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The STimulator of INterferon Genes (STING) is a transmembrane protein located at the endoplasmic reticulum, which serves as a molecular hub in response to cytoplasmic cyclic dinucleotide (CDN) second messengers such as cyclic GMP-AMP (cGAMP). Upon stimulation of cyclic GMP-AMP synthase (cGAS) by a wide array of pathogens that provide extracellular microbial DNA and by intracellular (nuclear and mitochondrial) DNA, the second messenger cGAMP is generated, which then binds directly to the STING protein resulting in the activation of the STING pathway. This activation by cytosolic DNA initiates a cascade of events, cGAS-STING-TBK1-IRF3 signaling, that induces type 1 IFN response, and eventually the signaling is terminated by degradation of pathway components and clearance of stimulatory DNA. Given the significant roles of STING as an adaptor for DNA sensors to pathogens and its involvement in immune sensing, tumor growth control, and control of autoinflammatory and autoimmune disorders, the STING pathway has become a very important drug target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses and initiates host immune responses against pathogens and cancer, but inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Credible evidence suggests that the cGAS-cGAMP-STING pathway makes fundamental contributions to at least three major cancer therapies: radiation therapy, chemotherapy, and immunotherapy, and it thus represents a promising drug target. Therefore, there is a need to identify lead compounds that effectively modulate human STING for further drug development. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the new concept of nanoluciferase (nanoluc) complementation where a small peptide (SmBiT) linked to cGAMP through a linker (cGAMP tracer) can be recognized by an anti cGAMP antibody that is linked to a nanoluc complementary fragment (LgBiT) resulting in bioluminescence upon the addition of nanoluc substrate. The presence of free cGAMP that is generated in the reaction competes with cGAMP tracer and decreases bioluminescence. The assay is sensitive and can detect less than 10 nM cGAMP and less than one ng of cGAS and thus, it is suitable for testing compounds that modulate cGAS activity. We will show data that correlate the activation of cGAS using less than 10 ng of dsDNA stimulation, and the activation of IFN response element reporter assays to detect cytokine release upon cGAS-STING activation. The assay is homogenous, HTS formatted and can be completed in less than one hour. Thus, this assay provides valuable contribution for investigating modulators of the STING pathway and facilitates the search for novel therapeutics. Citation Format: Said A. Goueli, Kevin Hsiao, Hui Wang, Matt Larsen. Detection and quantification of cellular GAMP using homogenous HTS formatted bioluminescent assay [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 2765.

Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

A direct method for measuring adenosine-triphosphate (ATP) in seawater was developed recently, in which commercial reagents are added directly to seawater. However, calibration is required if seawater quality changes (such as changes in salinity, pH, Mg2+, Fe3+) as the seawater matrix interferes with ATP measurement. In this research, a 0.1 μm filtration process is introduced to eliminate such interferences. In addition, a filter rinsing step with sterilized artificial seawater is proposed to eliminate interference of free ATP. The ATP-filtration method is fast (2 = 0.72, n = 100) with intact cell concentration. Microbial ATP concentration measured using the ATP-filtration method and the ATP-direct method were comparable. Microbial ATP measured along the treatment train of a full-scale seawater reverse osmosis (SWRO) plant decreased from 530 in the raw seawater to 10 ng-ATP/L after pre-treatment and to 0.5 ng-ATP/L in the SWRO permeate. The method was also applied to monitor bacterial growth potential (BGP) across the pre-treatment train of a (pilot) seawater desalination plant, where the removal of BGP through the media filtration and ultrafiltration was 44% and 7%, respectively.

Activating KRAS mutations present in many cancers are difficult to target with drugs until recently with the FDA approval of sotorasib (AMG-510), a KRAS G12C selective inhibitor. In an effort to study Ras inhibitors in cells and identify new compounds that inhibit Ras and its downstream signaling, assays such as western or ELISA are commonly used. These traditional immunoassays can be tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. We applied this bioluminescent and homogeneous “Add and Read” assay to analyze Ras signaling pathway activation and inhibition through multiple nodes of the pathway including detection of phosphorylated ERK. The assay was used to profile different allele specific inhibitors with cell lines harboring different activating KRAS mutations and obtained the predicted pharmacology and selectivity. The potency of PROTAC compound targeting selective degradation of KRAS G12C was also tested using Lumit and the result showed that maximal decrease in RAS signaling was achieved. Unlike current assays that require lengthy sample preparation and multiple washing steps, the Lumit immunoassay provides an alternative platform because it is homogeneous and allows to decipher signaling pathways activities in a fast and simple manner. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest such as Ras, and identify much needed inhibitors of its mutants. Citation Format: Hicham Zegzouti, Laurie Engel, Matthew Swiatnicki, Juliano Alves, Said A. Goueli. Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays [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 2977.

