Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma.

BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Mutant IDH Inhibits IFNγ-TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma.

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate-dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate-mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell-autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell-specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ-TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. SIGNIFICANCE: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587.

Screening with the activated protein C resistance assay yields significant savings in a patient population with low prevalence of factor V leiden.

Testing for factor V Leiden can be performed with a molecular assay or a test for activated protein C resistance. We noted that physicians in our institution tended to order the molecular test 80% of the time, but the prevalence of the mutation in our patient population was less than 10%. Consequently, we decided to introduce the activated protein C resistance assay in house and consistently use it for screening before the more expensive genetic test and to negotiate a discounted charge for the latter at a reference laboratory. After 6 years since these interventions began, the prevalence of an abnormal screening test result remained low (202/2,475 [8.2%]), even among white patients (10.9%). With this simple approach, the cost to test patients for factor V Leiden decreased by more than 90%, while the productivity of our laboratory increased by the introduction of a high-volume, fully automated assay.

An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structure.

BACKGROUND: A strength of Drosophila as a model system is its utility as a tool to screen for novel regulators of various functional and developmental processes. However, the utility of Drosophila as a screening tool is dependent on the speed and simplicity of the assay used. METHODS: Here, we use larval locomotion as an assay to identify novel regulators of skeletal muscle function. We combined this assay with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. The data from the screen were supported with characterization of the muscle pattern in embryos and larvae that had disrupted expression of the strongest hit from the screen. RESULTS: With this assay, we showed that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function, illustrating that mechanisms that reduce muscle function exist and that the larval locomotion assay is sufficiently quantitative to identify conditions that both increase and decrease muscle function. We extended the data from this screen and tested the mechanism by which the strongest hit, fascin, impacted muscle function. Compared to controls, animals in which fascin expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against fascin only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. CONCLUSIONS: These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development.

Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1alpha subunit and a constititutively expressed HIF-1beta subunit. Under hypoxic conditions, the HIF-1alpha subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1alpha-HIF-1beta heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl(2). Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1alpha and HIF-1-dependent transcription induced by hypoxia or CoCl(2). Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl(2), while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1alpha stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1alpha fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1alpha stabilization by CoCl(2). These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.

Emery-Dreifuss muscular dystrophy-linked genes and centronuclear myopathy-linked genes regulate myonuclear movement by distinct mechanisms.

Muscle cells are a syncytium in which the many nuclei are positioned to maximize the distance between adjacent nuclei. Although mispositioned nuclei are correlated with many muscle disorders, it is not known whether this common phenotype is the result of a common mechanism. To answer this question, we disrupted the expression of genes linked to Emery-Dreifuss muscular dystrophy (EDMD) and centronuclear myopathy (CNM) in Drosophila and evaluated the position of the nuclei. We found that the genes linked to EDMD and CNM were each necessary to properly position nuclei. However, the specific phenotypes were different. EDMD-linked genes were necessary for the initial separation of nuclei into distinct clusters, suggesting that these factors relieve interactions between nuclei. CNM-linked genes were necessary to maintain the nuclei within clusters as they moved toward the muscle ends, suggesting that these factors were necessary to maintain interactions between nuclei. Together these data suggest that nuclear position is disrupted by distinct mechanisms in EDMD and CNM.

The ligand-independent translocation assay: an enabling technology for screening orphan G protein-coupled receptors by arrestin recruitment.

Finding natural and/or synthetic ligands that activate orphan G protein-coupled receptors (oGPCRs) is a major focus in current drug discovery efforts. Transfluor is a cell-based GPCR screening platform that utilizes an arrestin-green fluorescent protein conjugate (arrestin-GFP) to detect ligand interactions with GPCRs. The assay is ideally suited for oGPCRs because binding of arrestin-GFP to activated receptors is independent of the interacting G protein. Before embarking on a high-throughput screen, it is important to know that the target oGPCR can actually bind arrestin-GFP. This information was thought to be inaccessible, however, as arrestin-GFP recruitment is an agonist-driven process. This chapter describes an assay that enables GPCRs to be validated in Transfluor in the absence of ligand. This assay, termed the ligand-independent translocation (LITe) assay, utilizes a modified G protein-coupled receptor kinase to bypass the requirement of ligand for initiating arrestin-GFP translocation. Using the LITe assay, one can determine if an oGPCR binds arrestin-GFP and if the response is quantifiable by high-content screening instruments. In addition, the assay expedites the development and identification of oGPCR stable cell lines with the best Transfluor properties. In this way, the assay provides criteria for selecting the best oGPCRs to move forward for a Transfluor screening campaign. Moreover, the assay can be used for quality control purposes during the orphan receptor screen itself by providing positive translocation responses for calculation of Z prime values. In summary, the LITe assay is a powerful new technology that enables a faster and more reliable path forward in the deorphanization of GPCRs with Transfluor.

High-content screening of known G protein-coupled receptors by arrestin translocation.

G protein-coupled receptors (GPCRs) have proven to be one of the most successful target classes for drug discovery. Accordingly, many assays are available to screen GPCRs, including radioactive-binding assays, second messenger signaling assays, and downstream reporter assays. One of the more novel approaches is the Transfluor technology, a cell-based assay that uses a detectable tag on a cytosolic protein, called arrestin, that is involved in the desensitization or inactivation of GPCRs. Monitoring the translocation of GFP-tagged arrestin from the cytosol to activated GPCRs at the plasma membrane measures the pharmacological effect of test compounds that bind the receptor target. Moreover, the Transfluor assay provides further, high-content information on the test compound itself and its effects on cell processes due to the fluorescent imaging of whole cells used in this screen. Screening known GPCRs with Transfluor against large compound libraries is best accomplished in cell lines stably expressing an optimum level of the target receptor. This chapter describes how to generate a clonal cell line stably expressing the known GPCR with suitable Transfluor properties. It then describes the steps involved in performing a Transfluor screen and discusses high content data resulting from the screen.

Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.

The authors demonstrate the use of a simple, universal G-protein-coupled receptor (GPCR) assay to screen for agonists for a specific GPCR. Cells stably expressing a green fluorescent protein (GFP)-labeled beta-arrestin fusion protein and the vasopressin V2 receptor (V2R) were used in a high-content screening (HCS) assay to screen a small peptide library for V2R agonists. Cells were treated with the peptides at a final concentration of 500 nM for 30 min. Agonist stimulation causes V2R internalization into endosomes. GFP-beta-arrestin remains associated with the V2R in endosomes, resulting in a fluorescent pattern of intracellular spots. Assay plates were automatically imaged and quantitatively analyzed using an HCS imaging platform and a fast turnkey image analysis application optimized for detection of receptor activation and intracellular spots. Hits were further evaluated to determine their potency. The combination of unique biology, automated high-content analysis, and a powerful means of validating hits results in better leads.

The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.

G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V(2) receptor (V(2)R), which binds arrestin with high affinity, and the beta(2)-adrenergic receptor (beta(2)AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V(2)R or the beta(2)AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V(2)R and beta(2)AR, respectively. EC(50) values agreed closely with K(d) values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC(50) doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z' values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.

The effect of lupus anticoagulant in the second-generation assay for activated protein C resistance.

The activated protein C resistance (APCR) assay is the test of choice to screen for factor V Leiden. We evaluated the effect of lupus anticoagulant on the baseline clotting time of the second-generation APCR assay with plasma samples from 54 patients to determine whether a falsely low APCR ratio could be predicted. We also assessed whether a modification of the assay could make it more reliable in the presence of strong lupus anticoagulants. Of 54 plasma samples, 5 yielded a false-positive APCR ratio, and all 5 had a prolonged baseline clotting time. Further dilution (1:40) of the plasma samples in factor V-deficient plasma led to correction of the APCR ratio and did not affect the sensitivity of the test for factor V Leiden. Our data support that the baseline clotting time is a good predictor of a false-positive APCR test result and should be checked before calculating the ratio. The modified APCR assay reliably identified the false-positive ratios and could be used to screen for factor V Leiden in samples with strong lupus anticoagulant.

Assessment of oral health needs and barriers to care in a Gullah community: Hollywood smiles.

OBJECTIVES: To assess the oral health (OH) needs and barriers to OH care in Gullah African American communities. METHODS: A community advisory board (CAB) was formed to guide the research study. Five focus groups (n = 27 participants) were conducted to explore the OH needs/barriers. Participants completed demographic surveys and participated in discussions facilitated by open-ended questions. All sessions were audio-recorded, transcribed and analyzed using NVivo8. RESULTS: Facilitators of OH included positive experiences and modeling. Fear and access to care were the most cited barriers. Tooth extraction was the dental treatment of choice. Intervention recommendations included improving clinic access, using the churches to socially influence receipt of OH care, providing group educational sessions with OH specialists, and having local "lay people" to provide support and help to navigate OH care systems. CONCLUSIONS: The design of a multilevel, culturally and locally relevant intervention may lead to a decrease in OH disparities in Gullah communities.

Desensitization of human CRF2(a) receptor signaling governed by agonist potency and ßarrestin2 recruitment.

The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong ßarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1µM) generated only weak ßarrestin2 recruitment. ßarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the ßarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a ßarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, ßarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response.

Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and beta-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses.

The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF(1)) receptor phosphorylation is required for beta-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to beta-arrestin recruitment of motifs in the CRF(1) receptor carboxyl terminus and third intracellular loop. beta-Arrestin-2 translocated significantly more rapidly than beta-arrestin-1 to agonist-activated membrane CRF(1) receptors in multiple cell lines. Although CRF(1) receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF(1) receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF(1) receptor internalization. To investigate molecular determinants mediating the robust beta-arrestin-2-CRF(1) receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF(1) receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired beta-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF(1) receptor phosphorylation but did not alter beta-arrestin-2 recruitment. Phosphorylation was abolished in a CRF(1) receptor possessing both mutations. Surprisingly, this mutant still recruited beta-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF(1) receptors. Our data reveal the involvement of at least the following two distinct receptor regions in beta-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF(1) receptor conformation. Deficient beta-arrestin-2-CRF(1) receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF(1) receptor signaling.

Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.

BACKGROUND: A cell-based assay system (Transfluor) has been developed for measurement of G-protein coupled receptor (GPCR) activity by using cells transfected to express a fusion protein of arrestin plus green fluorescent protein (GFP) and the target GPCR. Upon agonist stimulation, the arrestin-GFP translocates to and binds the activated GPCR at the plasma membrane. The receptor/arrestin-GFP complexes then localize in clathrin-coated pits and/or intracellular vesicles. This redistribution of arrestin-GFP into condensed fluorescent spots is useful for visually monitoring the active status of GPCRs and its quantitation is possible with certain types of digital image analysis systems. METHODS: We designed two lines of image processing algorithms to carry out quantitative measurement of the arrestin-GFP movement on an inverted version of laser scanning cytometry (iCyte) as an imaging platform. We used a cell line expressing arrestin-GFP and the wild-type beta2-adrenergic receptor or a modified version of this receptor with enhanced affinity for arrestin. Each cell line was challenged with various concentrations of agonist. RESULTS: A dose-dependent signal was measured and half-maximal effective concentration values were obtained that agreed well with results determined by other methods previously reported. CONCLUSIONS: The results indicate that the combination of Transfluor, iCyte, and our algorithms is suitable for robust and pharmacologically relevant GPCR ligand exploration.