Tumor Necrosis Factor Receptor Family Research Articles

The TNF-TNFR Family of Co-signal Molecules.

Costimulatory signals initiated by the interaction between the tumor necrosis factor (TNF) ligand and cognate TNF receptor (TNFR) superfamilies promote clonal expansion, differentiation, and survival of antigen-primed CD4+ and CD8+ T cells and have a pivotal role in T-cell-mediated adaptive immunity and diseases. Accumulating evidence in recent years indicates that costimulatory signals via the subset of the TNFR superfamily molecules, OX40 (TNFRSF4), 4-1BB (TNFRSF9), CD27, DR3 (TNFRSF25), CD30 (TNFRSF8), GITR (TNFRSF18), TNFR2 (TNFRSF1B), and HVEM (TNFRSF14), which are constitutive or inducible on T cells, play important roles in protective immunity, inflammatory and autoimmune diseases, and tumor immunotherapy. In this chapter, we will summarize the findings of recent studies on these TNFR family of co-signaling molecules regarding their function at various stages of the T-cell response in the context of infection, inflammation, and cancer. We will also discuss how these TNFR co-signals are critical for immune regulation and have therapeutic potential for the treatment of T-cell-mediated diseases.

Immunohistochemical Analysis of LGR5 and TROY Expression in Gastric Carcinogenesis Demonstrates an Inverse Trend

Background:Two of the Wnt signaling pathway target genes, tumor necrosis factor receptor family member (TROY) and leucine-rich G-protein coupled receptor (LGR5), are involved in the generation and maintenance of gastrointestinal epithelium. A negative modulatory role has recently been assigned to TROY, in this pathway. Here, we have examined their simultaneous expression in gastric carcinogenesis. Methods:Tumor and paired adjacent tissues of intestinal-type gastric cancer (GC) patients (n = 30) were evaluated for LGR5 and TROY expression by immunohistochemistry. The combination of the percentage of positively stained cells and the intensity of staining was defined as the composite score and compared between groups. The obtained findings were re-evaluated in a mouse model. Results:TROY expression in the tumor tissue was significantly lower than that of the adjacent tissue (2.5 ± 0.9 vs. 3.3 ± 0.9, p = 0.004), which was coincident with higher LGR5 expression (3.6 ± 1.1 vs. 2.7 ± 0.9, p = 0.001). This observation was prominent at stages II/III of GC, leading to a statistically significant mean difference of expression between these two molecules (p = 0.005). In the H. pylori infected-mouse model, this inverse expression was observed in transition from early (8-16 w) to late (26-50 w) time points, post treatment (p = 0.002). Conclusion:Our data demonstrates an inverse trend between TROY down-regulation and LGR5 up-regulation in GC tumors, as well as in response to H. pylori infection in mice. These findings support a potential negative modulatory role for TROY on LGR5 expression.

Treatment with AMG 420, an Anti-B-Cell Maturation Antigen (BCMA) Bispecific T-Cell Engager (BiTE®) Antibody Construct, Induces Minimal Residual Disease (MRD) Negative Complete Responses in Relapsed and/or Refractory (R/R) Multiple Myeloma (MM) Patients: Results of a First-in-Human (FIH) Phase I Dose Escalation Study

Treatment with AMG 420, an Anti-B-Cell Maturation Antigen (BCMA) Bispecific T-Cell Engager (BiTE®) Antibody Construct, Induces Minimal Residual Disease (MRD) Negative Complete Responses in Relapsed and/or Refractory (R/R) Multiple Myeloma (MM) Patients: Results of a First-in-Human (FIH) Phase I Dose Escalation Study

JNJ-64457107, a CD40 Agonist, Induces Cell Death in BCL6hiIRF4neg GCB Subtype of DLBCL

