Necrosis Factor Ligand Superfamily Research Articles

Novel therapeutic approach targeting mucosal APRIL in IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is a form of glomerulonephritis. Also known as Berger's disease, it is the most common type of primary glomerulonephritis worldwide and is characterised by IgA deposits in the glomerulus. IgA nephropathy expert Prof. Yusuke Suzuki, who is based at Jutendo University Faculty of Medicine in Japan, explains that studies suggest that a number of factors, including aberrently-glycosylated IgA1, or its immune complex with glycan-specific IgG and IgA may play key roles in the pathogenesis of the condition. 'A proliferation-inducing ligand (APRIL) is one of protein froms the tumour necrosis factor (TNF) ligand superfamily. Research suggests a key role for this ligand in the long-term survival of plasma cells in the bone marrow and lymphoid tissues including mucosae and it is being explored as a potential target for the development of therapeutic options for autoimmune diseases and B cell malignancies. 'Recent studies into the pathological role of APRIL in IgAN, including our own recent findings, have led us to consider a new therapeutic approach,' he outlines. 'Our data using human samples from patients with IgAN and targeting APRIL in animal models has provided proof of concept (POC) of the beneficial effects of neutralising APRIL in IgAN.' Based on this work, clinical trials of new drugs that target APRIL have so far demonstrated exciting possibilities as one of the most promising new treatments for IgAN. A number of pharmaceutical companies are currently working on this and other, related drugs to treat the condition.

Abstract 5621: Prevalence of GITR expression and pharmacodynamic (PD) biomarkers in syngeneic tumor models treated by a GITR agonist (GITRL-Fc)

Abstract GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the tumor necrosis factor (TNF) ligand superfamily. GITRL binds and activates the co-stimulatory surface receptor GITR, which promotes proliferation and activation of effector T cells (Teff) and inhibits suppressive activity of regulatory T cells (Treg). It is thus hypothesized that co-stimulation of GITR by agonist agents will promote anti-tumor immunity. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc) that shows robust single agent antitumor efficacy and immune effects in multiple syngeneic mouse models, suggesting its potential benefit in cancer immunotherapy To investigate the prevalence of GITR expression in human tumors, RNA-Seq data analyses of 33 tumor types in TCGA showed GITR is highly expressed in a subset of solid tumors, including head & neck, lung, breast, esophageal, and bladder cancers. In most solid tumors, GITR expression correlated poorly with T cell markers, implying that GITR may not be exclusive to immune cells and may be expressed in tumor cells as well. Similar findings emerged from RNA-Seq data analysis of patient-derived xenograft (PDX) samples from 24 tumor types. The gene expression data was corroborated by immunohistochemistry (IHC) analysis of GITR expression in 17 tumor types which showed that in addition to immune cells, GITR was expressed on tumor cell membranes. A multi-platform approach was taken to investigate GITRL-Fc pharmacodynamic (PD) biomarkers in tumors and in matched whole blood samples from mice bearing CT26 colon, 4T1 breast, and B16F10 melanoma carcinoma models. Global gene expression levels were profiled by microarray on treated and control tissues. We also monitored the changes of immune cell populations and cytokine secretions by flow cytometry, Luminex and IHC. Immune gene changes were more robust in tumors than in blood samples. In tumor samples, GITRL-Fc increased the gene expression associated with T cells, CD8 T cells, cytotoxicity, Th1 cells, interferon gamma (IFN-γ), natural killer cells, Teff cells, and T cell activation markers. These gene changes were validated by quantitative real-time PCR. Similarly, flow cytometry analysis showed that GITRL-Fc promoted activation of CD4+ effector cells, decreased Treg frequency, and increased the ratio of CD8+ T cell/Treg in the tumor. GITRL-Fc also modulated secretion of cytokines in splenocytes, including an increase in IFN-γ. Taken together, the PD biomarker changes in immune-related gene expression, immune cell populations, and cytokine secretions observed in these preclinical tumor models are consistent with GITRL-Fc mechanism of action and demonstrated target engagement of GITRL-Fc. Additional approaches, including in-silico sorting, to monitor rare immune cell populations in tumor samples will be discussed. Citation Format: Min Wang, Fiore Cattaruzza, Pete Yeung, Alayne Brunner, Erwan LeScolan, Yuwang Liu, Jennifer Cain, Gilbert OYoung, Earth Light Lowe, Belinda Cancilla, Rose Harris, Tim Hoey, Austin Gurney, John Lewicki, Gretchen Argast, Ann M. Kapoun. Prevalence of GITR expression and pharmacodynamic (PD) biomarkers in syngeneic tumor models treated by a GITR agonist (GITRL-Fc) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5621. doi:10.1158/1538-7445.AM2017-5621

