Expression Of NF-κB Target Genes Research Articles

T Cell Factor 4 Is a Pro-catabolic and Apoptotic Factor in Human Articular Chondrocytes by Potentiating Nuclear Factor κB Signaling

T cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors are downstream effectors of Wnt/β-catenin signaling, which has been implicated in the development and progression of osteoarthritis (OA). This study aimed to investigate the role of TCF/LEF transcription factors in human articular chondrocytes. Primary human osteoarthritic cartilage predominantly expressed TCF4 and to a lesser extent, LEF1 and TCF3 mRNA. Overexpression of TCF4, but not of TCF3 or LEF1, induced MMP-1, -3, and -13 expression and generic MMP activity in human chondrocytes. This was due to potentiating NF-κB signaling by a protein-protein interaction between TCF4 and NF-κB p65 activating established NF-κB target genes such as MMPs and IL-6. LEF1 competed with TCF4 for binding to NF-κB p65. IκB-α was able to counteract the effect of TCF4 on NF-κB target gene expression. Finally, we showed that TCF4 mRNA expression was elevated in OA cartilage compared with healthy cartilage and induced chondrocyte apoptosis at least partly through activating caspase 3/7. Our findings suggest that increased TCF4 expression may contribute to cartilage degeneration in OA by augmenting NF-κB signaling.

DDRGK1 Regulates NF-κB Activity by Modulating IκBα Stability

NF-κB is a ubiquitously expressed transcription factor that regulates a large number of genes in response to diverse physiological and pathological stimuli. The regulation of the transcriptional activity of NF-κB is often dependent on its interaction with IκBα. Proteins that bind to IκBα are critical regulators of NF-κB activity. DDRGK1 is a member of the DDRGK domain-containing protein family with unknown function. In this study, we showed that the depletion of DDRGK1 inhibits cell proliferation and invasion. Microarray analysis indicated that the expression of NF-κB target genes showed the most significant decrease after depleting of DDRGK1, suggesting that DDRGK1 may play an important role in the NF-κB signaling pathway. We further demonstrated that DDRGK1 interacts with IκBα and regulates its stability, thereby regulates the NF-κB transcriptional activity. Our findings identify DDRGK1 as an important regulator of the NF-κB pathway.

Stat2 loss leads to cytokine-independent, cell-mediated lethality in LPS-induced sepsis

Deregulated Toll-like receptor (TLR)-triggered inflammatory responses that depend on NF-κB are detrimental to the host via excessive production of proinflammatory cytokines, including TNF-α. Stat2 is a critical component of type I IFN signaling, but it is not thought to participate in TLR signaling. Our study shows that LPS-induced lethality in Stat2(-/-) mice is accelerated as a result of increased cellular transmigration. Blocking intercellular adhesion molecule-1 prevents cellular egress and confers survival of Stat2(-/-) mice. The main determinant of cellular egress in Stat2(-/-) mice is the genotype of the host and not the circulating leukocyte. Surprisingly, lethality and cellular egress observed on Stat2(-/-) mice are not associated with excessive increases in classical sepsis cytokines or chemokines. Indeed, in the absence of Stat2, cytokine production in response to multiple TLR agonists is reduced. We find that Stat2 loss leads to reduced expression of NF-κB target genes by affecting nuclear translocation of NF-κB. Thus, our data reveal the existence of a different mechanism of LPS-induced lethality that is independent of NF-κB triggered cytokine storm but dependent on cellular egress.

Abstract LB-141: Potent single agent activity of Ibrutinib (PCI-32765) in patients with chronic lymphocytic leukemia (CLL): clinical and translational results from an ongoing phase II study.

