Bioluminescence Reporter Assay Research Articles

A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

A Bioluminescence Reporter Assay for Retinoic Acid Control of Translation of the GluR1 Subunit of the AMPA Glutamate Receptor.

The present protocol describes a bioluminescence reporter assay developed to quantify the ability of synthetic agonists of retinoic acid receptors (RARs) to activate glutamate receptor subunit 1 (GluR1) translation. The reporter assay uses firefly luciferase under the control of the GluR1 5' untranslated region (5' UTR) which is bound by RARs to regulate its translation. This method is used to demonstrate the role of RARα in retinoic acid regulation of GluR1 translation. This method may also be used to screen drugs that influence RAR induction of GluR1 translation as an important mechanism controlling learning and memory in the brain.

291 Dual-specific antibodies blocking both PD-L1 and PD-L2 engagement of PD-1 restore anti-tumor immunity

BackgroundInhibition of T cell activation and effector function via engagement of the co-inhibitory receptor PD-1 is a critical mechanism enabling tumors to evade host immunity. The two ligands for PD-1, PD-L1 and PD-L2, can be expressed by a variety of immunosuppressive stromal cells, particularly of the myeloid lineage, endothelial cells, and by tumors themselves. In addition to PD-1, PD-L1 engages B7-1 in an additional co-inhibitory interaction. Blocking only PD-1 or only PD-L1 thus does not relieve all inhibitory components of this pathway. We hypothesized that bispecific antibodies blocking both PD-L1 and PD-L2 could more fully restore tumor-specific T cell activation and potentiate anti-cancer immunotherapy. Furthermore, we speculated that enhancing the cytotoxic effector function of these antibodies might further enhance their efficacy through the depletion of tumor cells and supportive stroma.MethodsWe investigated the capacity of monoclonal antibodies capable of bivalent binding to both PD-L1 and PD-L2 to restore the function of PD-1-suppressed T cells in vitro. To assess the in vivo therapeutic efficiency of bispecific PD-Ligand antibodies with ADCC capacities, mouse IgG2a and modified human IgG1 versions were generated. We assessed their ADCC activity in vitro using a bioluminescent reporter assay, and their therapeutic efficiency in vivo in syngeneic or human-cell derived tumors.ResultsThe bispecific antibodies we generated restore the function of PD-1-suppressed T cells in vitro with equivalent efficiency to the FDA approved PD-1 antibody Pembrolizumab. Moreover, our modified human bispecific antibodies lead to significantly higher FcγRIIa activation than FDA-approved clinical human IgG1 PD-L1 antibodies in vitro. In vivo, ADCC-capable PD-Ligand bispecific antibodies suppress the growth of U2940 lymphoma in immunodeficient mice more efficiently than Rituximab, and in a syngeneic model of PD-L1/PD-L2 double positive colon carcinoma, these antibodies demonstrate superiority to PD-1 blocking antibodies to limit tumor growth and increase survival. Furthermore, treatment with our bispecific antibodies increases T cell proliferation and cytotoxicity and reduces density of immunosuppressive myeloid stroma in vivo.ConclusionsADCC-capable PD-Ligand bispecific antibodies display higher therapeutic potential than existing anti-PD-1 antibodies and represent a new class of PD-1 pathway therapeutics with significant potential for the treatment of a variety of human cancers.AcknowledgementsI would like to thank Anupallavi Srinivasamani, PhD student in Michael Curran’s lab, who performed a considerable amount of the work on this project before I joined.