Abstract LB-154: Epigenetic silencing of triple-negative breast cancer hallmarks by Withaferin A

Abstract Background: Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. We found that the steroidal lactone Withaferin A (WA), isolated from the plant Withania somnifera, triggers active chromatin remodeling and elicits promising chemosensitizing effects in triple negative breast cancer (BC) cells. Here, we have further characterized potential involvement of epigenetic reprogramming by Withaferin A in TNBC treatment. Materials and methods: By Infinium HumanMethylation450 arrays, Epityper Mass array and CpG pyrosequencing, we determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines treated with a pharmacologically relevant concentration of 700 nM WA. In parallel, we performed ChIP analysis of histone mark H3K4Me3 to evaluate histone demethylation by JARID1B/KDM5B. Furthermore, epigenetic changes in response to WA were crosscompared with genomewide methylation changes and clinical breast cancer patient characteristics in The Cancer Genome Atlas database. Results: In contrast to the DNA hypomethylating agent 5-aza-2’-deoxycytidine (DAC), WA treatment of TNBC cells silences an epigenetic cancer network through gene-specific hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Remarkably, DNA hypermethylation of corresponding CpG sites in these genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ER)/progesterone receptor (PR) positive status in primary BC tumors. Increased expression levels of JARID1B, promoter-specific loss of active H3K4me3 chromatin marks and gain of DNA methylation suggest that WA-specific gene silencing involves epigenetic reprogramming of HER2/ER/PR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks with an improved therapeutic sensitivity. Conclusions: WA may represent a novel and attractive phytopharmaceutical to overcome therapy resistance in TNBC. Citation Format: Wim Vanden Berghe, Katarzyna Szarc vel Szic, Ken Declerck, René A.J. Crans, Jolien Diddens, Clarissa Gerhäuser. Epigenetic silencing of triple-negative breast cancer hallmarks by Withaferin A. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-154.

Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma

ABSTRACTAdeno-associated virus type 2 (AAV2) is a defective DNA virus that was previously considered to be non-pathogenic. We identified somatic AAV2 integration in a subset of 11 hepatocellular carcinomas (HCC) that mainly developed in normal liver without known etiology through recurrent insertional mutagenesis in cancer driver genes such as telomerase reverse transcriptase (TERT), cyclin A2 (CCNA2), cyclin E1 (CCNE1), tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10), and lysine (K)-specific methyltransferase 2B (KMT2B).

Antibody-based targeting of TNF-ligands for cancer therapy

The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamily constitute an important immunoregulatory axis pivotal for the correct execution of immune responses. TNF ligand and receptor family members among others are involved in induction of cell death in malignant cells as well as in providing co-stimulatory signals that help mount effective anti-cancer immune responses. This diverse and important regulatory role in immunity has sparked great interest in the development of TNFL/TNFR-targeted cancer immunotherapeutics. Here, I will discuss our cancer immunotherapeutic drug discovery and development program using selected examples of the TNF-ligand superfamily.

ET-45 * KNOCKDOWN OF TNFSF13B INDUCES GLIOMA STEM CELL APOPTOSIS

Tumor Necrosis Factor (Ligand) Super Family member 13B (TNFSF13B; BAFF) is primarily produced by myeloid lineage cells, activated T cells and dendritic cells, and up-regulates anti-apoptotic Bcl-2 family proteins. Approximately 20% of glioblastoma overexpress TNFSF13B, which may account for treatment resistance. A Kaplan-Meier curve from a query of The Cancer Genome Atlas (TCGA) showed that glioblastoma patients with increased expression (EXP > 2) of TNFSF13B have a significantly shorter overall survival (P = 0.025). By immunoblotting, TNFSF13B expression was differentially expressed in a panel of glioma stem cells (GSCs). GFP-tagged TNFSF13B shRNA lentivirus was used to knock down TNFSF13B in multiple cell lines. Erlotinib or lapatinib inhibited cell viability to a greater extent in shTNFSF13B cells compared to scramble control. After 1 µM erlotinib treatment for 3 days, cell viability was suppressed by 34% in shTNFSF13B GSC11 cells compared to scramble control cells. Lapatinib (0.1 µM for 3 days) reduced cell viability by 40% in shTNFSF13B GSC11 cells compared to controls. Apoptosis, assessed by Annexin V-based flow cytometry, revealed a 30% increase in Annexin V staining after erlotinib treatment (1 µM for 3 days) in shTNFSF13B GSC11 cells compared with controls. The level of anti-apopototic proteins BCL-XL and Bid markedly decreased which was concomitant with an increase in cleaved caspase 9 in shTNFSF13B cells treated with EGFR inhibitors. Using an apoptosis protein array, we observed that anti-apoptotic proteins Bcl-2, survivin, claspin and HIF-1α significantly decreased in shTNFSF13B cells compared with scramble cells after treatment of 1 µM erlotinib. Ongoing in vivo studies are validating the impact of targeting TNFSF13B in a glioma xenograft model. Our studies identified TNFSF13B as a potentially important mediator of glioma stem cell survival in vitro and suggest that TNFSF13B may be a potential therapeutic target to enhance the efficacy of anti-EGFR therapy.