Abstract Background: B-cell receptor (BCR) activation plays a pivotal role in the pathogenesis and for progression of CLL. Ibrutinib (PCI-32765), an irreversible inhibitor of Bruton's tyrosine kinase (BTK), can disrupt BCR signaling and has durable antitumor activity in CLL. We initiated a phase II single center trial to extend this experience and assess the impact of ibrutinib on tumor cells in vivo. Methods: Treatment naïve (TN) and relapsed/refractory (R/R) pts with CLL are treated with 420 mg ibrutinib daily. Pts are enrolled in 2 cohorts: 1) deletion (del) of chromosome 17p and 2) >65yo and treated until disease progression. Response is evaluated at 6 months (mo) and every 6 mo thereafter. 17p del was assessed by FISH cytogenetics. Spleen volume on computed tomography (CT) scans was computed using a General Electric Advanced Workstation Server. Results: With a median follow-up of 9 mo we report on the first 53 patients: for del 17p n=29; 15 TN, 14 R/R; and for >65yo n=24; 8 TN, 16 R/R. Estimated event free survival at 12 mo is 94%. Most adverse events were mild with non hematologic toxicities (regardless of causality) grade ≥3 in <13% of pts. Two deaths on study were not treatment related. At 6 mo 95% of pts (n=44 evaluable) had a nodal response (median reduction in lymph node size of 73%) and 52% of pts had a partial response (PR) by IWCLL criteria. One pt had progressive disease (presumed Richter's transformation). All pts had a decrease in splenomegaly. The median reduction of splenic volume computed from CTs was 55% (30-1716 ml). Tumor infiltration in bone marrow biopsies (n=26), decreased by a median 82% as assessed by immunohistochemistry for CD79a and the median ALC decreased from 79 k/µl (0.5-402) to 32 k/µl (0.8-167) at 6 mo for a median reduction of 62%. We evaluated the in vivo effects of ibrutinib using blood and tissue samples collected pre and during ibrutinib treatment. On treatment expression of genes known to be induced by B-cell receptor (BCR) activation (Herishanu et al 2011) was significantly reduced in CLL cells in the peripheral blood (PB) in all pts (n=15; median reduction 57%; P<.001). Consistent with inhibition of BCR signaling, we found concurrent reduction in PLCγ2 phosphorylation; a direct target of BTK. Patients also donated matched lymph node (LN) core biopsies pre-treatment and within the first 4 days on treatment. Ibrutinib inhibited BCR signaling in the LN (n=8, P=.004) to a similar degree as observed in the PB. Similarly, ibrutinib decreased expression of NF-κB target genes in PB and LN. In keeping with inhibition of BCR and NF-κB signaling surface markers of cellular activation such as CD38, CD69, and CD86 were significantly reduced by ibrutinib as measured by flow cytometry (P<.02). Finally, ibrutinib significantly reduced tumor proliferation as determined by the percentage of PB CLL cells expressing Ki67 (median reduction of 80%; P<.001). Conclusions: Ibrutinib as a single agent is well tolerated and highly effective in both TN and R/R pts achieving rapid disease control in blood, lymph nodes, spleen and bone marrow that is durable. Correlative studies on patient samples during ibrutinib treatment provide direct evidence for on target effects of ibrutinib in vivo demonstrating effective and sustained inhibition of BCR and NF-κB signaling in both PB and LNs. Ibrutinib deserves further investigation as targeted therapy for CLL. We thank our patients for participation and donation of blood and tissue samples. Supported by the Intramural Research Program of NIH. Citation Format: Adrian Wiestner, Sarah Herman, Rashida Mustafa, Janet Valdez, Jade Jones, Nakhle Saba, Andrew Lipsky, Diane C. Arthur, Gerald Marti, Francine Thomas, Irina Maric, Stefania Pittaluga, Xin Tian, Susan Soto, Georg Aue, Mohammed Z. Farooqui. Potent single agent activity of Ibrutinib (PCI-32765) in patients with chronic lymphocytic leukemia (CLL): clinical and translational results from an ongoing phase II study. [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 LB-141. doi:10.1158/1538-7445.AM2013-LB-141

Abstract 99: Identification of genes involved in NFκB driven resistance to endocrine treatment in breast cancer.