Genomic instability engages the cGAS (cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (stimulator of interferon genes) signaling (cGAS-cGAMP-STI NG) pathway on both sides of cancer either by being instrumental for antitumor immunity and blocking malignancy or by promoting inflammation-driven carcinogenesis and metastasis. The cGAS-STING axis also plays an essential role in initiating host immune defense against microbial invasion and STING-deficient mice are more susceptible to some viral infections. Credible evidence suggests that the cGAS-cGAMP-STI NG pathway makes fundamental contributions to at least three major cancer therapies, radiation therapy, chemotherapy, immunotherapy, and thus represents a promising pharmaceutical target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP) upon binding to DNA and initiating host immune responses against pathogens and cancer. However, inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Hence, STING is an attractive drug target for the treatment of STING-driven autoimmune and inflammatory disorders. Therefore, there is a need to identify lead compounds that effectively inhibit human STING for further drug development. An attempt to search for such molecules have been described recently with the report on a STING-specific inhibitor that showed high efficiency, specificity, and safety, paving the way for therapeutically manipulating STING-mediated clinical diseases. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the use of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) to release AMP which can be converted to ATP via a coupled enzyme reaction. The ATP generated can be quantified using luciferase/luciferin detection reagents. The assay is homogenous, HTS formatted and can be completed in less than one hour. The assay is sensitive and can detect as low as 10 nM cGAMP. We will show data on the use of agents that stimulate GAS activity such as dsDNA, and the use of reporter assays to detect cytokine release upon cGAS-STING activation. Citation Format: Said A. Goueli, kevin Hsiao. Monitoring the concentration of cGAMP using homogenous bioluminescent HTS-formatted assay [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 1402.

In this study, the removal of particulate, organic and biological fouling potential was investigated in the two-stage dual media filtration (DMF) pretreatment of a full-scale seawater reverse osmosis (SWRO) desalination plant. Moreover, the removal of fouling potential in two-stage DMF (DMF pretreatment) was compared with the removal in two-stage DMF installed after dissolved air floatation (DAF) (DAF-DMF pretreatment). For this purpose, the silt density index (SDI), modified fouling index (MFI), bacterial growth potential (BGP), organic fractions and microbial adenosine triphosphate (ATP) were monitored in the pretreatment processes of two full-scale SWRO plants. Particulate fouling potential was well controlled through the two stages of DMF with significant removal of SDI15 (&gt, 80%), MFI0.45 (94%) and microbial ATP (&gt, 95%). However, lower removal of biological/organic fouling potential (24–41%) was observed due to frequent chlorination (weekly) of the pretreatment, resulting in low biological activity in the DMFs. Therefore, neutralizing chlorine before media filtration is advised, rather than after, as is the current practice in many full-scale SWRO plants. Comparing overall removal in the DAF-DMF pretreatment to that of the DMF pretreatment showed that DAF improved the removal of biological/organic fouling potential, in which the removal of BGP and biopolymers increased by 40% and 16%, respectively. Overall, monitoring ATP and BGP during the pretreatment processes, particularly in DMF, would be beneficial to enhance biological degradation and lower biofouling potential in SWRO feed water.

Several potential growth methods have been developed to monitor biological/organic fouling potential in seawater reverse osmosis (SWRO), but to date the correlation between these methods and biofouling of SWRO has not been demonstrated. In this research, the relation between a new adenosine triphosphate (ATP)-based bacterial growth potential (BGP) test of SWRO feed water and SWRO membrane performance is investigated. For this purpose, the pre-treatment of a full-scale SWRO plant including dissolved air flotation (DAF) and two stage dual media filtration (DMF) was monitored for 5 months using BGP, orthophosphate, organic fractions by liquid chromatography coupled with organic carbon detection (LC-OCD), silt density index (SDI), and modified fouling index (MFI). Results showed that particulate fouling potential was well controlled through the SWRO pre-treatment as the measured SDI and MFI in the SWRO feed water were below the recommended values. DAF in combination with coagulation (1&ndash, 5 mg-Fe3+/L) consistently achieved 70% removal of orthophosphate, 50% removal of BGP, 25% removal of biopolymers, and 10% removal of humic substances. Higher BGP (100&ndash, 950 &micro, g-C/L) in the SWRO feed water corresponded to a higher normalized pressure drop in the SWRO, suggesting the applicability of using BGP as a biofouling indicator in SWRO systems. However, to validate this conclusion, more SWRO plants with different pre-treatment systems need to be monitored for longer periods of time.