CD40 is a member of the TNF receptor family that is expressed in multiple tumor types and normal cells including antigen presenting cells (APC), endothelium and platelets. CD40-CD40L signaling is one of the pivotal mechanisms essential for robust antigen presentation and adaptive immunity. JNJ-64457107 (JNJ-107) is an anti-CD40 IgG1 agonistic antibody that is currently in a Phase 1 clinical trial (NCT02829099). JNJ-107/ADC-1013 induced significant anti-tumor activity and immunological memory (Mangsbo 2015). It was also reported that a CD40 agonist, SGN-40, induced direct cytotoxicity in diffuse large B cell lymphoma (DLBCL) cell lines (Burlington 2011), and clinical responses have been noted in this indication (Advani 2009). However, not all DLBCL patients and DLBCL cell lines respond to SGN-40. Thus, a patient selection strategy in B cell malignancies may be beneficial. Here, we investigated the mechanism of action and potential biomarkers of response to JNJ-107 in DLBCL.Here, we show that JNJ-107 induced dose-dependent APC activation measured by upregulation of co-stimulatory molecules CD86, CD54, HLA-DR and CD23 on peripheral B cells in ex vivo whole blood and in vitro generated dendritic cells by flow cytometry. The highest APC activation was at 0.1-5 µg/ml of JNJ-107. We also found that JNJ-107 induced cell death of CD40 positive DLBCL cell lines representing both, germinal center B-cell (GCB) and activated B-cell (ABC) subtypes at similar doses. Cell death was measured by the Cell Titer-Glo assay and confirmed by staining with PI/AnnexinV. JNJ-107 at 1 µg/ml induced cell death (in 30-90% of cells) in 11/20 DLBCL cell lines (called “sensitive”) after 4 days of treatment, with 2 GCB lines (BJAB and SU-DHL4) showing >90% cell death. No DLBCL cell line proliferation was observed in response to JNJ-107.CD40 signals through the NF-kB pathway in DLBCL. We investigated, if basal expression of NF-kB signaling components predicts responses to JNJ-107. First, we assessed activation of the NF-kB pathway in DLBCL lines by Western blot before and after treatment with JNJ-107. We used p65 and p52 as markers of canonical and non-canonical NF-kB activation, respectively. We found that JNJ-107 predominantly activated non-canonical NF-kB signaling in DLBCL cell lines by inducing p100 cleavage and release of p52 (Figure 1). A lower rate of p52 induction was observed in JNJ-107 “sensitive” lines that had constitutive expression of p52 (Pfeiffer, OCI-Ly7). Thus, we did not observe strong correlation between p52 induction and cell death in response to JNJ-107.BCL6 and IRF4 are markers of cell of origin in DLBCL. BCL6 and IRF4 are constitutively expressed in GCB and ABC subtypes, respectively. Interestingly, “sensitive” GCB lines had higher BCL6 expression than resistant lines. BCL6 protein was rapidly degraded (within 16h) in “sensitive” GCB DLBCL lines stimulated with JNJ-107. BCL6 is a transcriptional repressor that is regulated by many factors including IRF4. IRF4 is induced downstream of CD40, and directly represses BCL6 expression by binding to its promoter (Saito 2007). Consistent with constitutive activation of NF-kB, ABC DLBCL lines constitutively expressed IRF4. Surprisingly, several GCB lines also constitutively expressed IRF4 and were unable to further upregulate IRF4 in response to JNJ-107. The latter lines showed a lower degree of cell death in response to JNJ-107 compared to lines negative for IRF4 expression (except Pfeiffer).In summary, APC activation in whole blood, direct cytotoxicity and non-canonical NF-kB activation in DLBCL cell lines were seen with CD40 agonist, JNJ-107. GCB cell lines with high BCL6 expression and negative for IRF4 (BCL6hiIRF4neg) were more sensitive to JNJ-107 induced cell death. Based on these findings, GCB DLBCL patients with functional CD40 signaling in the tumor cells (BCL6hiIRF4neg phenotype) may show enhanced anti-tumor responses.Figure 1. JNJ-107 potently induces non-canonical NF-kB signaling in DLBCL cell lines. DLBCL lines were treated with 0.1 µg/ml JNJ-107 for 15 min, then 5 µg/ml cross-linker, IgG F(ab')2 for 16h. p65 and p52 fold induction was determined by Western blot of whole lysates. Representative data of 2 independent experiments are shown. [Display omitted] DisclosuresRusbuldt:Janssen R&D: Employment. Smith:Janssen R&D: Employment. Balasubramanian:Janssen Research & Development: Employment, Equity Ownership. Borzillo:Janssen R&D: Employment. Alvarez Arias:Janssen R&D: Employment.

