIK/NF-κB cascade. We determined levels of ARC and cIAP1 by reverse-phase protein array in 511 samples obtained from patients with newly diagnosed AML and found that cIAP1 and ARC were inversely correlated (R = −0.225, P< 0.0001) further supporting the negative regulation of ARC by cIAP1 in primary AML samples.Data indicate that birinapant induces caspase-8-mediated cell death, but increases levels of ARC in AML cells which inhibits caspase-8 activation, suggesting that ARC is a resistance factor for birinapant-induced cell death. To further investigate this mechanism, we generated stable ARC-knock down (K/D) OCI-AML3 and Molm13 cells and stable ARC-overexpressing (O/E) KG-1 cells and treated these cells with birinapant or birinapant plus TNFα. We found what ARC-K/D OCI-AML3 and Molm13 cells were more sensitive and ARC-O/E KG-1 cells were more resistant to birinapant- or birinapant plus TNFα-induced apoptosis than their control cells. We reported previously that demethylating agents can enhance birinapant-induced apoptosis induction in AML cells. Examination of NIK and ARC levels in decitabine or 5-azacytidine treated AML cells showed that the demethylating agents indeed decreased NIK and ARC protein levels.Leukemia cells are in close contact with the bone marrow (BM) microenvironment in vivo that protects them from cell death induced by various therapeutic agents. Leukemia cells were co-cultured with BM-derived mesenchymal stromal cells (MSCs) in vitro to mimic in vivo conditions. We found that birinapant decreased cIAP1 and increased ARC levels also in MSCs co-cultured with AML cells. We generated stable ARC-K/D MSCs and treated KG-1, OCI-AML3, and Molm13 cells co-cultured with ARC-K/D or vector control MSCs with birinapant plus TNFα and primary AML patient samples co-cultured with ARC-K/D or vector control MSCs with birinapant. ARC-K/D MSCs provided AML cells with less protection than control MSCs against birinapant plus TNFα- or birinapant-induced apoptosis.Collectively, data demonstrate that ARC is regulated via the cIAP1/NIK signaling pathway and is a resistance factor for SMAC mimetic birinapant-induced cell death. ARC K/D sensitizes AML cells to SMAC mimetic-induced cell death and also suppresses MSC-mediated protection of AML cells against drug-induced apoptosis. Disclosures:No relevant conflicts of interest to declare.

Impact of NF-κB gene polymorphism on allograft outcome in Hispanic renal transplant recipients

BackgroundThe dimeric NF-κB transcription factors play critical roles in diverse cellular processes including adaptive and innate immunity, cell differentiation, proliferation and apoptosis. It regulates the expression of numerous genes that play a key role in the inflammatory response during kidney allograft rejection. This study aims to determine the association of NF-κB gene polymorphisms with allograft outcomes in the Hispanic renal transplant recipients. MethodsA total of 607 Hispanic renal transplant recipients at St. Vincent Medical Center between 2001 and 2010 were included in this study. The NF-κB genotypes were studied along with clinical data. In the case of NF-κB genotypes, the following single nucleotide polymorphisms (SNPs) were included: NF-κB1 (rs3774959, rs3774932, rs3774937, rs230526, rs230519), NF-κB2 (rs1056890, rs7897947, rs12769316) and NF-κB inducing kinase (NIK) (rs9908330, rs7222094). The association of each genotype with renal allograft survival and acute rejection was evaluated. ResultsNF-κB1 (rs3774937) CC genotype showed protective association with allograft rejection (OR=0.66, 95% CI=0.44–0.99, p=0.04). There was a significant increase in allograft survival time associated with the NF-κB1 (rs3774959) A allele (OR=0.76, 95% CI=0.60–0.98, p=0.03) while GG genotype was associated with a higher risk of graft failure (OR=1.51, 95% CI=1.02–2.21, p=0.03). There were no associations between polymorphic markers in NF-κB2 and NIK genes with allograft survival or acute rejection. Among non-genetic factors, we found that the use of tacrolimus, a deceased donor, delayed graft function and acute rejection were associated with allograft failure. ConclusionThe result of present study suggests that NF-κB1 gene polymorphisms may determine the incidence of acute rejection or graft survival among Hispanic allograft recipients.