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid(TNA) extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities., Graphical abstract Unlabelled Image

Monitoring cellular signaling events can help better understand cell behavior in health and disease. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple steps, and are not easily adaptable to high throughput screening (HTS). Here, we describe a new immunoassay approach based on bioluminescent enzyme complementation. This immunoassay takes less than two hours to complete in a homogeneous “Add and Read” format and was successfully used to monitor multiple signaling pathways’ activation through specific nodes of phosphorylation (e.g pIκBα, pAKT, and pSTAT3). We also tested deactivation of these pathways with small and large molecule inhibitors and obtained the expected pharmacology. This approach does not require cell engineering. Therefore, the phosphorylation of an endogenous substrate is detected in any cell type. Our results demonstrate that this technology can be broadly adapted to streamline the analysis of signaling pathways of interest or the identification of pathway specific inhibitors., Hwang et al. uses NanoLuc Binary Technology to develop a bioluminescent immunoassay for monitoring activation of multiple signaling pathways through specific nodes of phosphorylation. Their method takes less than two hours to complete in a homogeneous “Add and Read” format with no requirement for cell engineering.

The success of immunotherapy treatment in oncology ushered a new modality for treating a wide variety of cancers. However, lack of effect in some patients made it imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. It has been recently recognized that adenosine levels increased significantly in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressant and attenuating several effector T cell functions. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and producing AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. Towards the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. We have developed very sensitive and homogenous assays that enabled us to monitor the activity of both enzymes and demonstrate selectivity of known inhibitors as well as monoclonal antibodies. This should speed up screening for novel inhibitors leading to more effective cancer therapy

The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) was first reported in Wuhan, China in late 2019 resulting in infection of over 20 million and death of over one million world-wide as of October 2020. The infection by this virus is highly transmissible and can lead to severe pneumonia and death. The SARS-CoV-2 is an enveloped virus and belongs to the beta coronavirus family containing a large and complex positive-sense single stranded RNA genome (~30kb).It binds to human ACE2 receptor with high affinity, compared to the other CoVs, via its spike protein (S) and membrane fusion leading to the release of its genomic RNA that replicates and produce higher number of subgenomic RNAs. The viral RNA is then capped by nsp 14 and nsp10/16, two nonstructural proteins that catalyze the methylation of RNA to limit its degradation by cellular 5'-3'exonuclease and evade host immune system. These enzymes play critical role in translation of the viral RNA and its replication and thus leading to viral survival. We have used a bioluminescent high throughput enzyme assay for monitoring the activity of these enzymes in order to develop novel inhibitors which can be advanced for therapeutic treatment. We show that we can determine the activity of nanogram amounts of these enzymes and test the activity of these enzymes using different inhibitors, some of which are competitive with S-adenosyl methionine the canonical substrate for these enzymes. We also show the effect of inhibitors that compete with the oligonucleotide substrate. Finally, we tested the effect of a peptide modulator of COVID-2 on the activity of both nsp10/16 and nsp14. The assay is simple, homogenous, fast, and bioluminescent which provides a very robust platform for testing novel compounds on the viral methyltransferases leading to the generation of new drugs towards inhibiting the replication of COVID19 Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring COVID-19 RNA methyltransferases activities for developing therapeutic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1162.

Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activities. However, cost and logistical limitations prevent profiling activities from being performed in-house. We describe the development of an automated and flexible kinase profiling workflow that combines ready-to-use kinase enzymes and substrates in convenient eight-tube strips, a bench-top liquid handling device, ADP-Glo Kinase Assay (Promega, Madison, WI) technology to quantify enzyme activity, and a multimode detection instrument. Automated methods were developed for kinase reactions and quantification reactions to be assembled on a Gilson (Middleton, WI) PIPETMAX, following standardized plate layouts for single- and multidose compound profiling. Pipetting protocols were customized at runtime based on user-provided information, including compound number, increment for compound titrations, and number of kinase families to use. After the automated liquid handling procedures, a GloMax Discover (Promega) microplate reader preloaded with SMART protocols was used for luminescence detection and automatic data analysis. The functionality of the automated workflow was evaluated with several compound-kinase combinations in single-dose or dose-response profiling formats. Known target-specific inhibitions were confirmed. Novel small molecule-kinase interactions, including off-target inhibitions, were identified and confirmed in secondary studies. By adopting this streamlined profiling process, researchers can quickly and efficiently profile compounds of interest on site.

