Luciferase Reporter Cell Line Research Articles

Development of FluoAHRL: A Novel Synthetic Fluorescent Compound That Activates AHR and Potentiates Anti-Inflammatory T Regulatory Cells.

Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.

Abstract 75: Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies

Abstract Despite the considerable success of immune checkpoint therapies targeting T cells, a sizable proportion of patients experience resistance or relapse due to the immunosuppressive nature of the tumor microenvironment. Myeloid cells, a major component that suppresses effector lymphocytes, have emerged as an alternative and promising therapeutic target. However, there is a deep lack of widely accessible immunological models capable of representing the intricate three-way interaction between tumor cells, T cells, and myeloid cells. To address this need, we conducted a comprehensive protein profiling of human tumor and immune cell lines available at ATCC for various established and novel immune checkpoint molecules. Cell lines with high endogenous expression of the immune checkpoint proteins, such as programmed death-ligand 1 and 2 (PD-L1 and PD-L2), cluster of differentiation 155 (CD155), B7 homolog 3 (B7-H3), sialic acid-binding Ig-like lectin 10 (Siglec-10), or signal-regulatory protein alpha (SIRPα), were selected and constructed into luciferase reporter cell lines. For tumor reporter cell lines, a gamma interferon activation site (GAS) response element was placed upstream of the luciferase gene in the lentiviral vector, enabling the activation of the JAK-STAT signaling pathway within tumor cells to induce luciferase expression. In myeloid reporter cell lines, a nuclear factor kappa B (NF-κB) response element replaced GAS to monitor the activation of the NF-κB signaling pathway. In the presence of corresponding immune checkpoint inhibitors that enhance T cell-mediated anti-tumor activity, these reporter cell lines produce a bioluminescent signal based on luciferase expression. This signal can be easily detected and quantified to assess the efficacy of the inhibitor. Our data revealed that bioluminescence intensity in the tumor and myeloid reporter cell lines increased by >100-fold in a dose-dependent manner in response to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) stimulation, respectively, and by >50-fold in response to the conditioned media collected from activated primary T cells. Furthermore, in co-culture assays involving various combinations of immune cell and tumor cell types with corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated a significant increase in bioluminescence intensity. In conclusion, these newly established luciferase reporter cell lines offer an excellent ex vivo model for cancer immunotherapy. These cell lines naturally express immune checkpoint proteins, enabling the sensitive and reproducible monitoring of combinatorial responses from various immune cell types. Citation Format: Hyeyoun Chang, Alicia C. Walker, John G. Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 75.

Abstract 724: MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling

Abstract The development of novel antibody drugs is a lengthy journey. Many factors can lead to failures during clinical development. According to reports, off-target binding of antibodies could also be a significant cause. It has been stated that about 25% of preclinical candidates might have off-target issues. With the emergence of new modalities like CD3 bispecific antibodies, ADCs, and CAR-T therapies, early-stage off-target screening of antibodies has become even more crucial. Although techniques such as immunohistochemistry, ELISA, and flow cytometry (FACS) have been used for off-target testing of antibodies, these methods still lack sensitivity and specificity, and their throughput is limited. To address the need for antibody target deconvolution and receptor identification, we developed a novel membrane protein screening array-AB5000 (MPSA-AB5000). MPSA-AB5000 is a high-throughput cell-based platform for identifying the targets of antibodies and other ligands that bind to membrane proteins. MPSA-AB5000 is an excellent solution for antibody specificity screening, which enables early off-target screening and can de-risk the antibody drug development program. The MPSA-AB5000 makes use of the full coverage of unique human membrane proteins, including receptors, transporters, enzymes, miscellaneous, and others. Each membrane protein is expressed in live cells to ensure native conformation. In addition, the same membrane protein clones with a C-terminal epitope tag were constructed, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, MPSA-AB5000 uses luciferase reporter cell line detection. The wash-free strategy reduces false negatives, and signal amplification makes remarkable discrimination. Compared to similar technologies, we possess significant advantages in the following aspects: a. High sensitivity and sigh throughput: our technology platform offers not only high sensitivity, capable of detecting subtle interactions but also supports high-throughput screening, allowing for the evaluation of a large number of samples in a short timeframe. b. Avoidance of false negative signals: by eliminating washing steps, our method reduces the risk of false negative signals, ensuring reliable detection of non-specific binding. c. Comprehensive validation approaches: We employ various validation methods such as FACS and ELISA to eliminate false positive signals and enhance the accuracy and credibility of our results. All the advantages ensure that MPSA-AB5000 is more rapid, simple, and highly sensitive than traditional assays. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 724.