Identification of LuxR Family Regulators That Integrate Into Quorum Sensing Circuit in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is one of the most important food-borne pathogens that cause economic and public health problems worldwide. Quorum sensing (QS) is a way for the cell-cell communication between bacteria that controls a wide spectrum of processes and phenotypic behaviors. In this study, we performed a systematic research of LuxR family regulators in V. parahaemolyticus and found that they influence the bacterial growth and biofilm formation. We then established a QS reporter plasmid based on bioluminescence luxCDABE operon of Vibrio harveyi and demonstrated that several LuxR family regulators integrated into QS circuit in V. parahaemolyticus. Thereinto, a novel LuxR family regulator, named RobA, was identified as a global regulator by RNA-sequencing analyses, which affected the transcription of 515 genes in V. parahaemolyticus. Subsequent studies confirmed that RobA regulated the expression of the exopolysaccharides (EPS) synthesis cluster and thus controlled the biofilm formation. In addition, bioluminescence reporter assays showed that RobA plays a key role in the QS circuit by regulating the expression of opaR, aphA, cpsQ-mfpABC, cpsS, and scrO. We further demonstrated that the regulation of RobA to EPS and MfpABC depended on OpaR and CpsQ, which combined the QS signal with bis-(3′-5′)-cyclic dimeric GMP to construct a complex regulatory network of biofilm formation. Our data provided new insights into the bacterial QS mechanisms and biofilm formation in V. parahaemolyticus.

177Lu-Lilotomab Satetraxetan Has the Potential to Counteract Resistance to Rituximab in Non-Hodgkin Lymphoma.

Patients with non-Hodgkin lymphoma (NHL) who are treated with rituximab may develop resistant disease, often associated with changes in expression of CD20. The next-generation β-particle–emitting radioimmunoconjugate 177Lu-lilotomab-satetraxetan (Betalutin) was shown to up-regulate CD20 expression in different rituximab-sensitive NHL cell lines and to act synergistically with rituximab in a rituximab-sensitive NHL animal model. We hypothesized that 177Lu-lilotomab-satetraxetan may be used to reverse rituximab resistance in NHL. Methods: The rituximab-resistant Raji2R and the parental Raji cell lines were used. CD20 expression was measured by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was measured by a bioluminescence reporter assay. The efficacies of combined treatments with 177Lu-lilotomab-satetraxetan (150 or 350 MBq/kg) and rituximab (4 × 10 mg/kg) were compared with those of single agents or phosphate-buffered saline in a Raji2R-xenograft model. Cox regression and the Bliss independence model were used to assess synergism. Results: Rituximab binding in Raji2R cells was 36% ± 5% of that in the rituximab-sensitive Raji cells. 177Lu-lilotomab-satetraxetan treatment of Raji2R cells increased the binding to 53% ± 3% of the parental cell line. Rituximab ADCC induction in Raji2R cells was 20% ± 2% of that induced in Raji cells, whereas treatment with 177Lu-lilotomab-satetraxetan increased the ADCC induction to 30% ± 3% of that in Raji cells, representing a 50% increase (P < 0.05). The combination of rituximab with 350 MBq/kg 177Lu-lilotomab-satetraxetan synergistically suppressed Raji2R tumor growth in athymic Foxn1nu mice. Conclusion: 177Lu-lilotomab-satetraxetan has the potential to reverse rituximab resistance; it can increase rituximab binding and ADCC activity in vitro and can synergistically improve antitumor efficacy in vivo.

Rifamycin O, An Alternative Anti-Mycobacterium abscessus Agent.

Mycobacterium abscessus is the most difficult-to-treat nontuberculous mycobacteria because of its resistance to many antibiotics. In this study, we screened the Korea Chemical Bank library for a bioluminescent reporter assay to identify molecules capable of acting against M. abscessus. On application of the assay, rifamycin O showed excellent in vitro activity with a narrow range of the minimum inhibitory concentration required to inhibit the growth of 90% of the bacterium (MIC90 = 4.0–6.2 μM); its in vivo efficacy in the zebrafish (Danio rerio) infection model was comparable to that of rifabutin at 25 μM. Furthermore, rifamycin O did not show significant toxicity in cells and the zebrafish model. These results are the first in vivo indication that rifamycin O may be a drug candidate for treating M. abscessus infections.