The success of immune oncology for treating a wide variety of cancers ushered a new paradigm in cancer therapy. However, due to some limitations in some patients, it is imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. Recent recognition of significant increase in adenosine levels in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressant and attenuating several effector T cell functions have spurred interest in targeting ectonucleotidases that generate adenosine. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and producing AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. To facilitate the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. It is very sensitive, and the assays are homogenous and enabled us to monitor the activity of both purified enzymes and enzymes bound to the cellular membranes. Most significant is the ability to discern the selectivity of known small molecule inhibitors as well as selective monoclonal antibodies. Our recent expansion of testing of many small molecules and several antibodies have confirmed the utility of this approach for discovering novel therapeutics that might synergize other regiments such as chemotherapies, and in the design of targeting inhibitors. This should speed up screening for novel inhibitors leading to more effective cancer therapy Citation Format: Said A. Goueli, Kevin Hsiao, Hicham Zegzouti. Monitoring the activities of ectonucleotidases CD39 and CD73 and their specific modulation by antibodies and small molecules using homogenous HTS formatted platform [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 6305.

GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation, and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators has been challenging due to paucity of convenient assays. In this study, we describe a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Since Mg2+ plays a critical role in influencing the affinity of GTPases with guanosine triphosphate/guanosine diphosphate (GTP/GDP) and the process of nucleotide exchange, manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP that remains after the GTPase reaction to ATP and detection of the generated ATP using the luciferin/luciferase combination. The GTPase/GAP/GEF-Glo assay system enables monitoring of GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins when expressed in cells as fusion proteins by performing the assay in a pulldown format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds and its robustness was demonstrated by a high Z′-factor of 0.93 and CV of 2.2%. The assay system has a high dynamic range, formatted in a convenient add–mix–read, and applicable to high-throughput screening.

The modification of a diverse array of substrates by Fe(II)/2-oxoglutarate-dependent dioxygenases is central to the modulation of distinct biological processes such as epigenetics, hypoxic signaling, and DNA/RNA repair. Of these, JumonjiC domain-containing histone lysine demethylases (JMJCs) and prolyl hydroxylases are potential drug targets due to their relevance to human diseases. Thus, assays to interrogate this enzyme superfamily are needed to identify selective and potent inhibitors as leads for drug development and that could also be useful research tools. Since succinate is a common product to all Fe(II)/2-oxoglutarate-dependent dioxygenase reactions, a method that detects succinate would be suitable to all members of this enzyme superfamily. We therefore developed a bioluminescent and homogenous succinate detection assay and validated its use with diverse sets of enzyme classes. We evaluated the substrate specificities of these enzymes, their apparent kinetic constants, and inhibition profiles and mode of action of reported and novel inhibitors. Our results indicate that succinate detection is a useful readout for the monitoring of enzymatic activities with distinct substrate entities, as well as for the discovery of novel inhibitors. By investigating a large number of Fe(II)/2-oxoglutarate-dependent enzymes, this method could have a significant impact on the field of dioxygenase research.

The use of adenosine triphosphate (ATP) to monitor bacterial growth potential of seawater is currently not possible as ATP cannot be accurately measured at low concentration in seawater using commercially available luciferase-based ATP detection. The limitation is due to interference of salt with the luciferin–luciferase reaction, which inhibits light production. This research demonstrates that new reagents developed for (i) ATP extraction from microbial cells and (ii) ATP detection in seawater are able to reliably detect Microbial ATP as low as 0.3 ng L–1 in seawater. The luminescence signal of the new detection reagent is significantly higher (>20 times) than the luminescence signal of the freshwater reagent, when applied in seawater. ATP can now be used to monitor bacterial growth potential (BGP) through pre-treatment trains of seawater reverse osmosis (SWRO) plants. The level of detection of the new BGP test is significantly lower than the estimated threshold value required to prevent biofouling in SWRO systems. The new reagents have been used to monitor Microbial ATP in coastal North Sea water. Moreover, Microbial ATP has been applied to monitor the bacterial growth potential (using indigenous bacteria) through the pre-treatment train of an SWRO desalination plant. A significant reduction (>55%) of the bacterial growth potential was found through the dual media filtration with 4.5 mg-Fe(III) L–1 coagulant. Overall, the new reagents can detect low Microbial ATP concentrations in seawater and can be used to monitor bacterial growth potential in seawater desalination plants.