Abstract 2645: A high-throughput platform for targets screening by membrane protein library

Abstract The specificity of antibodies, CAR-T cells and other biologic therapeutics to their binding targets is important to evaluate drug quality. The non-specific binding may lead serious toxic side effects. Therefore, specificity detection is an important step in the evaluation process of drug development. At present, the non-specific binding of antibody drugs is often verified only after serious adverse reactions founding in clinical trials, which may result in drug safety accidents and the abandonment of previous studies. To address the need for antibody target deconvolution and receptor identification, we developed a novel approach “AB5000”. AB5000 is an excellent solution for antibody specificity screening which enable early off-target screening and can de-risk the antibody drug development program. The AB5000 makes use of the full coverage membrane protein, representing over 5465 unique human membrane proteins including receptors, transporters, enzymes, miscellaneous and other membrane protein. Gene vector was transfected into target cells. Each membrane protein introduces a C-terminal epitope tag, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, we use luciferase reporter cell line system which is more rapid, simple and highly sensitive than flow cytometry or other binding assays. The AB5000 has widely application in testing the specificity of antibodies, deconvolution of orphan antibodies and ligands, detection of CAR-T proteins. The AB5000 represents a rapid, comprehensive, and highly sensitive platform for identifying the membrane protein targets of a wide variety of antibodies, protein ligands, and functional interactions. It is a promising powerful tool for biotherapeutic modalities development. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. A high-throughput platform for targets screening by membrane protein library [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2645.

A novel cell-based assay for the high-throughput screening of epithelial–mesenchymal transition inhibitors: Identification of approved and investigational drugs that inhibit epithelial–mesenchymal transition

ObjectivesLung cancer with distant metastases is associated with a very poor prognosis, and epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Therefore, elucidation and inhibition of EMT signaling in lung cancer may be a new therapeutic strategy for improving the prognosis of patients. We constructed a high-throughput screening system for EMT inhibitors. Using this system, we aimed to identify compounds that indeed inhibit EMT. Materials and methodsWe generated a luciferase reporter cell line using A549 human lung cancer cells and E-cadherin or vimentin as EMT markers. EMT was induced by transforming growth factor β1 (TGF-β1), and candidate EMT inhibitors were screened from a library of 2,350 compounds. The selected compounds were further tested using secondary assays to verify the inhibition of EMT and invasive capacity of cells. ResultsValues obtained by the assay were adjusted for the number of viable cells and scored by determining the difference between mean values of the positive and negative control groups. Four compounds were identified as novel candidate drugs. Among those, one (avagacestat) and two compounds (GDC-0879 and levothyroxine) improved the expression of E-cadherin and vimentin, respectively, in epithelial cells. GDC-0879 and levothyroxine also significantly inhibited the invasive capacity of cells. ConclusionWe systematically screened approved, investigational, and druggable compounds with inhibitory effects using a reporter assay, and identified candidate drugs for EMT inhibition.

Using long, sequence-specific dsRNA to knockdown inducible protein expression and virus production via an RNAi-like mechanism

RNA interference (RNAi) is a powerful innate immune mechanism to knock down translation of specific proteins whose machinery is conserved from plants to mammals. The template used to determine which mRNA's translation is inhibited is dsRNA, whose origin can range from viruses (long dsRNA, ∼100–1000s bp) to host (micro(mi)RNA, ∼20mers). While miRNA-mediated RNAi is well described in vertebrates, the ability of long dsRNA to guide RNAi-mediated translation inhibition in vertebrates is controversial. Indeed, as long dsRNA is so effective at inducing type I interferons (IFNs), and IFNs down-regulate RNAi machinery, it is believed that IFN-competent cells are not capable of using long dsRNA for RNAi. In the present study the ability of long, sequence specific dsRNA to knock down both host protein expression and viral replication is investigated in IFN-competent rainbow trout cells. Before exploring RNAi effects, the optimal dsRNA concentration that would funnel into RNAi without triggering the IFN response was determined. After which, the ability of sequence specific long dsRNA to target knockdown via RNAi was evaluated in: (1) uninfected host cells using inducible luciferase gene expression and (2) host cells infected with chum salmon reovirus (CSV), frog virus 3 (FV3) or viral hemorrhagic septicemia virus genotype IVa (VHSV-IVa). Induced expression studies utilized RTG-P1, a luciferase reporter cell line, and dsRNA containing luciferase sequence (dsRNA-Luc) or a mis-matched sequence (dsRNA-GFP), and subsequent luminescence intensity was measured. Anti-CSV studies used dsRNA-CSVseg7 and dsRNA-CSVseg10 to target CSV segment 7 and CSV segment 10 respectively. Inhibition of virus replication was measured by viral titration and RT-qPCR. Taking advantage of the fact that long dsRNA can accommodate more sequences than miRNAs, the antiviral capability of dsRNA molecules containing both CSV segment 7 and segment 10 simultaneously was also measured. Target sequence appears important, as dsRNA-FV3MCP did not knock down FV3 titres, and while dsRNA-VHSV-N knocked down VHSV-IVa, dsRNA-VHSV-G and dsRNA-VHSV-M did not. This is the first study in fish to provide evidence that sequence specific long dsRNA induces potent gene expression silencing and antiviral responses in vitro via an RNAi-like mechanism instead of an IFN-dependent response.