Abstract Introduction: Breast cancer is a heterogeneous disease exemplified by patients with Estrogen Receptor (ER) positive and negative breast cancer. Treatment of patients with breast cancer is increasingly governed by this notion of heterogeneity. For example, patients with ER+ breast cancer are treated with endocrine therapy. Although endocrine treatment is the mainstay of therapy for patients with ER+ breast cancer, resistance to endocrine therapy is a major clinical problem. Recently we identified a 6-gene signature predictive of Tamoxifen sensitivity. In addition, our earlier work has shown the importance of the NFkB transcription factor family in determining ER function. In this study, we aim to elucidate the role of NFkB in endocrine therapy response. Materials and methods: Public gene expression data sets of breast cancer patients and cell lines have been analyzed. ER- (PAM50-model) and NFkB-activation signatures (Annunziata et al, Cancer Cell, 2007) were calculated for all samples. In addition, samples were classified using our Tamoxifen response gene signature. We selected 11 cell lines with presumed strong or weak activation of NFkB based on the analysis of NFkB target gene expression. These cell lines were analyzed for NFkB DNA-binding using the TransAM ELISA kits (Active Motif). ER and NFkB target gene expression was determined using qRT-PCR. In addition, the expression of the 6 endocrine therapy responsive genes (ABAT, ADAMDEC1, CLEC7A, ETS1, ITK and STC2) was evaluated. Results: When analyzing the NFkB activation signature in ∼700 samples from patients with breast cancer, we clearly observed an inverse correlation with the ER activation signature (R=-0.65, P<0.01). Also, we observed NFkB hyperactivation in ER- breast cancers (P<0.01). When restricting our analysis to ER+ samples, we demonstrated NFkB hyperactivation in samples from patients predicted by our 6-gene signature to be resistant to endocrine therapy. NFkB DNA-binding experiments confirmed the gene expression based NFkB activation status of eleven selected cell lines. In addition, NFkB DNA-binding was associated with elevated expression of ADAMDEC1 and ETS1 and reduced expression of ABAT (All Ps<0.05). Finally, the 6-gene signature predictive of Tamoxifen response showed an increased expression in cell lines with increased NFkB DNA-binding (P<0.01). Conclusion: In the present study we demonstrate that NFkB activation is associated with attenuated response to endocrine treatment in patients with breast cancer. Our data suggest that this association is due to a repressive effect of NFkB on ER but mechanistic data in support of this view are currently lacking. We propose that the panel of cell lines evaluated in part of the present study will prove to be helpful instrument in elucidating the role of the interaction between ER and NFkB in endocrine treatment resistance. Citation Format: Leen Sas, Peter Van Dam, Luc Dirix, Peter Vermeulen, Filip Lardon, Steven Van Laere. Identification of genes involved in NFκB driven resistance to endocrine treatment in breast cancer. [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 99. doi:10.1158/1538-7445.AM2013-99

Lentiviral Delivery of RNAi for In Vivo Lineage-Specific Modulation of Gene Expression in Mouse Lung Macrophages

Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings.

NS-398 enhances the efficacy of bortezomib against RPMI8226 human multiple myeloma cells

Bortezomib is commonly used in treating multiple myeloma (MM). However, a number of patients develop resistance to bortezomib over time. Cox-2 is overexpressed in MMcells and contributes to apoptosis resistance and MM development. In the present study, RPMI8226 MM cells were treated with the Cox-2 inhibitor NS-398 to investigate whether it enhanced the effect of bortezomib on MM. The results showed that NS-398 and bortezomib acted synergistically to inhibit growth, arrest the cell cycle at the G1phase and to induce the apoptosis of MM cells. NS-398 inhibited the NF-κB p65 protein levels and the expression of various NF-κB target genes, including cyclinD1, c-Myc, survivin and Bcl-2. In conclusion, NS-398 enhanced the efficacy of bortezomib against MM cells invitro and this was associated with the inhibition of NF-κB signaling. These findings suggest that the combined use of NS-398 and bortezomib may constitute a promising novel treatment protocol for MM patients.

Abrogation of galectin-4 expression promotes tumorigenesis in colorectal cancer

Although it has been well established that galectin-4 is selectively expressed by intestinal epithelial cells, the role of galectin-4 in colorectal cancer (CRC) development is, as yet, poorly understood. Here, we aimed to explore the role of galectin-4 in CRC development, both in vitro and in vivo. Galectin-4 expression was investigated in tissue specimens from patients with adenoma, carcinoma and ulcerative colitis (UC) using immunohistochemistry. Colorectal cancer-derived HT-29 cells, in which galectin-4 expression was knocked down, were established using shRNA. mRNA and protein expression levels of galectin-4 and several downstream cancer-related genes were analyzed using RT-PCR, qPCR array, Western blotting, and immunofluorescence assays. To investigate the effect of galectin-4 expression abrogation on tumorigenesis in vivo, xenograft assays were performed. Immunohistochemistry analyses showed high expression levels of galectin-4 in normal colon mucosa tissues. Conversely, the expression levels of galectin-4 were significantly lower in CRC samples and its precursor lesions with dysplasia or inflammation. We found that shRNA-mediated galectin-4 silencing increases cell proliferation and, concomitantly, activates NF-κB and STAT3 signaling along with IL-6 up-regulation. In addition, we found that shRNA-mediated galectin-4 silencing promotes the expression of NF-κB target genes and other cancer-related genes and, concomitantly, enhances the in vivo growth of xenografts. We show that abrogation of galectin-4 expression promotes cancer cell proliferation and, for the first time, provide evidence that down-regulation of galectin-4 elicits tumor promotion in vitro and in vivo through activation of IL-6/NF-κB/STAT3 signaling.

Epstein-Barr Virus Deubiquitinase Downregulates TRAF6-Mediated NF-κB Signaling during Productive Replication

Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication.