CD137 (4-1BB) Signalosome: Complexity Is a Matter of TRAFs.

CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer.

Cerebrospinal Fluid Level of Soluble CD27 Is Associated with Disease Severity in Neuromyelitis Optica Spectrum Disorder

Object: CD27 belongs to the tumor necrosis factor receptor family and is constitutively expressed on T cells. The concentration of cerebrospinal fluid (CSF) soluble (s)CD27 is elevated in patients with multiple sclerosis (MS). However, whether the level of CSF sCD27 is elevated in neuromyelitis optica spectrum disorder (NMOSD) remains unknown. The aim of this study was to measure the CSF concentration of sCD27 and to determine its relationship with NMOSD disease activity. Methods: CSF CXCL13 was measured by ELISA in neuromyelitis optica (NMO) (n = 31) and MS (n = 23) patients and in controls (CTLs) (n = 22). Results: The concentration of sCD27 was higher in the NMO group than in the MS (p = 0.082) and CTL (p = 0.002) groups, and there was a positive correlation with CSF IL-6 (p = 0.000) and a negative correlation with IL-10 (p = 0.073). In the NMO group, patients with higher sCD27 concentrations exhibited worse disease disability in their CSF (p = 0.006). Moreover, the sCD27 concentrations had a significantly positive correlation with the level of CSF total protein (p = 0.030). Furthermore, the patients positive for AQP4-IgG (n = 26) seemed to have higher levels of sCD27 in their CSF (p = 0.069) than those negative for AQP4-IgG (n = 5). Conclusions: We revealed that the level of CSF sCD27 was elevated in NMOSD and correlated with NMOSD disease activity.

Multi\u2013omic analysis of signalling factors in inflammatory comorbidities

BackgroundInflammation is a core element of many different, systemic and chronic diseases that usually involve an important autoimmune component. The clinical phase of inflammatory diseases is often the culmination of a long series of pathologic events that started years before. The systemic characteristics and related mechanisms could be investigated through the multi–omic comparative analysis of many inflammatory diseases. Therefore, it is important to use molecular data to study the genesis of the diseases. Here we propose a new methodology to study the relationships between inflammatory diseases and signalling molecules whose dysregulation at molecular levels could lead to systemic pathological events observed in inflammatory diseases.ResultsWe first perform an exploratory analysis of gene expression data of a number of diseases that involve a strong inflammatory component. The comparison of gene expression between disease and healthy samples reveals the importance of members of gene families coding for signalling factors. Next, we focus on interested signalling gene families and a subset of inflammation related diseases with multi–omic features including both gene expression and DNA methylation. We introduce a phylogenetic–based multi–omic method to study the relationships between multi–omic features of inflammation related diseases by integrating gene expression, DNA methylation through sequence based phylogeny of the signalling gene families. The models of adaptations between gene expression and DNA methylation can be inferred from pre–estimated evolutionary relationship of a gene family. Members of the gene family whose expression or methylation levels significantly deviate from the model are considered as the potential disease associated genes.ConclusionsApplying the methodology to four gene families (the chemokine receptor family, the TNF receptor family, the TGF– β gene family, the IL–17 gene family) in nine inflammation related diseases, we identify disease associated genes which exhibit significant dysregulation in gene expression or DNA methylation in the inflammation related diseases, which provides clues for functional associations between the diseases.