Large-Scale Identification of Multiple Classes of Host Defense Peptide-Inducing Compounds for Antimicrobial Therapy.

The rapid emergence of antibiotic resistance demands new antimicrobial strategies that are less likely to develop resistance. Augmenting the synthesis of endogenous host defense peptides (HDPs) has been proven to be an effective host-directed therapeutic approach. This study aimed to identify small-molecule compounds with a strong ability to induce endogenous HDP synthesis for further development as novel antimicrobial agents. By employing a stable HDP promoter-driven luciferase reporter cell line known as HTC/AvBD9-luc, we performed high-throughput screening of 5002 natural and synthetic compounds and identified 110 hits with a minimum Z-score of 2.0. Although they were structurally and functionally diverse, half of these hits were inhibitors of class I histone deacetylases, the phosphoinositide 3-kinase pathway, ion channels, and dopamine and serotonin receptors. Further validations revealed mocetinostat, a benzamide histone deacetylase inhibitor, to be highly potent in enhancing the expression of multiple HDP genes in chicken macrophage cell lines and jejunal explants. Importantly, mocetinostat was more efficient than entinostat and tucidinostat, two structural analogs, in promoting HDP gene expression and the antibacterial activity of chicken macrophages. Taken together, mocetinostat, with its ability to enhance HDP synthesis and the antibacterial activity of host cells, could be potentially developed as a novel antimicrobial for disease control and prevention.

High-Throughput Identification of Epigenetic Compounds to Enhance Chicken Host Defense Peptide Gene Expression

Enhancing the synthesis of endogenous host defense peptides (HDPs) has emerged as a novel antibiotic-free approach to infectious disease control and prevention. A number of epigenetic compounds have been identified as HDP inducers and several have proved beneficial in antimicrobial therapy. However, species-specific regulation of HDP synthesis is evident. In attempt to identify epigenetic compounds with potent HDP-inducing activity for poultry-specific application, we developed a stable luciferase reporter cell line, known as HTC/AvBD10-luc, following our earlier construction of HTC/AvBD9-luc. HTC/AvBD10-luc was developed through permanent integration of a chicken macrophage cell line, HTC, with a lentiviral luciferase reporter vector driven by a 4-Kb AvBD10 gene promoter. Using a high throughput screening assay based on the two stable cell lines, we identified 33 hits, mostly being histone deacetylase (HDAC) inhibitors, from a library of 148 epigenetic compounds. Among them, entinostat and its structural analog, tucidinostat, were particularly effective in promoting multiple HDP gene expression in chicken macrophages and jejunal explants. Desirably, neither compounds triggered an inflammatory response. Moreover, oral gavage of entinostat significantly enhanced HDP gene expression in the chicken intestinal tract. Collectively, the high throughput assay proves to be effective in identifying HDP inducers, and both entinostat and tucidinostat could be potentially useful as alternatives to antibiotics to enhance intestinal immunity and disease resistance.

Abstract LB536: Newly established CAR-T target antigen luciferase reporter cell lines facilitate CAR-T development

Abstract Adoptive T cell therapy is a type of immunotherapy in which T cells are genetically modified to fight tumors. It has emerged as an exciting new approach for cancer treatment. Chimeric antigen receptor (CAR)-T cells have displayed remarkable efficacy in treating malignant cancers, particularly liquid tumors. CAR-T cells have proven to be a new type of ’living’ therapeutic by harnessing the patients' immune system to recognize specific tumor associated antigens and redirect the engineered T cells to more specifically target tumor cells. A large number of research efforts have been put into developing new CAR structures to increase the scope of targeted cancers and anti-tumor efficacy. Evaluating the biofunction of CAR-T cells in vitro often involves a series of labor-intensive co-culture experiments and immunoassays. Reproducibility also remains a challenge during the validation of new CAR-T cells due to donor-to-donor variation and other factors. In this study, we present a panel of luciferase reporter tumor cell lines that can be utilized to examine the function of CAR-T cells. The panel of selected human tumor cell lines naturally express high levels of CAR-T target antigens on cell surface, such as CD19, CD20 and Her2. After introduction of a Lenti-LUC2 luciferase reporter into the parental cell lines, single cell cloning was performed to isolate stable clones with high luciferase expression. To verify the performance of the target Luciferase reporter cell lines, we used commercially available CAR-T cells expressing CD19, CD20, HER2 and empty vector-transduced T cells as controls in co-culture experiments. The cytotoxicity of the CAR-T cells against target tumor cells was measured using both a luciferase assay and the xCELLigence potency assay. Our results demonstrate that the luciferase reporter system is a relatively simple, robust, and highly sensitive means to measure biological processes. The luciferase reporter cell lines provide an advantage in measuring target cell killing without having to use radioactive 51Cr release assay or pre-labeling the cells in CAR-T functional evaluation. In addition, these reporter cell lines were characterized and authenticated using cell morphology, growth kinetics, and STR analysis. The expression stability of both the target antigen and luciferase was verified by comparing the low passage and the high passage reporter cells. In summary, the well-characterized CAR-T target antigen luciferase reporter cell lines from ATCC provide excellent tools for studying CAR-T biofunction and validating new CAR-T agents for cancer immunotherapy. Citation Format: John Foulke, Brian Della Fera, Luping Chen, Fang Tian. Newly established CAR-T target antigen luciferase reporter cell lines facilitate CAR-T development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB536.