Final analysis: Randomized, blinded, placebo-controlled phase II trial of sorafenib with and without mapatumumab in patients with advanced hepatocellular carcinoma (HCC).

4029 Background: TRAIL is a member of the tumor necrosis factor ligand super family that induces programmed cell death primarily in tumor cells (including liver tumors) through TRAIL death receptor...

BAFF serum levels in myasthenia gravis: effects of therapy

B lymphocyte activating factor (BAFF) belonging to the tumour necrosis factor (TNF) ligand super-family is a key survival factor for B lymphocytes [1]. In transgenic mice, BAFF overproduction is associated with lymphocytic disorders and autoimmunity [2], and increased serum BAFF levels are found in human autoimmune diseases [3]. Myasthenia gravis (MG) is an antibody-mediated disease of neuromuscular transmission. Most patients have serum antibodies against the acetylcholine receptor (AChR). Anti-AChR positive MG is associated with thymus alterations, such as follicular hyperplasia and thymoma [4]. Thymus hyperplasia is characterized by expanded thymus perivascular spaces with infiltration of T and B lymphocytes and germinal centre formation; BAFF was found to be expressed in the MG hyperplastic thymus where it could foster a microenvironment permissive to B-cells [5]. Our aim was to evaluate serum BAFF levels in MG patients in different phases of their disease and the effects of therapy. We tested serum samples from 66 patients, 52 women and 14 men (M/W: 1/3.7) aged 14–74 years (mean 39) and 41 healthy controls, 32 women and 9 men (M/W: 1/3.5), aged 20–66 years (mean 41). Patient population included: 61 anti-AChR positive (42 early-onset, 11 lateonset and 8 thymoma) and 5 early-onset anti-AChR negative MG cases. No patient had anti-muscle specific kinase (MuSK) antibodies. All patients but two had generalized disease. Thymectomy was performed on all thymoma cases and 42/47 early-onset MG patients; a typical thymus hyperplasia was found in 35/42 of these cases. BAFF levels were measured in 52 patients before immunosuppressive (IS) treatment; in 13 of these cases, who had undergone thymectomy with histological findings of thymus hyperplasia, we evaluated serial samples taken before and 5–33 years after thymectomy; 14 patients were under IS therapy (mostly with prednisone) at the inclusion. BAFF levels were measured in duplicate by quantitative sandwich enzyme immunoassay using commercial Quantikine kit (R&D Systems, USA). The normal range for the BAFF assay was defined by the geometric mean BAFF level ?2 SD in controls. Statistical analysis was performed by paired and unpaired Student t test and ANOVA. All patients and controls gave informed consent to the study. Mean serum BAFF levels were significantly higher in the MG population (938.8 ± 284 pg/mL) than in controls (797 ± 192 pg/mL) (p = 0.005). Overall, 20/66 patients (30.3%) had BAFF levels above the normal range. BAFF values did not correlate with disease severity. In 33 MG samples taken before any treatment the mean BAFF value was 953 ± 268 pg/mL, and it did not differ in 19 samples from patients who had undergone thymectomy and had never received IS therapy (1,060 ± 266 pg/mL) (p = 0.14). In the 13 patients in whom serial samples (before and after thymectomy) were tested, serum BAFF levels did not significantly change after thymectomy (p = 0.22) (Fig. 1) with no clearly correlation with clinical outcome. On the other hand, in MG patients under IS therapy, BAFF This study has been approved by the local ethics committee and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

Hif-2alpha mediates UV induced apoptosis through a novel ATF3 dependent death pathway