Suppression of NF-κB/p65 Inhibits the Proliferation in Oral Squamous Cancer Cells

Object: Hypoxia occurs when oxygen tension drops below normal limits, and malignant tumors often experience hypoxia, which activates the expression of genes through oxygen-sensitive transcription factors, including the hypoxiainducible factor (HIF) and the nuclear factor-κB (NF-κB). NF-κB pathway represents an attractive therapeutic target in both cancer cells and ischemic cells, which involves immune response. To investigate the expression of NF-κB target genes in oral squamous cancer carcinoma (OSCC) under hypoxia, we performed cDNA plate array analyses of 23 NF-κB-regulated genes in different cell lines. Our aim was to clarify the functions of NF-κB in OSCC under hypoxia. Results: We conducted an NF-κB reporter assay to examine NF-κB activation under hypoxia. This luciferase-based reporter assay showed that hypoxia induced NF-κB activation after 24 h of hypoxia. We also found that vascular endothelial growth factor-C (VEGF-C) of NF-κB-regulated genes was upregulated in both cell lines under hypoxia. We then tested the influence of NF-κB/p65 knockdown on these cells and NF-κB/p65 knockdown inhibited the proliferation of OSCC cells by colony assay. Conclusion: Based on these findings, we postulate that one mechanism by which hypoxic OSCC cells may involve NF-κB-mediated upregulation. Our results also indicate that the knockdown of NF-κB/p65 subunit lead to growth inhibition during hypoxia in OSCC cells. These results suggest that knockdown of NF-κB/p65 subunit could be a potential therapeutic option for patients with OSCC.

INOS Activation Regulates β-catenin Association with Its Partners in Endothelial Cells

BackgroundSignals that disrupt β-catenin association to cadherins may influence the translocation of β-catenin to the nucleus to regulate transcription. Post-translational modification of proteins is a signalling event that may lead to changes in structural conformation, association or function of the target proteins. NO and its derivatives induce nitration of proteins during inflammation. It has been described that animals treated with NO donors showed increased permeability due to modulation of VE-cadherin/catenin complex. We, therefore, aim to evaluate the effect of iNOS activation on the expression, nuclear localisation and function of β-catenin in endothelial cells.Methodology/Principal FindingsExpression, nuclear localisation, post-translational modifications and function of β-catenin was analysed by cell fractionation, immunoprecipitation, immunoblots, QRT-PCR and permeability assays in murine endothelial cells (H5V). Influence of macrophage activation on expression of VE-cadherin/p120-catenin/β-catenin complex in co-cultured H5V cells was also assessed. Activation of macrophages to produce NO provoked a decrease in VE-cadherin/p120-catenin/β-catenin expression in H5V cells. Phosphorylation of β-catenin, p120-catenin and VE-cadherin, and reduction in the barrier properties of the cell monolayer was associated with iNOS induction. Moreover, high NO levels provoked nitration of β-catenin, and induced its translocation to the nucleus. In the nucleus of NOS activated cells, nitration levels of β-catenin influenced its association with TCF4 and p65 proteins. High levels of NO altered β-catenin mediated gene expression of NFκB and Wnt target genes without affecting cell viability.ConclusionsNOS activity modulates β-catenin post-translational modifications, function and its association with different partners to promote endothelial cell survival. Therapeutic manipulation of iNOS levels may remove a critical cytoprotective mechanism of importance in tumour angiogenesis.

Dll4-Fc, an Inhibitor of Dll4-Notch Signaling, Suppresses Liver Metastasis of Small Cell Lung Cancer Cells through the Downregulation of the NF-κB Activity

Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc-overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling.

NF-κB Activation in Mesenchymal Stromal Cells Mediates Leukemia Cell Chemoresistance