5894The biased angiotensin receptor 1-ligand TRV023 attenuates atherosclerosis through selective inhibition of Gq-mediated signaling

Abstract Introduction Antagonists of the angiotensin II receptor type 1 (AT1) belong to the most successful treatments of cardiovascular disease. Besides G-protein-independent β-arrestin-mediated signaling, AT1 preferencially signals via G-proteins of the families Gq and G13. Conventional AT1-antagonists equally inhibit all of these pathways, even though the beneficial effects are generally attributed to the inhibition of Gq only. While data on β-arrestin signaling in the vascular disease context are conflicting, we found an excessive pathological remodeling and dramatic exacerbation of atherosclerosis upon interference with G13-mediated signaling. Against this background, selective inhibition of the Gq-pathway seems desirable. Thus, based on the novel concept of ligand-biased signaling, we aimed to identify biased AT1-antagonists that selectively inhibit the Gq-pathway, while allowing for continued signaling through G13. Methods and results We systematically profiled known AT1-ligands and identified the angiotensin analogue Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH (TRV023) as a Gq-selective AT1-antagonist: TRV023 has recently been described as a β-arrestin-biased ligand; however its signaling properties regarding distinct G-protein isoforms have never been defined. Indeed, using bioluminescence reporter assays, we found that TRV023 inhibits Gq-signaling at least as potently as losartan and telmisartan, but unlike the conventional antagonists, it does not impair β-arrestin-signaling (Fig. 1A). Intriguingly, while the conventional antagonists effectively inhibited induction of a G13 luciferase reporter, TRV023 did not significantly interfere with G13-mediated signaling, indicating Gq-selective antagonistic effects. According to our hypothesis, these G-protein-biased signaling properties could render TRV023 superior to conventional antagonists in the treatment of vascular disease. In order to test this hypothesis, we exposed ApoE-deficient mice to one of two different atherosclerosis models: A novel model for accelerated atherosclosis based on partial carotid ligation and western diet, or a conventional atherosclerosis model based on 12 weeks of western diet. Using osmotic pumps, mice were continuously treated with vehicle control, TRV023 or losartan in doses that proved equipotent with respect to blood pressure lowering effects. In line with our hypothesis, TRV023 but not losartan significantly attenuated plaque development in both atherosclerosis models (Fig. 1B). Figure 1 Conclusion Our studies have identified TRV023 as a Gq-biased AT1-antagonist and indicate that selective inhibition of AT1-dependent Gq-mediated signaling by biased ligands may be a promising approach for the treatment of atherosclerosis. Hence, TRV023 qualifies as a drug candidate, particularly since it has already proven to be safe and well-tolerable in clinical phase I and II trials in the context of acute heart failure. Acknowledgement/Funding Else Kröner-Fresenius-Foundation

A Bioluminescence Reporter Assay for Retinoic Acid Control of Translation of the GluR1 Subunit of the AMPA Glutamate Receptor

Retinoic acid (RA) regulates numerous aspects of central nervous system function through modulation of gene transcription via retinoic acid receptors (RARs). However, RA has important roles independent of gene transcription (non-genomic actions) and in the brain a crucial regulator of homeostatic plasticity is RAR control of glutamate receptor subunit 1 (GluR1) translation. An assay to quantify RAR regulation of GluR1 translation would be beneficial both to study the molecular components regulating this system and screen drugs that influence this critical mechanism for learning and memory in the brain. A bioluminescence reporter assay was developed that expresses firefly luciferase under the control of the GluR1 5′ untranslated region bound by RAR. This assay was introduced into SH-SY5Y cells and used to demonstrate the role of RARα in RA regulation of GluR1 translation. A screen of synthetic RAR and RXR ligands indicated that only a subset of these ligands activated GluR1 translation. The results demonstrate the practicality of this assay to explore the contribution of RARα to this pathway and that the capacity of RAR ligands to activate translation is a quality restricted to a limited number of compounds, with implications for their RAR selectivity and potentially their specificity in drug use.