Epi-Aszonalenin B from Aspergillus novofumigatus inhibits NF-κB activity induced by ZFTA-RELA fusion protein that drives ependymoma

Nuclear factor-kappa B (NF-κB) signaling is an intracellular signaling pathway involved in inflammatory responses and the pathogenesis of various cancers, including ependymoma, which is a rare and chemotherapy-resistant glioma. Several isoforms of fusion proteins that consist of a nuclear protein, zinc finger translocation associated (ZFTA), and RELA (ZFTA-RELA), an NF-κB-signaling effector transcription factor, cause excessive activation of the NF-κB signaling pathway and result in supratentorial ependymomas (ST-EPN-RELA). As inhibitors of NF-κB activity induced by ZFTA-RELA are expected to be therapeutic agents for ST-EPN-RELA, we established an NF-κB responsive luciferase reporter cell line that expresses the most common isoform of ZFTA-RELA in a doxycycline-dependent manner. Using this reporter cell line, we screened fungus extracts for compounds that inhibit the NF-κB activity induced by ZFTA-RELA expression and identified aszonalenin, an alkaloid from Aspergillus novofumigatus. We also purified analogs of aszonalenin, namely acetylaszonalenin and epi-aszonalenin B and C. In a luciferase assay using cells constitutively expressing luciferase (counter assay), acetylaszonalenin and epi-aszonalenin C showed non-specific inhibition of the luciferase activity. Aszonalenin and epi-aszonalenin B inhibited the NF-κB responsive luciferase activity by expressing ZFTA-RELA more strongly than the luciferase activity in the counter assay. The upregulation of endogenous NF-κB responsive genes, such as CCND1, ICAM1, and L1CAM, by ZFTA-RELA expression was inhibited by epi-aszonalenin B, but not by aszonalenin. This study suggests that epi-aszonalenin B may be a lead compound for the therapeutic development of ST-EPN-RELA.

Potent Nrf2-inducing, antioxidant, and anti-inflammatory effects and identification of constituents validate the anti-cancer use of Uvaria chamae and Olax subscorpioidea

BackgroundUvaria chamae (UC) and Olax subscorpioidea (OS) roots are included in traditional anti-cancer remedies and some studies have identified their chemopreventive/chemotherapeutic potential. This study aimed to identify some cellular/molecular mechanisms underlying such potential and the associated chemical constituents.MethodsEffect on the viability of cancer cells was assessed using the Alamar Blue assay; ability to modulate oxidative stress was assessed using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay; potential to modulate Nuclear factor erythroid 2-related factor like-2 (Nrf2) activity was assessed in the AREc32 luciferase reporter cell line; and anti-inflammatory effect was assessed using lipopolysaccharide-induced nitric oxide release model in the RAW264.7 cells (Griess Assay). Chemical constituents were identified through liquid chromatography-mass spectrometry (LC-MS).ResultsExtracts up to 100 μg/ml were non-toxic or mildly toxic to HeLa, AREc32, PC3 and A549 cells (IC50 > 200 μg/ml). Each extract reduced basal and peroxide-induced levels of reactive oxygen species (ROS) in HeLa cells. OS and UC activated Nrf2, with UC producing nearly four-fold induction. Both extracts demonstrated anti-inflammatory effects. Chamanetin, isochamanetin, isouvaretin, uvaricin I and other compounds were found in U. chamae root extract.ConclusionAs Nrf-2 induction, antioxidant and anti-inflammatory activities are closely linked with chemoprevention and chemotherapy of cancers, the roles of these plants in traditional anti-cancer remedies are further highlighted, as is their potential as sources of drug leads.