Abstract 3518Within the bone marrow (BM) microenvironment, BM mesenchymal stromal cells (BM-MSC) provide leukemia cells with a rich environment that serves as a sanctuary and protects them from chemotherapeutic agents. Interactions between leukemia cells and BM-MSC are thought to change the behavior of both stroma and leukemia cells resulting in an increased resistance to standard drugs. To discover interaction-induced changes in BM-MSCs that might promote microenvironment-mediated chemoresistance we used genome-wide gene expression profiling to study normal-donor BM-MSCs co-cultured with REH cells, a pre-B acute lymphoblastic leukemia (ALL) cell line. Upregulated genes in normal-donor BM-MSCs by co-culture included many cytokines and chemokines and implicated activation of NF-κB in BM-MSCs. Similar results were obtained by co-culturing normal-donor BM-MSCs with OCI-AML3 cells, an acute myeloid leukemia (AML) cell line. Increased expression of NF-κB target genes in BM-MSCs was also induced by co-culture with primary cells from ALL and AML patients, suggesting that NF-κB activation is a common consequence of leukemia-stroma interaction. Blocking canonical-pathway NF-κB activation by overexpressing a super-repressor form of IκBα in BM-MSC, significantly reduced stroma-mediated chemoresistance to vincristine (VCR) of leukemia cells in vitro and in vivo using an extramedullary bone marrow model. When small-molecule IKKβ inhibitors (MLN3316 and CDDO-Me) were used in co-cultures, blocking canonical-pathway NF-κB activation in ALL cell lines (REH or RS4;11) as well as BM-MSCs, the apoptotic effects of VCR on leukemia cells were further increased. Conclusion:results indicate that leukemia cells activate NF-κB in stromal cells and that such activation is in turn essential to promote survival of leukemia cells during chemotherapy. Targeting NF-κB in BM-MSC may ameliorate stroma-mediated chemoresistance and help in eliminating BM-resident leukemia cells. Disclosures:No relevant conflicts of interest to declare.

Chemical Biology Strategy Reveals Cell Lineage- and Cell Differentiation-Specific Small Molecule Inhibitors of NF-κB.

AbstractAbstract 2421Members of the nuclear factor-kB (NF-κB) family of transcription factors play important roles in cell-signaling involved in host-defense, immune responses, inflammation, and cancer. Multiple stimuli operating through different receptors are capable of activating NF-κB, including the Tumor Necrosis Factor (TNF) family cytokines, Toll-like receptors (TLRs), Nucleotide-binding Leucine Rich Repeat Proteins (NLRs), DNA-damaging agents, and others. Drugs that selectively block activation of NF-κB through one or more of these receptors could have activity against various types of cancer in which activation of NF-κB plays a role. We used a chemical biology strategy to identify novel chemical inhibitors of cell pathways responsible for NF-κB activation. Screening of a large chemical library (>330K compounds from NIH) was conducted using human 697 pre-B-cell leukemia cells with a stably integrated NF-κB-luciferase reporter gene and stimulating the Carma/Bcl-10/MALT pathway using protein kinase C (PKC) activators (PMA/Ionomycin). Confirmed hits were counter-screened against HEK293T-NF-κB-luciferase cells treated with TNF-a, with the goal to eliminate non-pathway specific inhibitors of NF-κB. Among the hit compounds, we identified oxadiazole- and oxazole-based chemical probes as potential cell lineage- and cell differentiation-specific inhibitors of NF-κB (“NLDSi” = NF-κB lineage, differentiation-specific inhibitors). Using a panel of cell lines with stably integrated NF-κB-driven luciferase reporter genes, NLDSi compounds suppressed PMA/Ionomycin-induced NF-κB activity in a concentration-dependent manner (IC50=0.1–1.0 μM) in the pre-B-cell leukemia cell-line 697, but not in the T-cell leukemia line JURKAT, the myeloma cell-line RPMI8226, or HEK293 epithelial cells. These compounds also suppressed NF-κB activity (measured by reporter gene) in 697 cells stimulated with BAFF or treated with doxorubicin. Examining NF-κB target gene expression by q-RT-PCR (mRNA level) and immunoblotting (protein level) showed that NLDSi compounds reduced PMA/Ionomycin-induced TRAF1 and A20 expression in 697 cells and in the pre-B-cell leukemia cell line REH, but not in several mature B-cell lines, including BJAB, Daudi, OCILy3, and OCILy10. NLDSi compounds also inhibited NF-κB target gene expression in pre-B-cell lines stimulated with BAFF or treated with lipopolysaccharide (LPS) or doxorubicin (Dox). Nevertheless we found these compounds could suppress of PMA/Ionomycin-induced expression of TRAF1 and A20 gene in the primary leukemia cells of patients with chronic lymphocytic leukemia (CLL). Taken together, the compounds described here appear to selectively suppress NF-κB activation in the B-cell lineage at specific stages of differentiation. As such, these small molecules could serve as chemical probes for uncovering novel targets that regulate NF-κB activity and potentially lead to development of therapies that selectively block certain types of NF-κB activation in pre-B cell ALL, CLL, or other hematologic malignancies. (Supported by NIH grants X01 MH077633-1, U54–005033, and P01-CA-81534). Disclosures:No relevant conflicts of interest to declare.