Isolation of photoprotective signal transduction mutants by systematic bioluminescence screening in Chlamydomonas reinhardtii

In photosynthetic organisms, photoprotection to avoid overexcitation of photosystems is a prerequisite for survival. Green algae have evolved light-inducible photoprotective mechanisms mediated by genes such as light-harvesting complex stress-related (LHCSR). Studies on the light-dependent regulation of LHCSR expression in the green alga Chlamydomonas reinhardtii have revealed that photoreceptors for blue light (phototropin) and ultraviolet light perception (UVR8) play key roles in initiating photoprotective signal transduction. Although initial light perception via phototropin or UVR8 is known to result in increased LHCSR3 and LHCSR1 gene expression, respectively, the mechanisms of signal transduction from the input (light perception) to the output (gene expression) remain unclear. In this study, to further elucidate the signal transduction pathway of the photoprotective response of green algae, we established a systematic screening protocol for UV-inducible LHCSR1 gene expression mutants using a bioluminescence reporter assay. Following random mutagenesis screening, we succeeded in isolating mutants deficient in LHCSR1 gene and protein expression after UV illumination. Further characterization revealed that the obtained mutants could be separated into 3 different phenotype groups, the “UV-specific”, “LHCSR1-promoter/transcript-specific” and “general photoprotective” mutant groups, which provided further insight into photoprotective signal transduction in C. reinhardtii.

Bioluminescent reporter assay for monitoring ER stress in human beta cells

During type 1 diabetes development, cells in the islets of Langerhans engage adaptive mechanisms in response to inflammatory signals to cope with stress, to restore cellular homeostasis, and to preserve cell function. Disruption of these mechanisms may induce the formation of a repertoire of stress-induced neoantigens, which are critical in the loss of tolerance to beta cells and the development of autoimmunity. While multiple lines of evidence argue for a critical role of the endoplasmic reticulum in these processes, the lack of tools to specifically monitor beta cell stress hampers the development of therapeutic interventions focusing on maintaining endoplasmic reticulum homeostasis. Here we designed and evaluated a stress-induced reporter in which induction of stress correlates with increased light emission. This Gaussia luciferase-based reporter system employs the unconventional cytoplasmic splicing of XBP1 to report ER stress in cells exposed to known ER-stress inducers. Linking this reporter to a human beta cell-specific promotor allows tracing ER-stress in isolated human beta cells as well as in the EndoC-βH1 cell line. This reporter system represents a valuable tool to assess ER stress in human beta cells and may aid the identification of novel therapeutics that can prevent beta cell stress in human pancreatic islets.

Abstract 2549: Development of CAR T-cell therapy targeting glypican-3 in liver cancer