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD

The success of immunotherapy treatment in oncology ushered a new modality for treating a wide variety of cancers. However, lack of effect in some patients made it imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. It has been recently recognized that adenosine levels increased significantly in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressant and attenuating several effector T cell functions. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and producing AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. Towards the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. We have developed very sensitive and homogenous assays that enabled us to monitor the activity of both enzymes and demonstrate selectivity of known small molecule inhibitors as well as selective monoclonal antibodies. This should speed up screening for novel inhibitors leading to more effective cancer therapy Citation Format: Said Goueli, kevin Hsiao. Biochemical and cell-based activities of ectonucleotidases CD39 and CD73 using a homogenous HTS formatted platform [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B138. doi:10.1158/1535-7163.TARG-19-B138

Many enzymatic reactions use Adenosine triphosphate (ATP) as a substrate and produce adenosine monophosphate (AMP) as the common product of these reactions. AMP also functions as a modulator of many enzymes and a key cellular metabolite regulating energy homeostasis and signal transduction. For example, by activating AMP-Dependent protein kinase (AMPK), it maintains the energy charge ratio at optimal level during metabolic activities; and its concentration is altered during disregulated disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates in a homogenous and high through-put formatted form. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by first depleting the remaining residual ATP substrate and converting AMP produced into ATP, the later can be quantified using luciferase/luciferin and generating light. We also tested this assay to measure direct activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP-Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono- and poly-ADP-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system we can measure the activities of sulfotransferases. In summary, we have developed a very strong platform to monitor AMP concentrations that is applicable to diverse enzymatic reactions. Citation Format: Said A. Goueli, Kevin Hsiao, Subhanjan Mondal. Monitoring AMP Concentration,the universal product of diverse enzymatic reactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2151.

Since the introduction of Herceptin and Rituximab in 1986, therapeutic antibodies have gained tremendous momentum in the treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions that may result in compromised bioactivity and safety profile. The nonenzymatic post-translational modification of both deamidation of asparagine and isomerization of aspartate residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current methods (HPLC and liquid chromatography and mass spectrometry) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel, and are not amenable to high-throughput scaling. We have developed a robust, homogenous, high-throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions, such as new formulations, storage periods, and temperature.

Aim: To develop a homogenous, nonradioactive, antibody-free and universal assay for diverse families of methyltransferases and monitor the activity of these enzymes in a high-throughput format. Materials & methods: The assay conditions are optimized for monitoring the enzymatic activity of a broad range of methyltransferases regardless of the chemical structure or nature of the enzyme substrate in a low- and high-throughput-formatted protocols. The assay detects S-adenosyl-l-homocysteine, the universal reaction products of all methyltransferases. Results: We demonstrate the utility of using this protocol to determine the activity of DNA, protein methyltransferases and also to determine kinetic parameters of several inhibitors using purified enzymes. The assay is sensitive (20–30 nM of S-adenosyl-l-homocysteine) and robust. Conclusion: The methyltransferase Glo is nonradioactive, antibody-free and homogenous, universal assay to determine enzyme activity of diverse families of methyltransferases. The assay is formatted to meet the requirements of high-throughput screening in drug discovery programs searching for modulators of methyltransferases.

Signaling pathways activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences, causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential to better understand normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, nonhomogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two-subunit system based on NanoLuc luciferase that was successfully demonstrated for protein-protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF-κB, BTK and JAK/STAT). In very few steps we detected the expected biologic response of these pathways either by monitoring BTK autophosphorylation, assessing phosphorylation and degradation of IκB or phosphorylation of STAT3 upon activation of the cells with TNFα, or IL6, respectively. We also tested different small- or large-molecule inhibitors of these pathways or their corresponding node kinase (BTK, IKK, or JAKs) and obtained the expected pharmacology. The bioluminescent system presented here has many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis, allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors. Citation Format: Hicham Zegzouti, Brian Hwang, Nidhi Nath, Said Goueli. Deciphering key cancer and inflammation signaling pathways with homogeneous bioluminescent cell-based kinase activity assays [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 3446.