Validation of FOXM1 As a Therapeutic Target in Acute Myeloid Leukemia

Objectives:Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces genes critical in executing the mitotic program. The FOXM1 regulatory network is a major predictor of adverse survival outcomes across 18000 human tumor expression signatures (Gentles. Nat Med 2015). FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines the cellular localization of FOXM1. In the OCI-AML3 cell line and in AML primary samples with mutant alleles of NPM1, we have shown that FOXM1 and mutant NPM are re-localized to the cytoplasm (Khan. Leukemia 2017). We postulated that nuclear export of FOXM1 may underlie the chemotherapy responsiveness in NPM1mut patients. We conducted a multi-institution study to investigate the nuclear expression level of FOXM1 as a predictor of chemotherapy resistance in AML and validate it as a therapeutic target.Methods:Adult patients diagnosed with intermediate-risk AML were identified at Northwestern Memorial and the University of Illinois Hospitals. A total of 102 patients were included in the study with equivalent representation from each institution. Clinical data were collected by chart review, and corresponding bone marrow biopsy samples were retrieved under an IRB approved protocol. Tissue sections were stained with a FOXM1 antibody and scanned images were analyzed using HALO 2.0 software. All biopsy specimens were reviewed by a hematopathologist for adequacy. The percentage of total nuclei expressing FOXM1, nuclear FOXM1 intensity (optical density), and the FOXM1 nuclear:cytoplasmic ratio were quantified. Statistical analysis was performed by the Design and Analysis Core, UIC.Results:Patient characteristics are summarized in Table 1. Out of 102 patients, 77 (75%) achieved a complete remission with or without count recovery (CR/CRi). We used lines of induction therapy needed to achieve remission as a measure of chemotherapy resistance. Patients were stratified as needing 1 or >1 line of induction therapy. Induction therapy included cytarabine and anthracycline in 82 cases and hypomethylator in 19 cases.We found that patients needing >1 line of induction therapy had substantially higher levels of nuclear FOXM1 in their diagnostic bone marrow biopsy compared to patients who responded to first-line therapy (20.4% vs. 11.4% positive nuclei; p= 0.066). In logistic regression analysis, the percentage of FOXM1 positive nuclei (OR 1.85 for 10% increase in positive nuclei, p=0.02) and average nuclear intensity (OR 2.18 for 0.1 U increase in OD, p=0.05) were able to predict resistance to first-line chemotherapy. Due to variation in post-induction therapies, the institutions were considered separately for survival analysis. Nuc:Cyto FOXM1 ratio was able to predict inferior overall survival in a single institution cohort analyzed (n=45) in a model incorporating standard clinicopathologic prognostic factors. Multivariate regression analysis is shown in Table 2.We then proceeded to validate nuclear FOXM1 as a therapeutic target in AML. Short hairpin knockdown of FOXM1 in AML cell lines results in decreased colony size and number (KG-1 shFOXM1: 0.35 normalized to control, p=0.01; MV4-11 shFOXM1: 0.61, p=0.03). We have shown that proteasome inhibitors stabilize HSP70 which is a negative regulator of FOXM1 (Halasi. JBC 2016). The novel proteasome inhibitor ixazomib was tested for in vitro effect on FOXM1 in AML cells. Ixazomib inhibits the transcriptional activity of FOXM1 using a luciferase reporter cell line with inducible FOXM1. Treatment with ixazomib significantly decreases FOXM1 mRNA and inhibits FOXM1 protein expression in AML cell lines and patient samples (n=12) resulting in increased apoptosis by caspase cleavage. This is consistent with the FOXM1 auto-regulation loop (Halasi. Cell Cycle 2009). The therapeutic efficacy of ixazomib in AML was then tested in vivo. Single agent treatment with ixazomib compared to vehicle in an murine xenograft model of AML showed significant reduction in tumor volume after 3 weeks of treatment (0.89cm3 vs 1.62cm3, n=8 per group) and downregulation of FOXM1 expression.Conclusion:Our work shows for the first time that nuclear FOXM1 confers chemotherapy resistance in a clinically homogenous group of intermediate risk AML patients. Moreover we show that ixazomib, a well-tolerated novel proteasome inhibitors has therapeutic efficacy in AML models that correlates with suppression of FOXM1. [Display omitted] DisclosuresKhan:Teva: Speakers Bureau; Takeda: Research Funding; Gilead: Consultancy.

Cells to Systems Screening for Thyroid Hormone Disrupting Chemicals in the Brain

Precisely timed and regulated thyroid hormone (TH) levels are essential for vertebrate development and metabolism. To detect environmental chemicals that disrupt TH action, we developed an integrated luciferase reporter cell line in rat pituitary GH3 cells that is TH responsive through activation of endogenous thyroid hormone receptors (TRs). High throughput screening as part of the Tox21 program identified several non-TH related positive hits as RXR agonists, a supposed “silent” heterodimer partner for TRs. These data showed RXR agonists can affect TR signaling at least in pituitary cells. We extended these results to our sensitive and specific in vivo model for TH action, Xenopus laevis metamorphosis. We previously demonstrated that RXR agonists like organotins and the pharmaceutical bexarotene, strongly potentiate (and RXR antagonists inhibit) TH induced metamorphic programs, at the morphological, cellular (e.g. apoptosis or proliferation), transgenic reporter gene levels, and transcriptomic responses in tadpole tails. We have now extended this analysis to include RNA-Seq experiments over distinct time points in the tadpole brain, a common target of TH in humans and frogs, revealing specific gene sets particularly affected by TH and RXR ligands working in concert. Very few genes were affected by RXR ligands alone. The remarkable overlap between the environmental toxicant tributyltin and the synthetic and specific RXR ligand bexarotene regulated transcriptomes provides strong evidence that they have a common molecular target in multiple tissues. We have also created germline mutations in all RXR family members in Xenopus tropicalis (alpha, beta and gamma), and both copies of the duplicated TR beta gene in Xenopus laevis via genome editing approaches. This will allow us to further investigate TR-RXR heterodimer function across tissues and developmental timeframes, and in response to known and suspected TR and RXR ligands. Our studies revealed an unanticipated degree of TR and RXR ligand interactions in vitro and in vivo, highlighting a surprising role of RXRs as avenues for TH endocrine disruption, including the brain.