Abstract Background: Glypican-3 (GPC3) is a candidate therapeutic target in hepatocellular carcinoma (HCC). We have generated the HN3 and hYP7 antibodies that recognize the N-terminus and C-terminus of GPC3, respectively. Here, we engineered human T cells that express GPC3-specific chimeric antigen receptors (CARs) and evaluated their potential for the treatment of HCC. Methods: The GPC3-specific CARs (CAR.HN3 and CAR.hYP7) contain a CD3ζ chain and the 4-1BB co-stimulatory endodomain along with a truncated human EGFR polypeptide (huEGFRt) that retains the cetuximab epitope. A set of GPC3 positive-liver cancer cell lines including Hep3B, HepG2 and Huh7 were engineered to express the firefly luciferase gene. These cells were co-cultured with GPC3 CAR T cells at various ratios for 24 hours. A bioluminescent luciferase reporter assay was then used to evaluate the cytolytic activity of effector CAR T cells. We also evaluated the anti-tumor activity of CAR T cells in xenograft models with intraperitoneal injection of luciferase-expressing HCC cells into NOD/SCID/IL2gnull (NSG) mice. The tumor growth was measured via bioluminescence tumor imaging. Results: We found that the CAR.hYP7 cells targeting the C-terminus of GPC3 showed the highest cytolytic activity against GPC3-positive HCC cells. In two HCC intraperitoneal xenograft models (Hep3B and HepG2), luciferase-expressing HCC cells preferentially grew on the mouse liver. More importantly, we found that a single intraperitoneal treatment with a high dose of CAR.hYP7 cells (40 million per mouse) exhibited sustainable antitumor efficacy in the Hep3B model and all of the mice survived after 9 weeks post CAR T-cell treatment without recurrence. The serum levels of alpha fetoprotein (AFP) in mice treated with a high dose of CAR.hYP7 cells were less than 50 ng/ml, the threshold value for HCC diagnosis. Additionally, a single intraperitoneal treatment with a low dose of CAR.hYP7 cells (10 million per mouse) caused regression of liver tumor xenografts. In the low dose CAR.hYP7 group, Hep3B tumors in the mice grew locally and restricted to the fat tissues far from the mouse liver, indicating that CAR T cells prevented tumors from seeding and growing in the liver. Although CAR.HN3 cells targeting the N- terminus of GPC3 also exhibited high cytotoxicity to HCC cells in vitro, no significant Hep3B tumor growth inhibition was seen in the mice treated with CAR.HN3 cells. Conclusion: Our work suggests that GPC3-targeting CAR T cells, in particular CAR.hYP7, is a promising therapeutic intervention for liver cancer that can be translated to human use. Citation Format: Dan Li, Nan Li, Yifan Zhang, Haiying Fu, Madeline B. Torres, Qun Wang, Tim F. Greten, Mitchell Ho. Development of CAR T-cell therapy targeting glypican-3 in liver cancer [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 2549.

ASC acts in a caspase-1-independent manner to worsen acute pneumonia caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa expresses a type III secretion system (T3SS) that activates the host inflammasome-mediated immune response. We examined the role of inflammasome activation in severe infection outcomes. We infected C57BL/6 (B6) mice lacking inflammasome components ASC or caspase-1/11 with a highly virulent strain of P. aeruginosa, PSE9, using a mouse model of pneumonia. We evaluated inflammasome activation in vitro by infecting bone marrow-derived macrophages (BMDMs) with PSE9 and measuring cell death and release of inflammasome-dependent cytokines IL-18 and IL-1β. A bioluminescent reporter assay was used to detect activity of caspase-1 and caspase-3/7 in BMDMs from B6 and ASC-deficient mice.Results/Key Findings. ASC-/- mice exhibited significantly improved survival relative to caspase-1/11-/- mice and B6 mice, demonstrating that ASC and caspase-1/11 play differential roles in P. aeruginosa infection. We found that ASC-/- BMDMs exhibited significantly reduced cell death relative to B6 BMDMs, while caspase-1/11-/- BMDMs were resistant to cell death. IL-18 and IL-1β were both detected from supernatants of infected B6 BMDMs, but cytokine release was abrogated in both ASC-/- and caspase-1/11-/- BMDMs. We detected a 2.5-fold increase in the activation of caspase-3/7 in PSE9-infected B6 BMDMs, but no increase in infected ASC-/- BMDMs. Cell death, cytokine release and caspase-3/7 activity were dependent on a functional T3SS. Collectively, these results are consistent with a model whereby the T3SS apparatus of P. aeruginosa activates the caspase-1-dependent inflammasome and caspase-3/7 through an ASC-dependent mechanism. This activation may have implications for the outcomes of P. aeruginosa infections.