CD40 signaling—germinal center b cells go at their own pace

CD40 signaling—germinal center b cells go at their own pace

Osteoprotegerin and Myocardial Fibrosis in Patients with Aortic Stenosis

Left ventricular myocardial fibrosis in patients with aortic stenosis (AS) confers worse prognosis. Plasma osteoprotegerin (OPG), a cytokine from the TNF receptor family, correlates with the degree of valve calcification in AS, reflecting the activity of the tissue RANKL/RANK/OPG (receptor activator of nuclear factor κΒ ligand/RANK/osteoprotegerin) axis, and is associated with poorer outcomes in AS. Its association with myocardial fibrosis is unknown. We hypothesised that OPG levels would reflect the extent of myocardial fibrosis in AS. We included 110 consecutive patients with AS who had undergone late-gadolinium contrast enhanced cardiovascular magnetic resonance (LGE-CMR). Patients were characterised according to pattern of fibrosis (no fibrosis, midwall fibrosis, or chronic myocardial infarction fibrosis). Serum OPG was measured with ELISA and compared between groups defined by valve stenosis severity. Some 36 patients had no fibrosis, 38 had midwall fibrosis, and 36 had chronic infarction. Patients with midwall fibrosis did not have higher levels of OPG compared to those without fibrosis (6.78 vs. 5.25 pmol/L, p = 0.12). There was no difference between those with midwall or chronic myocardial infarction fibrosis (6.78 vs. 6.97 pmol/L, p = 0.27). However, OPG levels in patients with chronic myocardial infarction fibrosis were significantly higher than those without fibrosis (p = 0.005).

Up-regulation of TNF Receptor-associated Factor 7 after spinal cord injury in rats may have implication for neuronal apoptosis

TNF receptor-associated factor 7 (TRAF7), is an E3 ubiquitin ligase for several proteins involved in the activation of TLR-dependent NF-kappaB signaling. TRAF7 links TNF receptor family proteins to signaling pathways, thus participates in regulating cell death and survival mediated by TNF family ligands. To date, the biological function of TRAF7 after spinal cord injury (SCI) is still with limited acquaintance. In this study, we have performed an acute SCI model in adult rats and investigated the dynamic changes of TRAF7 expression in the spinal cord. Our results showed that TRAF7 was up-regulated significantly after SCI, which was paralleled with the levels of the apoptotic protein active caspase-3. Immunofluorescent labeling showed that TRAF7 was co-localizated with active caspase-3 in neurons. To further investigate the function of TRAF7, an apoptosis model was established in primary neuronal cells. When TRAF7 was knocked down by specific short interfering RNA (siRNA), the protein levels of active caspase-3 and the number of apoptotic primary neurons were significantly decreased in our study. Taken together, our findings suggest that the change of TRAF7 protein expression plays a key role in neuronal apoptosis after SCI.

The Pathogenesis of Necroptosis-Dependent Signaling Pathway in Cerebral Ischemic Disease.

Necroptosis is the best-described form of regulated necrosis at present, which is widely recognized as a component of caspase-independent cell death mediated by the concerted action of receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3). Mixed-lineage kinase domain-like (MLKL) was phosphorylated by RIPK3 at the threonine 357 and serine 358 residues and then formed tetramers and translocated onto the plasma membrane, which destabilizes plasma membrane integrity leading to cell swelling and membrane rupture. Necroptosis is downstream of the tumor necrosis factor (TNF) receptor family, and also interaction with NOD-like receptor pyrin 3 (NLRP3) induced inflammasome activation. Multiple inhibitors of RIPK1 and MLKL have been developed to block the cascade of signal pathways for procedural necrosis and represent potential leads for drug development. In this review, we highlight recent progress in the study of roles for necroptosis in cerebral ischemic disease and discuss how these modifications delicately control necroptosis.