Establishment of a reporter system for monitoring activation of the ER stress transducer ATF6β

ATF6 has two isoforms, ATF6α and ATF6β, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional roles of ATF6α in response to ER stress have been well-studied, those of its paralogue ATF6β are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF6β activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF6β. This system expresses a chimeric protein containing a synthetic transcription factor followed by the transmembrane domain and C-terminal luminal domain of ATF6β. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF6β.

Injectable hydrogels functionalized with antibody fragments as intra-articular cytokine sinks neutralizing pro-catabolic cytokines

Purpose: Intra-articular (IA) injection with biologics like cytokine neutralizing antibodies to treat osteoarthritis (OA) have been explored, but so far success is limited. One of the key obstacle for the efficacy of intra-articular therapies is the short retention time of these biologics in the joint space due to rapid clearing by the synovial membrane. To overcome these limitations, we developed a new concept that potentially can increase the therapeutic value of pro-inflammatory cytokine neutralizing antibody fragments in the management of OA. The concept is based on the use of injectable hydrogels functionalized with the variable domain of single chain heavy chain only antibodies (VHHs) for cytokine capture. VHHs can be easily isolated using phage-display technology, recombinantly engineered and produced in yeast. These hydrogels can either be pre-crosslinked in microgels before injection or injected as polymer-VHH conjugates that cross link in a hydrogel in situ. Here, hyaluronic acid was used as model polymer after substitution with tyramine and maleimide groups. The tyramine moiety is used in an enzymatic cross-linking reaction to yield stable macromolecular hydrogel networks. The maleimide group is used for thiol-maleimide chemistry coupling a recombinantly engineered VHH with a free Cysteine in its C-terminal tail to the polymer backbone. We hypothesized that this strategy to functionalize hydrogels with the VHH can retain biological activity of neutralizing VHH. To proof the concept of these so-called cytokine sinks we have selected an anti-TNFα VHH that effectively neutralizes TNFα, which plays an essential role in cartilage degradation in inflammatory arthritis. Methods: To achieve directed conjugation of the VHH to the polymer, we have introduced an unpaired cysteine by using recombinant DNA technology in the C-terminus of the anti-TNFα VHH. Afterwards, the modified VHH was produced in yeast and purified. The conjugation of the VHH to the hyaluronic acid was performed by incubating the VHH with tyramine and maleimide functionalized hyaluronic acid. Characterization of binding affinity for both the non-conjugated VHH and conjugated VHH were measured by ELISA. hydrogels functionalized with the VHH was prepared by mixing the hyaluronic acid-VHH conjugates with enzyme peroxidase and H2O2. Biological activity of the conjugated VHH and hydrogels functionalized with the VHH was measured using an NFκb responsive luciferase reporter cell line after stimulation with TNFα. An equimolar concentration of non-conjugated VHH was used as a control. Results: We show successful conjugation of the VHH to the polymer backbone. The VHH functionalized polymer could be used for making stable hydrogels after tyramine mediated cross linking. This modification did not affect the biological activity of the VHH. Using an NFκb responsive luciferase reporter cell line we demonstrated that hydrogels functionalized with the VHH efficiently inactivated TNFα and that this inhibition was comparable to inhibition by an equimolar concentration of non-conjugated VHH. Conclusions: We successfully developed a biocompatible and efficient way to couple VHH to hyaluronic acid. These conjugates could be used for in situ generation of cytokine sinks capable of capturing different pro-catabolic cytokines in treatment of OA. Our Results demonstrate that cytokine sinks have great potential for neutralizing inflammatory pro-catabolic cytokines after intra-articular injection by increasing the retention time of neutralizing antibody fragments in the joint cavity.