Recombinant IFN-\u03b3 from the bank vole Myodes glareolus: a novel tool for research on rodent reservoirs of zoonotic pathogens

Rodent species like Myodes glareolus and Microtus spp. are natural reservoirs for many zoonotic pathogens causing human diseases and are gaining increasing interest in the field of eco-immunology as candidate animal models. Despite their importance the lack of immunological reagents has hampered research in these animal species. Here we report the recombinant production and functional characterization of IFN-γ, a central mediator of host’s innate and adaptive immune responses, from the bank vole M. glareolus. Soluble dimeric recMgIFN-γ was purified in high yield from Escherichia coli. Its activity on M. glareolus and Microtus arvalis kidney cell lines was assessed by immunofluorescent detection of nuclear translocation and phosphorylation of the transcription factor STAT1. RecMgIFN-γ also induced expression of an IFN-γ-regulated innate immunity gene. Inhibition of vesicular stomatitis virus replication in vole cells upon recMgIFN-γ treatment provided further evidence of its biological activity. Finally, we established a recMgIFN-γ-responsive bank vole reporter cell line that allows the sensitive titration of the cytokine activity via a bioluminescence reporter assay. Taken together, we report valuable tools for future investigations on the immune response against zoonotic pathogens in their natural animal hosts, which might foster the development of novel animal models.

Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay

A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0–10.0) and temperatures up to 100 °C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3 mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B. cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems.

Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery.

The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative "actives" for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.

Genome-Wide Screen Reveals Rhythmic Regulation of Genes Involved in Odor Processing in the Olfactory Epithelium.

Odor discrimination behavior displays circadian fluctuations in mice, indicating that mammalian olfactory function is under control of the circadian system. This is further supported by the facts that odor discrimination rhythms depend on the presence of clock genes and that olfactory tissues contain autonomous circadian clocks. However, the molecular link between circadian function and olfactory processing is still unknown. To elucidate the molecular mechanisms underlying this link, we focused on the olfactory epithelium (OE), the primary target of odors and the site of the initial events in olfactory processing. We asked whether olfactory sensory neurons (OSNs) within the OE possess an autonomous circadian clock and whether olfactory pathways are under circadian control. Employing clock gene-driven bioluminescence reporter assays and time-dependent immunohistochemistry on OE samples, we found robust circadian rhythms of core clock genes and their proteins in OSNs, suggesting that the OE indeed contains an autonomous circadian clock. Furthermore, we performed a circadian transcriptome analysis and identified several OSN-specific components that are under circadian control, including those with putative roles in circadian olfactory processing, such as KIRREL2-an established factor involved in short-term OSN activation. The spatiotemporal expression patterns of our candidate proteins suggest that they are involved in short-term anabolic processes to rhythmically prepare the cell for peak performances and to promote circadian function of OSNs.

A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation.

Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. Two stabilization assays of P23H mutant opsin were developed and validated, one based on β-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin. Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.

Abstract 3727: Drug screen on six co-cultured GBM cell lines using a multiplex luciferase assay

Abstract Despite the addition of temozolomide (TMZ) to the glioblastoma (GBM) treatment regimen, disease outcome is still poor and additional therapeutic options are necessary. One major obstacle in the GBM treatment is TMZ resistance of the tumor cells resulting in relapse of the disease with no alternative treatments known. In an effort to discover additional treatment modalities we are performing drug screens on GBM cell lines, using wild-type variants and acquired TMZ resistant variants of these cell lines. To be able to screen a range of different cells, we adapted a multiplex bioluminescence reporter assay developed in our lab. This bioluminescent reporter assay is based on the Gaussia luciferase (Gluc) fused to an epitope tag. Different cell lines can be equipped with Gluc fused to different epitope tags (Gluc-tag) so that each cell lines expresses Gluc and a cell line specific epitope tag. Antibody immunoprecipitation allows us to separate the different Gluc-tags and quantify the relative activities of the different cell lines. Activity of each specific Gluc-tag represents the relative number of a specific cell line in the mixed population. Since Gaussia luciferase and the epitope tag is secreted by the cells, we can detect the cell line specific reporters ex vivo in the culture medium or animal blood. Using this novel method in a drug screen we aim to identify additional subtype specific therapy strategies for GBM braintumors. Citation Format: Sjoerd van Rijn, Lotte Hiddingh, Thomas Wurdinger, Ravi Narayan. Drug screen on six co-cultured GBM cell lines using a multiplex luciferase assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2014-3727