Abstract LB-152: Local selivery of GITR agonistic antibody induces a stronger antitumor immunity than systemic administration

Abstract The field of cancer immunotherapy is expanding rapidly with the success of an antagonistic antibody against CTLA-4 and PD-1/PD-L1. However, since approximately half of patients do not respond to the therapies even the combination regimen, the development of novel checkpoint inhibitors is desired for the recurrent or refractory patients. Recently, newer targets including select members of the tumor necrosis factor receptor (TNFR) family, including 4-1BB, OX40 and glucocorticoid-induced tumor necrosis factor receptor (GITR), are gathering attention. GITR is expressed at a low basal level on naïve murine T cells and at a very low level on human T cells, whereas a GITR ligand (GITRL) was abundantly expressed in murine dendritic cells and macrophages. Multiple studies have shown that GITR-GITRL interaction can provide a co-stimulatory signal to both CD4+ and CD8+ naïve T cells, enhancing proliferation and effector function, particularly in the setting of suboptimal T cell receptor stimulation. In contrast, murine and human Tregs constitutively express GITR, and it had been shown that activation of GITR signaling by GITR ligand or agonistic antibody inhibit the suppressive activity of Tregs. To enhance the antitumor effect of immune stimulatory reagents, we have been focusing on the intratumoral administration route. Since the GITR agonistic Ab directly activates effector T cells and suppresses Tregs, the increase of Ab concentration in tumors and surrounding tissues including lymph nodes by the intratumoral route may enhance only the tumor-infiltrating T cells and break the tumor-specific immune-tolerant microenvironment, sparing the unexpected effect on the systemic immune system. In this study, first, in a murine colon cancer model, we showed that the intratumoral delivery of Ab significantly increased the number of effector T cells infiltrated into tumors, and suppressed tumor growth more effectively than the intraperitoneal injection did. Then, we found that the injection of Ab into the peritumoral area induced a systemic antitumor immunity at a similar level to the intratumoral injection. Therefore, we hypothesized that the transfer of locally administrated Ab into tumor-draining lymph nodes (TDLNs) plays an important role in inducing an effective immunity. In fact, intratumorally or peritumorally injected Ab was detected in TDLNs, and resection of Ab-injected TDLNs significantly reduced GITR Ab-mediated systemic tumor immunity. Intratumoral injection showed less number of auto-reactive T cells in the spleen than the intraperitoneal injection did. Furthermore, the AH-1 tetramer assay and ELISpot assay showed that the combination of GITR Ab with anti-PD-1 Ab synergistically enhanced an induction of antitumor immunity, leading to eradication of the tumor. Intratumoral delivery of GITR Ab is a promising approach to induce an effective immunity than the systemic delivery. Citation Format: Kazunori Aoki, Kenta Narumi, Chihiro Shibasaki, Reina Miyakawa. Local selivery of GITR agonistic antibody induces a stronger antitumor immunity than systemic administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-152.

Abstract 4938: OX40 agonist antibody-based combination therapy with PI3Kβ selective inhibitor enhances T cell immunity