Modern immunotherapy approaches focusing on bolstering T cell responses and augmenting cell mediated immunity with final tumor destruction have ushered a new era and a turning point in cancer treatment. This was demonstrated by recent successes of several immunotherapeutic regimes, such as monoclonal antibody blocking of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), which boosted the development of this treatment modality. Although much success have been generated with multiple tumors at different stages, tumors can deploy multiple mechanism to allow their survival by avoiding immunesurveillance. Thus, identification of alternate immunosuppressive pathways that can be targeted to enhance tumor clearance are disparately needed. It is well known that adenosine produced in the tumor microenvironment (TME) hampers the immune reaction towards cancer cells by disabling the cytotoxic anti-tumor immune response while enhancing the proliferation and polarization of immunosuppressive cells and the neovascularization that support tumor growth. It is also known that modulation of adenosine levels in the TME limits tumor growth and improves anti-tumor immune activity. Therefore, targeting the enzymes that produce adenosine and adenosine receptors as potential biomarkers for treatment response and tumor progression can provide a novel therapeutic strategy that synergize with other check point immunoblockade antibodies. Towards this goal we have developed a novel assay to monitor the activity of the two enzymes responsible for generating extracellular adenosine in the TME, namely, ectonucleotidase triphosphate diphosphohydrolase 1 (CD39) and ecto 5'-nucleotidase (CD73). Since adenosine monophosphate (AMP) is generated from adenosine triphosphate (ATP) and adenosine diphosphate (ADP) by CD 39 followed by generation of adenosine (Ado) from AMP by CD73, we monitored the activity of both enzymes in different tumor cell lines with varied levels of both enzymes. We were able to correlate the activity of the ecto enzymes with their protein level on the cell surface and carried out enzyme characterization using both antibodies and small molecules selective for their targets. Our results clearly show that small molecule inhibitors of CD73 provide a better strategy to combat adenosine levels while small molecules towards CD39 were shown to be promiscuous since they inhibit other enzymes such as kinases. For targeting CD39, a better strategy might be the use of antibodies since they are selective to their cognate enzyme protein. Because the therapeutic potential of targeting CD39 and CD73 is not dependent solely on their nucleotidase enzyme activity but also due to their non-enzymatic role in tumor progression, antibodies might provide an alternative approach for targeting these enzymes. Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring CD73 and CD39 ectonucleotidase activities and their modulation by antibodies and small molecules inhibitors [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 3871.

Kinases continue to be one of the most important targets in today's drug discovery efforts. Following the identification of lead compounds through screening efforts, it is important to profile these leads against other kinases within that family, as well as from other families, to ascertain potential off-target effects. Because many kinase assays require the use of different substrates, optimization time and costs during profiling can be prohibitive. Here we demonstrate the versatility of a luminescent ADP accumulation assay, where one set of reagents can be used for a wide variety of kinases with differing K(m app) for ATP and substrates. Assay sensitivity allows for the use of low enzyme concentrations and small percent ATP conversion levels while still maintaining high signal:background ratios. We have used a simple, inexpensive automated pipetting system to automate the entire process from enzyme optimization through generation of compound IC(50) values. Agreement with literature values proves this combination of chemistry and instrumentation provides a simple, yet robust solution for automated kinase profiling.

ADP-Glo is a novel bioluminescent, homogeneous assay for monitoring ADP producing biochemical reactions and thus it is an ideal assay for detecting enzyme activity using a wide variety of substrates. It is a universal assay that can be used with protein kinases, lipid kinases, sugar kinases, and many more kinases as well as ATPases. Because of its high sensitivity, it is suitable for monitoring enzyme activities at very early substrate conversions requiring very low amount of enzymes. Furthermore, as the assay is applicable to a broad range of ATP and substrate concentrations, it is optimal for enzymes that require high ATP and substrate concentrations. This is critical since inhibitor potency has to be demonstrated at the cellular level where ATP is present at millimolar concentrations. ADP-Glo is performed in 2 steps upon completion of kinase reaction: a combined termination of kinase reaction and depletion of remaining ATP in the first step, and conversion of generated ADP to ATP and the newly produced ATP to light output using luciferase/luciferin reaction in the second step. The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the kinase activity. Due to its high signal to background and luminescent readout, this assay is less susceptible to generation of false hits and thus it is applicable to not only primary and secondary screening but also kinase profiling.