CK2 Kinase Inhibitors Display Anti-Myeloma Effects and Antagonize Osteoclast Activity in Models of Multiple Myeloma Bone Marrow Microenvironment

AbstractAbstract 444 Background.Multiple myeloma (MM) plasma cell growth in the bone marrow (BM) microenvironment is fueled by survival signals delivered by the surrounding non-malignant cells (stromal and other types) and through contacts with the extracelllar matrix. Interactions of MM cells with osteoclasts and osteoblasts generate a milieu, in which bone resorption and bone loss occur more rapidly than bone deposition. Novel agents, such as bortezomib and lenalidomide, which target the MM BM microenvironment, have shown unprecedented anti-myeloma efficacy in part due to their ability to somewhat revert these microenvironmental alterations. However, often resistance occurs also to novel drugs and the disease progresses. We have described that targeting protein kinase CK2 with chemical inhibitors or RNA interference causes MM cell death, increases the sensitivity to chemotherapeutics and compromises the NF-κB and STAT3 activity (Piazza FA et al. 2006, Blood; 108: 1698). We also found that CK2 inhibitors synergize with Hsp90 inhibitors (Manni S et al. 2012, Clinical Cancer Res; 18: 1888) and bortezomib (Manni S et al., Blood (2011 ASH Annual Meeting Abstracts); 118; 1849) in inducing MM cell death. Moreover, a phase I clinical trial is ongoing in USA (ID: NCT01199718) testing the oral CK2 inhibitor CX4945 (Cylene Pharmaceuticals, CA, USA) in MM patients. Purpose.We investigated whether and how CK2 inhibition with ATP-competitive CX4945 and tTBB inhibitors could affect the growth of MM cells and of osteoprogenitors in models of BM microenvironemnt. The aim of the study was to provide further insights into the mechanism of action of CK2 inhibitors also in the MM microenvironment, in particular on the stromal cell-mediated MM cell survival and on the unbalanced bone metabolism. We ultimately aimed at generating original data useful for the design of novel rational combination therapies incorporating CK2 inhibitors in the therapy of MM and of MM-bone disease. Methods.MM plasma cells from patients and MM cell lines were cultured in the presence of BM stromal cells obtained from MM patients or BM stromal cell lines or in the presence of osteoclasts. ATP-competitive CK2 inhibitors were added to the co-cultures or to cultures of osteoblast cell lines or progenitors. Cell growth was evaluated with different means and signaling pathways were studied in MM plasma cells and in the stromal cells. NF-κB target gene expression and DNA binding was tested with microplate arrays. For osteoclast generation, CD14+ peripheral blood monocytes were stimulated in alpha-MEM medium with 10% FBS plus RANKL (60ng/ml) plus M-CSF (25ng/ml) for 28 days; early-osteoblasts colonies were obtained from BM cells stimulated under appropriate conditions. Results.CK2 inhibition with CX4945 or tTBB caused apoptosis of MM cells (either freshly isolated from patients or cell lines) cultured on patient-derived mesenchymal stromal cells (MSC) or on the BM stromal cell line HS-5. The inhibitors did not significantly affect MSC viability. A reduction of NF-κB activity evaluated in MM cells was found upon CK2 inhibition, with a parallel reduction of the production of NF-κB-dependent cytokines. When assayed on osteoprogenitors, CX4945 displayed an inhibitory effect on osteoclast formation from CD14+ monocytes even at low concentrations (1 μM up to 7 μM, comparable with the effects of zolendronate 1 μM), whereas it inhibited the formation of osteoblasts from BM colonies at day 14 at fairly higher concentrations (>5 μM). Moreover, CX4945 inhibited osteoblast proliferation at even higher concentration (>7.5 μM). The anti-myeloma effect of CK2 inhibitors was present also when MM cells (INA-6 cell line) were cultured in the presence of osteoclasts generated from CD14+ monocytes. Conclusions.Our study shows that inhibition of CK2 could profoundly affect the growth of MM cells in models of BM microenvironment while substantially sparing the normal cellular stromal counterparts and osteoblasts and suggests that CK2 inhibitors could be exploited to target the hyperactivity of osteoclast seen in MM bone disease. Disclosures:Giuliani:Celgene: Research Funding.