Abstract OX40 (CD134), a tumor necrosis factor (TNF) receptor family member, plays a critical role in initiating signaling cascades required for full activation of tumor-reactive T cells. Due to its distinct mechanism of action, the use of OX40 agonist-based combinations is emerging as a novel avenue to improve the effectiveness of cancer treatments. Our recently published studies demonstrate that oncogenic activation of the PI3K pathway by loss of the expression of tumor suppressor PTEN, imposes an immunosuppressive microenvironment in favor of tumor immune evasion. These results highlight the therapeutic potential of combinational therapy using OX40 agonists and PI3K inhibitors in cancer patients. In this study, we sought to evaluate the antitumor efficacy of the combination of an OX40 agonist antibody and a PI3Kβ selective inhibitor, GSK2636771, in tumors with PTEN loss. By using a genetically engineered mouse (GEM) model of melanoma, which can spontaneously develop tumors with PTEN loss, we observed that combination therapy using anti-OX40 and GSK2636771 significantly delayed tumor growth and improved survival time of mice bearing PTEN loss tumors. This combinational treatment was also well tolerated in experimental mice. Unlike the combination of GSK2636771 and immune checkpoint blockers, this combinational treatment did not result in an increase in the number of CD8+ tumor-infiltrating T cells, but significantly enhanced the percentage of Ki67+ CD8+ T cells at the tumor site when compared to either treatment alone. These results suggest that GSK2636771 treatment can synergize with OX40 agonists to augment effector functions of tumor-reactive T cells. To further confirm this synergistic effect, we measured serum levels of 45 cytokines/chemokines in tumor-bearing mice receiving anti-OX40 alone or in combination with GSK2636771. Serum levels of CCL4, CXCL10 and IFN-γ, which are mainly produced by memory and/or effector T cells, were significantly increased in mice in the combination cohort in comparison to the monotherapy cohorts. More importantly, using a vaccine mouse model, we demonstrate that GSK2636771 in combination with anti-OX40 did not significantly impair the generation and maintenance of memory T cells. Taken together, our results suggest that the combinational approach of an OX40 agonist antibody and GSK2636771 may induce robust and durable antitumor T cell immunity. Our study also provides a rationale to explore the clinical activity of an OX40 agonist antibody in combination with GSK2636771 in cancer patients with PTEN loss tumors. Citation Format: Weiyi Peng, Chunyu Xu, Brenda Melendez, Heather Jackson, Jodi A. McKenzi, Leila J. Williams, Yuan Chen, Rina M. Mbofung, Sara E. Leahey E. Leahey, Greg Lizee, Michael A. Davies, Niranjan Yanamandra, Patrick Hwu. OX40 agonist antibody-based combination therapy with PI3Kβ selective inhibitor enhances T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4938.

Abstract 2716: Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies in vivo

Abstract Members of the tumor necrosis factor (TNF) superfamily of costimulatory receptors have emerged as promising targets for modulation of tumor infiltrating lymphocytes. Among these, 4-1BB (CD137) has a prominent role in promoting survival and expansion of cytotoxic CD8 T cells as well as activation of CD4 T cells, NK cells, and antigen presenting cells. The clinical relevance of 4-1BB agonism is currently being evaluated for two antibodies: urelumab (IgG4) and utomilumab (IgG2). However, it has been established that for the trimeric TNF receptor family, the homotrimeric ligands are superior activators compared to bivalent agonistic antibodies. Here we describe a prototype 4-1BB agonist consisting of three human 4-1BB ligands linked together as a single polypeptide chain and fused to the C-terminus of human IgG1 Fc (STIM-41BBL). Compared to a clinically relevant human 4-1BB antibody, STIM-41BBL shows increased activation of 4-1BB in a NF-kappa B reporter cell line. STIM-4-1BBL also shows augmented cytokine release compared to the antibody in CD8 T cells. Furthermore, we have developed a mouse STIM-41BBL and like the human counterpart, the ligand fusion induced greater cytokine release than an agonistic mouse 4-1BB antibody (IgG2a) in CD8 T cells. We have also introduced mutations (D265A/N297A) to the mouse 4-1BB antibody to make it effectorless in order to better approximate the clinical 4-1BB antibodies. Subsequently, we demonstrate that the murine STIM-41BBL had greater anti-tumor activity than the 4-1BB antibody in a MC38 tumor model. These results support the continued development of a 4-1BB ligand based therapeutic and suggests the greater potential of a STIM-41BBL fusion to a targeting antibody for more precise activation of immune system. Citation Format: Eric Tam, Galina Craig, Sara Ghassemifar, Alexander Koshkaryev, Sara Movassaghian, Cormac Cosgrove, James Sampson, Daryl Drummond, Andreas Raue. Development of 4-1BB agonists: Ligand fusion shows greater anti-tumor activity than antibodies in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2716.

Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTβR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

CD70 Modulates Thymocyte Development and Type 1 Diabetes in NOD Mice

CD70 is a tumor necrosis factor (TNF) superfamily member expressed by activated T cells, B cells, dendritic cells, NK cells and mature macrophages. It interacts with CD27, a TNF receptor family member on T cells that regulates T cell activation, proliferation and survival. Type 1 diabetes (T1D) is caused by T cell mediated beta cell destruction, however, T1D-prone NOD mice developed accelerated disease in the absence of CD70. This was associated with altered proportions of double negative vs. double positive thymocytes in CD70 knockout mice, indicating that CD70 modulates global thymopoiesis. Also affected, but in opposing directions, was the development of regulatory T cells (Treg) and natural killer T (NKT) cells, two immunoregulatory lymphocyte subsets that are functionally and numerically deficient in NOD mice. Tregs frequencies were reduced by 23% and 32% in CD70 heterozygous (CD70-/+) and homozygous (CD70-/-) knockout mice. In contrast, NKT cell frequencies were respectively increased by 48% and 61% in CD70-/+ and CD70-/- mice compared to standard NOD mice. The higher level of NKT cells in CD70 knockout mice was unexpectedly associated with reduced levels of thymic CD1d, a molecule that is required for NKT cell development. Collectively, our results show that CD70 controls the development of several T cell populations that are known to modulate T1D development in NOD mice and that, in the absence of CD70, T1D is exacerbated. These finding may facilitate the development of clinical therapeutics aimed at enhancing the stimulatory function of CD70 as a way to suppress disease. Disclosure C. Ye: None. J. Driver: None. M.V. Wiles: None. D. Serreze: None.

Improved Osteogenesis by HVEM-Expressing Allogenic Bone Marrow-Derived Mesenchymal Stem Cells in an Immune Activation Condition and Mouse Femoral Defect Model.

Use of allogeneic mesenchymal stem cells (allo-MSCs) in bone tissue engineering strategies can overcome the limitations associated with autologous MSCs, but unfortunately, the immunogenicity of allo-MSCs leads to a high rate of rejection, unless immunosuppressive agents are used. B and T lymphocyte attenuator (BTLA) is a newly discovered immunoglobulin superfamily inhibitory receptor, and Herpesvirus-entry mediator (HVEM), a member of the tumor necrosis factor receptor family, is the only ligand of BTLA. Both BTLA and HVEM are widely expressed in B and T lymphocytes and other immune cells and play significant roles in the negative regulation of an immunoreaction. Therefore, we hypothesized that MSCs could be modified to maintain their bone differentiation ability through negative regulation of the immune response, and to test this hypothesis, we generated HVEM-expressing MSCs and tested their potential for osteogenic differentiation and bone repair in a simulated immune activation condition in vitro and in a mice femoral defect model. We found that osteogenic differentiation of allo-MSCs was decreased significantly in the activated immune microenvironment and that HVEM expression by allo-MSCs inhibited the immune response, resulting in improved osteogenic differentiation in vitro and new bone formation by allo-MSCs in a mouse femoral defect model. Our results also preliminarily suggested that the mechanism by which HVEM-expressing allo-MSCs overcome inflammation and enhance osteogenesis may be related to inhibition of interleukin-17. Overall, the data obtained in the present study provide support for the further development of HVEM-modified allo-MSCs as potentially ideal seed cells for bone tissue engineering applications.

Crystal structures of the human 4-1BB receptor bound to its ligand 4-1BBL reveal covalent receptor dimerization as a potential signaling amplifier