The lipid second messengers phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) and sphingosine 1-phosphate (S1P) are well recognized to play important roles in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. Disruption of lipid signaling pathways often leads to human cancers, making lipid kinases attractive drug targets. In order to develop novel drugs against these enzymes, an assay that monitors their activity and amenable to high-throughput scale for screening large number of compounds is essential. The newly developed ADP-Glo assay is such an assay that measures kinase activity of lipid kinases by detecting the formation of ADP using a highly robust and sensitive bioluminescence approach. We evaluated this technology for studying lipid kinases, class I PI3 kinases, and sphingosine kinases and we show that the assay exhibits good tolerance to different lipids substrates. It generates kinetic parameters for substrates and inhibitors similar to those reported in the literature using other published assay formats. The sensitivity and robustness of this assay allow the detection of 5% of substrate conversion with Z' values >0.7 making it attractive for high-throughput screening (HTS) applications. It is noteworthy that ADP-Glo assay addresses the need for a single integrated platform to comprehensively measure all classes of lipid and protein kinases. The selected inhibitors of lipid kinases can be screened against the panel of desired protein kinases, making ADP-Glo assay a simple, inexpensive platform for HTS and profiling of lipid kinases.

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.

The advancement of a kinase inhibitor throughout drug discovery and development is predicated upon its selectivity towards the target of interest. Thus, profiling the compound against a broad panel of kinases is important for providing a better understanding of its activity and for obviating any off-target activities that can result in undesirable consequences. To assess the selectivity and potency of an inhibitor against multiple kinases, it is desirable to use a universal assay that can monitor the activity of all classes of kinases regardless of the nature of their substrates. The luminescent ADP-Glo kinase assay is a universal platform that measures kinase activity by quantifying the amount of the common kinase reaction product ADP. Here we present a method using standardized kinase profiling systems for inhibitor profiling studies based on ADP detection by luminescence. The kinase profiling systems are sets of kinases organized by family, presented in multi-tube strips containing eight enzymes, each with corresponding substrate strips, and standardized for optimal kinase activity. We show that using the kinase profiling strips we could quickly and easily generate multiple selectivity profiles using small or large kinase panels, and identify compound promiscuity within the kinome.

The advancement of a kinase inhibitor throughout drug discovery and development is predicated upon its selectivity towards the target of interest. Thus, profiling the compound against a broad panel of kinases is important for providing a better understanding of its activity and for obviating any off-target activities that can result in undesirable consequences. To assess the selectivity and potency of an inhibitor against multiple kinases, it is desirable to use a universal assay that can monitor the activity of all classes of kinases regardless of the nature of their substrates. The luminescent ADP-Glo kinase assay is a universal platform that measures kinase activity by quantifying the amount of the common kinase reaction product ADP. Here we present a method using standardized kinase profiling systems for inhibitor profiling studies based on ADP detection by luminescence. The kinase profiling systems are sets of kinases organized by family, presented in multi-tube strips containing eight enzymes, each with corresponding substrate strips, and standardized for optimal kinase activity. We show that using the kinase profiling strips we could quickly and easily generate multiple selectivity profiles using small or large kinase panels, and identify compound promiscuity within the kinome.

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular gro...

Drug safety is of significant important importance in the pharmaceutical industry indicating that minimal side effects constitute a major requirement in drug development. Thus, after rigorous scree...