BET bromodomain inhibitors PFI-1 and JQ1 are identified in an epigenetic compound screen to enhance C9ORF72 gene expression and shown to ameliorate C9ORF72-associated pathological and behavioral abnormalities in a C9ALS/FTD model

BackgroundAn intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), referred to as C9ALS/FTD. No cure or effective treatment exist for C9ALS/FTD. Three major molecular mechanisms have emerged to explain C9ALS/FTD disease mechanisms: (1) C9ORF72 loss-of-function through haploinsufficiency, (2) dipeptide repeat (DPR) proteins mediated toxicity by the translation of the repeat RNAs, and more controversial, (3) RNA-mediated toxicity by bidirectional transcription of the repeats that form intranuclear RNA foci. Recent studies indicate a double-hit pathogenic mechanism in C9ALS/FTD, where reduced C9ORF72 protein levels lead to impaired clearance of toxic DPRs. Here we explored whether pharmacological compounds can revert these pathological hallmarks in vitro and cognitive impairment in a C9ALS/FTD mouse model (C9BAC). We specifically focused our study on small molecule inhibitors targeting chromatin-regulating proteins (epidrugs) with the goal of increasing C9ORF72 gene expression and reduce toxic DPRs.ResultsWe generated luciferase reporter cell lines containing 10 (control) or ≥ 90 (mutant) G4C2 HRE located between exon 1a and 1b of the human C9ORF72 gene. In a screen of 14 different epidrugs targeting bromodomains, chromodomains and histone-modifying enzymes, we found that several bromodomain and extra-terminal domain (BET) inhibitors (BETi), including PFI-1 and JQ1, increased luciferase reporter activity. Using primary cortical cultures from C9BAC mice, we further found that PFI-1 treatment increased the expression of V1-V3 transcripts of the human mutant C9ORF72 gene, reduced poly(GP)-DPR inclusions but enhanced intranuclear RNA foci. We also tested whether JQ1, an BETi previously shown to reach the mouse brain by intraperitoneal (i.p.) injection, can revert behavioral abnormalities in C9BAC mice. Interestingly, it was found that JQ1 administration (daily i.p. administration for 7 days) rescued hippocampal-dependent cognitive deficits in C9BAC mice.ConclusionsOur findings place BET bromodomain inhibitors as a potential therapy for C9ALS/FTD by ameliorating C9ORF72-associated pathological and behavioral abnormalities. Our finding that PFI-1 increases accumulation of intranuclear RNA foci is in agreement with recent data in flies suggesting that nuclear RNA foci can be neuroprotective by sequestering repeat transcripts that result in toxic DPRs.

The IL-1promoter-driven luciferase reporter cell line THP-G1b can efficiently predict skin-sensitising chemicals.

IL-1functions as an essential pro-inflammatory mediator for the sensitisation of allergic contact dermatitis (ACD). However, studies conducted to date have typically used a limited number of haptens and examined their effects only on murine ACD or murine dendritic cells (DCs). It therefore remains unclear whether IL-1α and/or IL-1β is produced in ACD induced by haptens other than those commonly used in mouse ACD models, and whether they are essential for sensitisation leading to ACD in humans. In addition, it is unclear whether human DCs also produce IL-1α or IL-1β after stimulation by haptens in general. Here, we first demonstrated that 10 haptens (3 extreme, 1 strong, 3 moderate and 3 weak) increased both IL-1α mRNA and IL-1β mRNA expression by the human monocyte cell line THP-1, a commonly used surrogate of DCs in in vitro skin sensitisation tests. Next, we constructed an in vitro skin sensitisation test using a stable IL-1β reporter cell line, THP-G1b, and evaluated whether 88 haptens and 34 non-haptens increase IL-1β reporter activity. We found that 94% of 77 haptens evaluated after considering their applicability domain and solubility in the chosen media stimulated reporter activity. These studies demonstrated that most haptens, irrespective of their potency, increased IL-1β mRNA expression by THP-1 cells, confirming that human DCs also produce IL-1β after stimulation by most haptens. The luciferase assay using THP-G1b cells is thus another skin sensitisation test based on the adverse outcome pathway with reasonable performance.

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

BackgroundIn-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.MethodsTo identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium (enterotoxigenic E. coli) and a Gram-positive bacterium (Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.ResultsA total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening, with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment. Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression, showing a minimum 10-fold increase in IPEC-J2, 3D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants, but not 3D4/31 cells. In addition to pBD3, other porcine HDP genes such as pBD2, PG1-5, and pEP2C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene- and cell type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3D4/31 macrophages (P < 0.05) against both Gram-negative and Gram-positive bacteria.ConclusionsOur results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

Abstract 1018: Luciferase expressing monocyte reporter cell lines as a predictive human cell-based model for in vitro immune activation studies