Abstract 2555: A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies

Abstract Programmed death-1(PD-1) and its ligand (PD-L1) are important immunotherapy targets for cancer. PD-1 serves as a negative costimulatory receptor on various cell types, including T and B cells as well as myeloid-derived cells. Its ligand PD-L1 (B7-H1) is not expressed by normal epithelial tissues, but it is aberrantly expressed on a wide array of human cancers including melanoma, non-small cell lung carcinoma, breast cancer, pancreatic cancer, etc. Engagement of PD-1 by its ligands PD-L1 expressed on tumor cells associated with poorer prognosis by disabling the host antitumor response. The most recent investigational anti-PD-1 immunotherapy drugs such as nivolumab from BMS and lambrolizumab (MK-3475) from Merck have all demonstrated significant overall survival rate in patients with advanced melanoma. Here we developed a cell based bioluminescent reporter assay that can be used for rapid quantifying function of PD-1 or PD-L1 therapeutic antibodies as measured by activation of NFAT signaling pathway. For this, Jurkat T-cell line stably expressing NFAT-luciferase reporter and human PD-1 was generated. Raji stably expressing human PD-L1 was generated as antigen presenting cells (APC). by co-cultivating the two cell lines in the present of CD3 and IgG, Raji cells fully activate Jurkat NFAT pathway via CD28 binding to CD80 (B7-1) and CD86 (B7-2) endogenously expressed on Raji cells. PD-1 signaling in Jurkat cells following engagement of PD-L1 ectopically expressed on Raji cells inhibits T cell function, and results in NFAT pathway inhibition. Blockade of PD-L1 using anti-PD-L1 mAb reversed NFAT pathway activation. Citation Format: Mei Cong, Natasha Karassina, Jey Cheng, Frank Fan. A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2555. doi:10.1158/1538-7445.AM2014-2555

Circadian Clock Characteristics Are Altered in Human Thyroid Malignant Nodules

The circadian clock represents the body's molecular time-keeping system. Recent findings revealed strong changes of clock gene expression in various types of human cancers. Due to emerging evidence on the connection between the circadian oscillator, cell cycle, and oncogenic transformation, we aimed to characterize the circadian clockwork in human benign and malignant thyroid nodules. Clock transcript levels were assessed by quantitative RT-PCR in thyroid tissues. To provide molecular characteristics of human thyroid clockwork, primary thyrocytes established from normal or nodular thyroid tissue biopsies were subjected to in vitro synchronization with subsequent clock gene expression analysis by circadian bioluminescence reporter assay and by quantitative RT-PCR. The expression levels of the Bmal1 were up-regulated in tissue samples of follicular thyroid carcinoma (FTC), and in papillary thyroid carcinoma (PTC), as compared with normal thyroid and benign nodules, whereas Cry2 was down-regulated in FTC and PTC. Human thyrocytes derived from normal thyroid tissue exhibited high-amplitude circadian oscillations of Bmal1-luciferase reporter expression and endogenous clock transcripts. Thyrocytes established from FTC and PTC exhibited clock transcript oscillations similar to those of normal thyroid tissue and benign nodules (except for Per2 altered in PTC), whereas cells derived from poorly differentiated thyroid carcinoma exhibited altered circadian oscillations. This is the first study demonstrating a molecular makeup of the human thyroid circadian clock. Characterization of the thyroid clock machinery alterations upon thyroid nodule malignant transformation contributes to understanding the connections between circadian clocks and oncogenic transformation. Moreover, it might help in improving the thyroid nodule preoperative diagnostics.