Total protein kinase activity associated with nuclei of rat ventral prostate was extracted and fractionated with the aid of DEAE-Sephadex column chromatography. The flow-through peak contained kinase activities towards dephosphophosvitin, lysine-rich histone, and nonhistone proteins as phosphoprotein substrates. These activities were not stimulated by addition of cAMP, although the cAMP-dependent protein kinase inhibitor reduced the lysine-rich histone kinase activity by 70% or more, suggesting that it might represent the catalytic subunit of the cAMP-dependent histone kinase. By contrast, the inhibitor produced a stimulation of kinase activities toward dephosphophosvitin and nonhistone proteins by 15% and 50%, respectively. The bound fraction on the DEAE Sephadex column was eluted in two peaks of protein kinase activity, one at 0.15–0.20 M (NH4)2SO4, and the other at 0.25–0.30 M (NH4)2SO4. The first peak contained activity only toward nonhistone proteins, whereas the second had activity toward dephosphophosvitin, lysine-rich histone, and nonhistone proteins. None of these was inhibited by the cAMP-dependent protein kinase inhibitor. The effect of orchiectomy on the activity of the various protein kinase fractions was determined. The kinase activity toward dephosphophosvitin, in the flow-through peak, was reduced by about 20% and 40% at 24 and 48 h post-orchiectomy, respectively, and in the bound peak by 33% and 50%, under the same circumstances. Little change was observed in the kinase activity toward lysine-rich histone in the flow-through peak or in the bound peak at 24 h post-orchiectomy. However, at 48 h post-orchiectomy, it increased by 250% in both fractions. This particular kinase activity toward lysine-rich histone might be localized to nucleoplasm rather than chromatin. The protein kinase active toward nonhistone proteins present in the flow-through peak did not change at 24 h post-orchiectomy, but declined by 50% at 48 h post-orchiectomy. Of the two bound nonhistone protein kinase activities, that eluting at 0.25–0.30 M (NH4)2SO4 was reduced by over 80% within 24 h post-orchiectomy, whereas that eluting at 0.15–0.20 M increased by 100% at 24 h and 200% at 48 h post-orchiectomy. These data demonstrate a differential response of prostatic nuclear-associated protein kinases to androgen deprivation.

The study of kinases and their role in cellular regulation continues to expand as the human genome is sequenced and new kinases are identified as expression products of newly discovered genes. Reagents and assay systems that allow for sensitive, accurate, and high-throughput analysis of both purified kinases as well as crude extracts will enhance the characterization of these important cellular components and will speed the identification of appropriate therapeutic targets and the development of new and more effective treatments.

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinases (MAPKs), is essential for cellular proliferation and differentiation, and thus there exists great interest to develop specific and selective inhibitors of this enzyme. Whereas small molecule inhibitors PD098095 and U0126 have been used to study MAPK/ERK kinase (MEK), their target selectivity has been questioned recently. The cross-reactivity of ATP-directed inhibitors with other protein kinases prompted us to develop structure-based selective peptide inhibitors of ERK activation. Based on a MEK1-derived peptide, we developed inhibitors of ERK activation in vitro and in vivo. The inclusion of either an alkyl moiety or a membrane-translocating peptide sequence facilitated the cellular uptake of the peptide inhibitor and prevented ERK activation in 4-phorbol 12-myristate 13-acetate-stimulated NIH 3T3 cells or nerve growth factor-treated PC12 cells in a concentration-dependent manner. In addition, cell-permeable peptides inhibited ERK-mediated activation of the transcriptional activity of ELK1. The peptides did not have an inhibitory effect on the activity of two other closely related classes of MAPKs, c-Jun amino-terminal kinase or p38 protein kinase. Thus, these peptides may serve as valuable tools for investigating ERK activation and for selective investigation of ERK-mediated responses. With the knowledge of other kinase interacting domains, it would be possible to design cell-permeable inhibitors for investigating diverse cellular signaling mechanisms and for possible therapeutic applications.

The covalent modification of histone proteins, DNA and RNA by Fe(II)/2-oxoglutarate-dependent dioxygenases are key to the modulation of biological processes such as epigenetics, hypoxic signaling and DNA/RNA repair. Of these, JumonjiC domain-containing histone lysine demethylases (JMJCs), the ten-eleven-translocation (TET) DNA dioxygenases, the ALKB DNA/RNA hydroxylases and the prolyl hydroxylases EGLN1-3 have generated increased interest as potential drug targets for the treatment of a number of pathological conditions, including cancer. Since succinate is a common product to all Fe(II)/2-oxoglutarate-dependent dioxygenases, we determined the substrate specificities, the apparent kinetic constants for several JMJCs and members of the dioxygenase superfamily (i.e.: TET1, EGLN1, ALKBH3 and FTO), as well as inhibition profiles of reported inhibitors using a novel bioluminescent and homogenous succinate detection assay. Our results demonstrate that succinate detection is a useful strategy for the characterization of multiple Fe(II)/2-oxoglutarate-dependent dioxygenases with distinct substrate requirements, enabling the investigation of a large number of enzymes that cannot be evaluated in a miniaturized or high-throughput manner with the methods currently available. Citation Format: Juliano Alves, Said Goueli, Hicham Zegzouti. In vitro characterization of several enzymes of the Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily using a universal succinate detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1399. doi:10.1158/1538-7445.AM2017-1399