Abstract Cancer immunotherapy has emerged as an exciting new approach for cancer treatment, and immuno-oncology is one of the fastest growing fields in oncology. The development of the immunomodulatory drugs and biologics dictate a clear need for human cell-based model to evaluate immune activation. THP-1 cell line is one of the best surrogate models for in vitro human monocytes. Luciferase reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes through in vitro bioluminescence measurements. Here we report on the generation of a panel of THP-1 luciferase reporter cell lines that have been transduced with a Luc2P plasmid containing the response element of various transcription factors, which include NF-κB, GAS, NFAT, ISRE, AP-1, and CRE. After introduction of the pLenti-RE-LUC2P plasmid into the parental THP-1, single cell cloning was performed to isolate stable clones with the best signal to noise ratio of luciferase signals upon stimulation. THP-1 NF-κB reporter showed greater than 30 folds increase in bioluminescence signals while stimulated by TNFα or LPS. THP-1 GAS reporter responded with high sensitivity to IFN-γ which allowed signal fold change to be greater than 100 folds. We also found that IFN-α can stimulate GAS through cross-talk between STAT1 and STAT2 signaling which showed a greater than 10 folds change in bioluminescence signal. In addition, these cell lines were characterized and authenticated using cell morphology, growth kinetics, and STR analysis. The growth of these clones was comparable to that of parental THP-1, and the STR analysis showed that the derived luciferase reporter cell clones were 100% identical to the parental THP-1 cell line STR profile. The selected transcription factors play critical roles in regulating immune reactions, antiviral responses and inflammations. In addition to allowing the study of specific signaling pathway activity, these THP-1 reporter cell lines can be used to exam various immune response conditions and monocyte activation during immuno-oncology drug discoveries. For example, we show here that the THP-1 NF-κB reporter cells were not only highly sensitive to the stimulation of toll-like receptors and pro-inflammatory cytokine such as TNF-α in a dose-dependent manner, but also responded to the simulation of common damage-associated molecular pattern (DAMP) such as HMGB1, which is released during cancer cell death. Meanwhile, the activation of THP-1 GAS reporter cells were examined not only by cytokine IFN-γ simulation alone, but also were used in co-culture system with activated T cells to evaluate IFN-γ release during various immune-oncology drug treatments. In summary, luciferase expressing monocytes are valuable tools for studying signal transduction pathway activation, screening of compounds to find activators of signal transduction pathways, and efficacy evaluation of inflammatory effect of new drugs and chemicals. Citation Format: Brian Della Fera, Haiyun Liu, John Foulke, Fang Tian. Luciferase expressing monocyte reporter cell lines as a predictive human cell-based model for in vitro immune activation studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1018.

Abstract 4320: Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival

Abstract 75% of women suffering from ovarian cancer are diagnosed at late stage of the disease often associated with cancer cell infiltration into the peritoneal cavity. Standard therapy is tumor resection and subsequent platin-based chemotherapy. Relapse is frequently observed caused by persisting cancer cells. Persister cells comprise a therapy-tolerant subpopulation of cancer cells and repeated therapies presumably select for increased tolerance. The present project aims to find molecular markers for the identification and targeted eradication of persister ovarian cancer cells. Persister cells of the cisplatin-sensitive high-grade serous ovarian cancer cell line, OVCAR-3, were selected after cisplatin incubation with clinically relevant doses. Individual clones were isolated from control or cisplatin challenged OVCAR-3 cells. A subpopulation of the resulting clonal persister cells showed a distinct morphological phenotype characterized by increased migration and high viability in short- and long-term survival after cisplatin treatment. A significant upregulation (p&amp;lt;0.001) of the endoplasmic reticulum (ER) stress marker, ATF3, was identified by RNA sequencing and this ER stress phenotype is maintained for more than 20 passages. Additionally, the ovarian cancer OVCAR-8 luciferase reporter cell line was transduced with the “Watermelon” (WM) library [1]. The WM library is a complex barcode library that enables simultaneous tracing of cell lineage and the cellular transcriptional and proliferative states. To study cancer relapse in a CDX model, WM-labelled OVCAR-8-Luc cells were injected into mice and pulse-treated with carboplatin. Single cell RNA sequencing of 900 cells isolated from solid metastases, ascites and treatment-naïve mice revealed an increase in multiple ER stress markers including ATF3, ATF4, JUN and XBP1 in the most abundant solid relapse lineages. Furthermore, analysis of clinical data from GEO, EGA and TCGA showed that low ATF3 expression is associated with increased 5-year-survival of patients with high-grade serous ovarian cancer (Logrank p=6.3e-6). We found that increased ER stress correlates with survival of cancer persister cells and incidence of metastases. This phenomenon was observed in cell lines, a CDX model and clinical data from human ovarian cancer samples. We propose ER stress response, exemplified by ATF3, as a molecular marker to identify and therapeutic targeting of ovarian cancer persister cells. [1] Metabolic switching underlies the ability of cancer persister cells to cycle under drug treatment, Oren Y et al., unpublished Citation Format: Kathrin Böpple, Meng Dong, Markus Kleih, Andrea Gaißler, Yaara Oren, Whitney S. Henry, Moshe Oren, Yael Aylon, Bernd Winkler, Aviv Regev, Robert A. Weinberg, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4320.