Human (h)4-1BB (TNFRSF9 or CD137) is an inducible tumor necrosis factor receptor (TNFR) superfamily member that interacts with its cognate ligand h4-1BBL to promote T lymphocyte activation and proliferation. h4-1BB is currently being targeted with agonists in cancer immunotherapy. Here, we determined the crystal structures of unbound h4-1BBL and both WT h4-1BB and a dimerization-deficient h4-1BB mutant (C121S) in complex with h4-1BBL at resolutions between 2.7 and 3.2 Å. We observed that the structural arrangement of 4-1BBL, both unbound and in the complex, represents the canonical bell shape as seen in other similar TNF proteins and differs from the previously reported three-bladed propeller structure of 4-1BBL. We also found that the binding site for the receptor is at the crevice formed between two protomers of h4-1BBL, but that h4-1BB interacts predominantly with only one ligand protomer. Moreover, h4-1BBL lacked the conserved tyrosine residue in the DE loop that forms canonical interactions between other TNFR family molecules and their ligands, suggesting h4-1BBL engages h4-1BB through a distinct mechanism. Of note, we discovered that h4-1BB forms a disulfide-linked dimer because of the presence of an additional cysteine residue found in its cysteine-rich domain 4 (CRD4). As a result, h4-1BB dimerization, in addition to trimerization via h4-1BBL binding, could result in cross-linking of individual ligand-receptor complexes to form a 2D network that stimulates strong h4-1BB signaling. This work provides critical insights into the structural and functional properties of both h4-1BB and h4-1BBL and reveals that covalent receptor dimerization amplifies h4-1BB signaling.

Clinical significance of the TNF-α receptors, TNFRSF2 and TNFRSF9, on cell migration molecules Fascin-1 and Versican in acute leukemia

In human hematologic malignancies, some of the TNF receptor family members are up-regulated and have the ability to evoke reactions favoring tumor progression. Moreover, cell migration molecules, Fascin-1 and Versican are involved in proliferation, migration and invasion of cancer cells. They are linked to many human cancers. Therefore, we conducted this study to evaluate both the plasma and leukocytes concentrations of tumor necrosis factor receptor super family 2 (TNFRSF2), TNFRSF9, Fascin-1 and Versican in patients with acute leukemia, as well as to correlate these values with clinical features and treatment outcome. Therefore, forty-eight patients with acute myeloid leukemia (AML), thirty-two patients with acute lymphoblastic leukemia (ALL) and fifteen control subjects were included. TNFRSF2, TNFRSF9, Fascin-1 and Versican were measured in plasma and leukocytes of all subjects by enzyme-linked immunosorbent assay. We found that plasma TNFRSF9 was highly elevated in ALL and AML as compared with the control group. In addition, AML patients who failed to achieve complete remission showed a significant increase in leukocytes TNFRSF9 level. TNFRSF2 is significantly increased in plasma and leukocytes of ALL patients when compared with the control group and AML patients. Fascin-1 significantly increased in AML, but not in ALL cases. Plasma and leukocytes levels of Versican significantly increased in AML compared to both control and ALL subjects. Plasma Versican correlated with poor response to induction of chemotherapy in AML cases. In conclusion, TNFRSF2 and TNFRSF9 could act as a possible prognostic biomarkers for the outcomes of ALL patients and TNFRSF9 could be a potential target in AML. Versican may be used as a diagnostic biomarker and as a predictor of the response to chemotherapy in AML. In addition, plasma Fascin-1 is a potential biomarker for AML.

The Ectodysplasin receptor EDAR acts as a tumor suppressor in melanoma by conditionally inducing cell death.

Ectodysplasin receptor EDAR is seen as a typical Tumor Necrosis Factor receptor (TNFR) family member known to interact with its ligand Eda-A1, and signaling mainly through the nuclear factor-kappaB (NF-κB) and c-jun N-terminal kinases pathways. Mutations in genes that encode proteins involved in EDAR transduction cascade cause anhidrotic ectodermal dysplasia. Here, we report an unexpected pro-apoptotic activity of EDAR when unbound to its ligand Eda-A1, which is independent of NF-κB pathway. Contrarily to other death receptors, EDAR does recruit caspase-8 to trigger apoptosis but solely upon ligand withdrawal, thereby behaving as the so-called dependence receptors. We propose that pro-apoptotic activity of unbound EDAR confers it a tumor suppressive activity. Along this line, we identified loss-of-pro-apoptotic function mutations in EDAR gene in human melanoma. Moreover, we show that the invalidation of EDAR in mice promotes melanoma progression in a B-Raf mutant background. Together, these data support the view that EDAR constrains melanoma progression by acting as a dependence receptor.