Spleen Tyrosine Kinase Inhibitor Fostamatinib Blocks B Cell Receptor Signaling and Reduces Viability of BCR Subtype Diffuse Large B Cell Lymphoma

AbstractAbstract 3720Spleen tyrosine kinase (SYK) plays a key role in B cell receptor mediated survival in certain B cell malignancies including Diffuse Large B Cell Lymphoma (DLBCL). Therefore, targeting SYK represents an emerging therapeutic approach for the treatment of DLBCL. Indeed, fostamatinib, an orally available SYK inhibitor, has shown promising clinical activity in non-Hodgkin lymphoma (Friedberg et al. 2010 Blood 115: 2578). However, the overall response rate for novel targeted agents in unselected relapsed refractory DLBCL patients remains 25–30%, highlighting the opportunity for development of better treatment strategies. Here, we used preclinical models to study the mechanism underlining the efficacy of R406, the metabolic active form of fostamatinib. R406 was characterized in a heterogeneous panel of 17 DLBCL cell lines including both ABC and GCB subtypes. Overall, the cellular selectivity of R406, particularly in ABC-subtype DLBCL cell lines, was consistent with other BCR-targeted agents such as BTK inhibitor PCI-32765 (ibrutinib) and PI3 kinase δ inhibitor CAL-101. This strongly suggests that R406 functions through inhibition of BCR-mediated survival signaling. Furthermore, phospho-flow analysis and Western Blotting have demonstrated the effect of R406 on both basal and anti-IgG/M stimulated BCR signaling. Specifically, R406 decreases phosphorylation of proximal BCR pathway regulators BLNK, PLCγ2, as well as key players in downstream effect pathways such as ERK, AKT, RPS6, 4EBP and STAT3. To investigate whether the inhibition of BCR signaling by R406 affects cell viability, flow-cytometry based apoptosis analysis was employed. R406 induced apoptosis in sensitive cell lines in a dose-dependent manner. Mechanistically, treatment with R406 reduced MCL1 protein level and down-regulated Bfl-1 expression in sensitive, but not resistant, cell lines, which may partially contribute to the observed efficacy. Given the critical role of NFκB-dependent survival signaling in ABC subtype DLBCL, we then investigated the effect of R406 on this pathway by monitoring NF-κB target gene expression via quantitative real time PCR. Consistent with previous publication (Davis et al. 2010, Nature 463: 88), R406 significantly blocked chronic active BCR-induced NFκB signaling in sensitive cell lines with CD79A/B mutations, leading to downregulation of IL-6/IL-10 and subsequent suppression of JAK/STAT3 signaling. In contrast, we observed little effect of R406 in cell lines with downstream activating mutations of the NFκB pathway (such as CARD11, A20 mutations). Therefore, the molecular nature of NF-κB pathway lesions may serve as a predictive marker for R406 responsiveness in ABC subtype DLBCL cell lines. Furthermore, in exploration of a common indicator of sensitivity to R406 in both ABC and GCB subtypes, we have confirmed that response to R406 is dependent on surface expression of a functional BCR and presence of a intact BCR signaling cascade (Chen et al. 2008 Blood 111: 2230). These cells are thus characterized as “B-cell Receptor active” or BCR subtype. Work is currently underway to further elucidate the characteristics of the BCR subtype, which may serve as a general selective marker for R406’s efficacy in DLBCL cell lines. In conclusion, we have demonstrated that R406 functions as a BCR antagonist and reduces viability in DLBCL through inhibition of NFκB-mediated survival signals and downregulation of MCL-1. In addition, we have confirmed in ABC subtype DLBCL cell lines, similar to other BCR-targeted agents, R406 sensitivity is correlated with CD79A/B mutations whereas A20 or CARD11 mutations render cells resistance. Eventually, we proposed that BCR classification may serve as a broader selection maker for all DLBCL cell lines. Disclosures:Zhang:AstraZeneca: Employment, Equity Ownership. Parmentier:AstraZeneca: Employment, Equity Ownership. Lai:AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Passino:AstraZeneca: Employment, Equity Ownership. Powell:AstraZeneca: Employment, Equity Ownership. Devereaux:AstraZeneca: Employment, Equity Ownership. Byth:AstraZeneca: Employment, Possible shareholder Other.

Influenza A virus-encoded NS1 virulence factor protein inhibits innate immune response by targeting IKK

The IKK/NF-κB pathway is an essential signalling process initiated by the cell as a defence against viral infection like influenza virus. This pathway is therefore a prime target for viruses attempting to counteract the host response to infection. Here, we report that the influenza A virus NS1 protein specifically inhibits IKK-mediated NF-κB activation and production of the NF-κB induced antiviral genes by physically interacting with IKK through the C-terminal effector domain. The interaction between NS1 and IKKα/IKKβ affects their phosphorylation function in both the cytoplasm and nucleus. In the cytoplasm, NS1 not only blocks IKKβ-mediated phosphorylation and degradation of IκBα in the classical pathway but also suppresses IKKα-mediated processing of p100 to p52 in the alternative pathway, which leads to the inhibition of nuclear translocation of NF-κB and the subsequent expression of downstream NF-κB target genes. In the nucleus, NS1 impairs IKK-mediated phosphorylation of histone H3 Ser 10 that is critical to induce rapid expression of NF-κB target genes. These results reveal a new mechanism by which influenza A virus NS1 protein counteracts host NF-κB-mediated antiviral response through the disruption of IKK function. In this way, NS1 diminishes antiviral responses to infection and, in turn, enhances viral pathogenesis.