In this study, we describe a novel activating transcription factor 3 (ATF3)-dependent death pathway triggered by ultraviolet (UV) irradiation. We demonstrate that ATF3 contributes to UV-induced apoptosis through the regulation of hypoxia inducible factor (Hif)-2alpha expression, which in turn induces the expression of proapoptotic genes, such as Caspase7 or TRAIL (tumor necrosis factor (ligand) superfamily, member 10). Gain of function of Hif-2alpha as well as ATF3 is sufficient to trigger cell death, whereas loss of function of both proteins drastically inhibits UV-induced apoptosis. Repression of Hif-2alpha strongly impairs ATF3-mediated death, providing evidences that Hif-2alpha is the major death effector of ATF3. In addition, Hif-1alpha, already known as a proapoptotic gene, upon UV irradiation, is not able to compensate for the lack of Hif-2alpha expression, thereby confirming the major contribution of Hif-2alpha in UV-mediated cell death. We further demonstrate that this cascade of gene activation depends on p38 and c-Jun N-terminal kinase (JNK) activity. Impairment of such a pathway is likely to contribute to oncogenesis by promoting survival of cells that could accumulate severe chromosomal alterations.

Immunosuppressive therapy modulates T lymphocyte gene expression in patients with systemic lupus erythematosus.

To evaluate the T-cell large-scale differential gene expression in systemic lupus erythematosus (SLE) patients presenting with glomerulonephritis we studied SLE patients before and after immunosuppressive treatment. Large-scale gene expression of peripheral blood mononuclear T cells was evaluated using cDNA microarray nylon membranes containing 5184 cDNAs. Data were analysed using the SAM and Cluster and Treeview software. When untreated patients were compared to healthy individuals, 38 genes, most of them located on chromosomes 1, 3, 6, 17 and 19, were repressed, and when untreated patients were compared to treated ones, 154 genes, located on chromosomes 1, 6, 7, 12 and 17, were induced. In terms of biological function of coded proteins, the differentially expressed genes were associated with apoptosis, cell cycle, chromosomal scaffold, cytokine/chemokine, DNA repair/replication, Golgi/mitochondrial proteins, mRNA processing, signalling molecules and tumour suppressors. Two autoantigen genes related to RNA splicing (small nuclear riboprotein 70,000 MW-U1 SNR, and splicing factor 3a, 60,000 MW), and the tetranectin-plasminogen-binding protein were repressed. The Fc fragment of immunoglobulin G low affinity IIb, apoptotic protease activating factor-1, two subunits of cytochrome c, caspase 8, complement C5a, HLA-DRA, HLA-DQB1, transforming growth factor-beta receptor II, small nuclear ribonucleoprotein polypeptide N (Sm protein N) genes, heterogeneous nuclear riboprotein-C, and argininosuccinate lyase genes, among others, were induced. A total of 10 genes were repressed in untreated patients and induced in treated ones, among them tumour necrosis factor (ligand) superfamily member 9, tumour protein p53, mannosidase alpha class IA, and CD22. Although some of these differentially expressed genes are typically expressed in B cells, CD22 and CD32 have also been reported in T cells and may provide regulatory signals to B cells. Assessment of differential gene expression may provide hybridization signatures that may identify susceptibility, diagnostic and prognostic markers of SLE.

Analysis of differentially expressed lung metastasis-associated proteins in adenoid cystic carcinoma cell lines

To analyze differentially expressed metastasis-associated proteins in Adenoid cystic carcinoma cell lines of human salivary gland by proteomics. Protein expression alterations of ACC-2 and ACC-M cells were described by 2-D gels. After image analysis by software, proteins of interest were excised from the gels and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometer. 12 protein spots showed significantly differential expression patterns between two cell lines. In the identified protein candidates, transketolase, modulator recognition factor 2, Dim1p homolog, splicing factor (arginine/serine-rich 9) and v-Ha-ras l oncogene were all lowly expressed in the poorly metastatic ACC-2 cell and significantly upregulated in highly metastatic ACC-M cell, while type I collagen pro alpha and tumor necrosis factor (ligand) superfamily member 4 showed a high expression in ACC-2 cells and a low expression in ACC-M cells. Pirin (spot 6) just appears in ACC-2 cell and was not detectable in ACC-M cell, while retinal homeobox protein was just detected in ACC-M cell and did not appear in ACC-2 cell. The proteins may be involved in the adenoid cystic carcinoma lung metastasis through different mechanisms. Our work may contribute to discover diagnostic markers and therapeutic targets.