Nuclear Factor κB Activation Impairs Ependymal Ciliogenesis and Links Neuroinflammation to Hydrocephalus Formation

Hydrocephalus formation is a frequent complication of neuropathological insults associated with neuroinflammation. However, the mechanistic role of neuroinflammation in hydrocephalus development is unclear. We have investigated the function of the proinflammatory acting inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling system in neuroinflammatory processes and generated a novel mouse model that allows conditional activation of the IKK/NF-κB system in astrocytes. Remarkably, NF-κB activation in astrocytes during early postnatal life results in hydrocephalus formation and additional defects in brain development. NF-κB activation causes global neuroinflammation characterized by a strong, astrocyte-specific expression of proinflammatory NF-κB target genes as well as a massive infiltration and activation of macrophages. In this animal model, hydrocephalus formation is specifically induced during a critical time period of early postnatal development, in which IKK/NF-κB-induced neuroinflammation interferes with ependymal ciliogenesis. Our findings demonstrate for the first time that IKK/NF-κB activation is sufficient to induce hydrocephalus formation and provides a potential mechanistic explanation for the frequent association of neuroinflammation and hydrocephalus formation during brain development, namely impairment of ependymal cilia formation. Therefore, our study might open up new perspectives for the treatment of certain types of neonatal and childhood hydrocephalus associated with hemorrhages and infections.

Prolonged Postovulatory Proinflammatory Signaling in the Fallopian Tube Epithelium May Be Mediated through a BRCA1/DAB2 Axis

To assess inflammation-related gene expression in nonmalignant fallopian tube epithelium (FTE) from BRCA1/2 mutation carriers and control patients obtained during the luteal and follicular phase, and to determine the impact of BRCA1 and disabled homolog 2 (DAB2) on NF-κB-mediated proinflammatory signaling. A list of inflammation-related and NF-κB-responsive genes was compiled through gene set enrichment and PubMed database search, corresponding probes identified, and unpaired t tests conducted to identify differentially expressed genes in previously profiled FTE samples. ES2 and A549 cells were cotransfected with DAB2- or BRCA1-targeting siRNA and an NF-κB-responsive luciferase reporter, treated with TNF-α and luciferase activity determined. To determine whether DAB2 or BRCA1 alters mRNA expression of NF-κB target genes, cells were transfected with siRNA, treated with TNF-α, and harvested for total RNA extraction and quantitative real-time PCR. A subset of BRCA1-mutated luteal phase samples previously found to group with adnexal high-grade serous carcinomas (HGSCs) differentially expressed 124 inflammation-associated probesets relative to remaining FTE samples. These samples also differentially expressed 264 probes relative to other luteal phase samples exposed to the same postovulatory environment. Both BRCA1- and DAB2-targeting siRNA increased TNF-α-induced NF-κB activity and mRNA expression of NF-κB-dependent target gene SOD2 relative to nontargeting siRNA, suggesting that both proteins repress proinflammatory signaling. These data provide evidence of elevated proinflammatory signaling in a subset of BRCA1-mutated luteal phase FTE, consistent with an altered response to ovulation-associated cytokines. Furthermore, both BRCA1 and DAB2 affect NF-κB activity, indicating a novel link between BRCA mutation status, ovulation, and predisposition to HGSC.

Differential impact of oleate, palmitate, and adipokines on expression of NF-κB target genes in human vascular smooth muscle cells

It is widely accepted that obesity is a major risk factor for the development of atherosclerosis. In this context, adipose tissue produces a variety of adipokines and releases free fatty acids, contributing to a chronic-low grade inflammation state implicated in vascular complications. In this study, we investigated the role of adipokines, oleic acid (OA), palmitic acid (PA), and the combinations on activation of NF-κB target genes in human vascular smooth muscle cells (SMC) to assess the hypothesis of synergistic interactions between these molecules. Adipocyte-conditioned medium (CM), generated from human adipocytes, in combination with low concentrations of OA, but not PA, induces SMC proliferation and activation of the transcription factor NF-κB in a synergistic way. Combined treatment of CM and OA further regulates a set of downstream NF-κB target genes including angiopoietin-1, activin A, and MMP-1, all critically involved in SMC dysfunction. This suggests that the lipotoxic potential of fatty acids is substantially enhanced by the presence of adipocyte-derived factors.