L-DOPA Is an Endogenous Ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation

Retinoblastoma treatment: impact of the glycolytic inhibitor 2-deoxy-d-glucose on molecular genomics expression in LHBETATAG retinal tumors

Purpose: The purpose of this study was to evaluate the effect of 2-deoxy-D-glucose (2-DG) on the spatial distribution of the genetic expression of key elements involved in angiogenesis, hypoxia, cellular metabolism, and apoptosis in LHBETATAG retinal tumors. Methods: The right eye of each LHBETATAG transgenic mouse (n = 24) was treated with either two or six subconjunctival injections of 2-DG (500 mg/kg) or saline control at 16 weeks of age. A gene expression array analysis was performed on five different intratumoral regions (apex, center, base, anterior-lateral, and posterior-lateral) using Affymetrix GeneChip Mouse Gene 1.0 ST arrays. To test for treatment effects of each probe within each region, a two-way analysis of variance was used. Results: Significant differences between treatment groups (ie, 0, 2, and 6 injections) were found as well as differences among the five retinal tumor regions evaluated (P \u3c 0.01). More than 100 genes were observed to be dysregulated by ≥2-fold difference in expression between the three treatment groups, and their dysregulation varied across the five regions assayed. Several genes involved in pathways important for tumor cell growth (ie, angiogenesis, hypoxia, cellular metabolism, and apoptosis) were identified. Conclusions: 2-DG was found to significantly alter the gene expression in LHBETATAG retinal tumor cells according to their location within the tumor as well as the treatment schedule. 2-DG’s effects on genetic expression found here correlate with previous reported results on varied processes involved in its in vitro and in vivo activity in inhibiting tumor cell growth

Intravitreal injection analysis at the Bascom Palmer Eye Institute: evaluation of clinical indications for the treatment and incidence rates of endophthalmitis

To report the incidence of endophthalmitis, in addition to its clinical and microbiological aspects, after intravitreal injection of vascular-targeting agents. A retrospective review of a consecutive series of 10,142 intravitreal injections of vascular targeting agents (bevacizumab, ranibizumab, triamcinolone acetonide, and preservative-free triamcinolone acetonide) between June 1, 2007 and January 31, 2010, performed by a single service (TGM) at the Bascom Palmer Eye Institute. One case of clinically-suspected endophthalmitis was identified out of a total of 10,142 injections (0.009%), presenting within three days of injection of bevacizumab. The case was culture-positive for Staphylococcus epidermidis. Final visual acuity was 20/40 after pars plana vitrectomy surgery. In this series, the incidence of culture-positive endophthalmitis after intravitreal injection of vascular agents in an outpatient setting was very low. We believe that following a standardized injection protocol, adherence to sterile techniques and proper patient follow-up are determining factors for low incidence rates

Abstract 4262: PRAME as a biomarker for a new molecular subclass of uveal melanoma

Abstract Introduction: Uveal melanoma is the most common primary intraocular tumor of the eye and has a propensity for fatal hematogenous metastasis. Gene expression profiling is routinely used in clinic to classify uveal melanoma into prognostic subgroups: Class 1 (low metastatic risk) and Class 2 (high metastatic risk). Approximately 40% of Class 2 patients metastasize, which accounts for 85% of metastasis. The remaining 15% of patients that metastasize are Class 1 based on gene expression profiling, however this study demonstrates that the Class 1 patients that metastasize (Class1met+) can be defined as a new molecular subclass, distinct from Class 1 patients that do not metastasize (Class1met-) and Class 2 patients. Methods: Tumor samples from Class1met+ and Class1met- patients with at least 1 year of follow-up underwent histopathologic, cytogenetic and transcriptomic analyses. Results: In a comparison of Class1met+ and Class1met- tumors, the only significant difference was a larger mean basal tumor diameter in Class1met+ tumors. Cytogenetic analyses revealed more extensive copy number gains and losses in Class1met+ tumors. The most significant differentiating features in Class1met+ tumors included 1q gain, 6p gain, 6q loss, and 8q gain. Using principal component analysis, the Class1met+ tumors clustered separately from the Class1met- tumors, indicating the existence of significant transcriptomic differences between the two subgroups. Strikingly, the most highly differentially expressed gene in Class1met+ tumors was preferentially expressed antigen in melanoma (PRAME); mean mRNA expression of PRAME in Class1met+ tumors was 61-fold higher than in Class1met- tumors. Discussion: We define a new subtype of uveal melanoma and demonstrate that gene expression of PRAME can distinguish between Class1met+ patients and Class1met- patients. Citation Format: Matthew G. Field, Christina L. Decatur, J. William Harbour. PRAME as a biomarker for a new molecular subclass of uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4262. doi:10.1158/1538-7445.AM2015-4262</jats:p

Abstract 2332: The glucose analog 2-DG selectively inhibits AKT and ERK in endothelial cells via interference of N-linked glycosylation and induces endothelial cell apoptosis in vitro and in vivo

Abstract BACKGROUND: As every step in tumor angiogenesis is an energy consuming process, targeting endothelial (EC) glucose metabolism is a rational strategy for angiogenesis inhibition. We recently showed that interference with endothelial glucose metabolism by the glucose analog 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis in vitro and in vivo, at concentrations lower than those required to induce tumor cell cytotoxicity. The objectives of the current study are to further understand the basis for EC sensitivity to 2-DG in vitro and in vivo. METHODS: Time dependent effects of 2-DG were studied on EC tube formation at 4, 8 and 18 hrs. Apoptosis was assessed by the TUNEL assay at 24, 48 and 72 hours. The effects of low antiangiogenic concentrations (0.6 mM) of 2-DG on ERK, AKT and mTOR pathways were assessed in endothelial (HUVEC) and cancer (MDA-MB-231, HT-29, 786-0) cells by western blot. The in vivo effects of intraocular 2-DG administration on newly forming (CD-105) and total (lectin) tumor vessels were evaluated in the LHBETATAG retinoblastoma model. In vivo EC apoptosis after 2-DG treatment was assessed by TUNEL/CD31 staining of treated tumors. RESULTS: Inhibition of HUVEC capillary formation by 2-DG occurred as early as 8 hours after treatment, and the effects were clear at 18 hours compared to controls. EC apoptosis was non-significantly increased at 24 hours, and was prominent at 48 and 72 hours. Antiangiogenic 2-DG concentrations (0.6 mM) significantly inhibited phosphorylation of Akt473, S6 and Erk at 24 hours. On the other hand, 2-DG did not affect these pathways in MDA-MB-231 or HT-29 cells, while they occurred at a lower level in 786-0 cells. Mannose successfully reversed AKT, S6 and ERK inhibition only in HUVECs, but not in 786-0 cells, suggesting that the EC selective inhibition of ERK and AKT are mediated by inhibition of N-linked glycosylation, while other mechanisms mediate the effects in cancer cells. Importantly, these effects were 2-DG specific, as other glucose analogs (FDG) and the glycolytic inhibitor oxamate did not significantly affect ERK or AKT. Intraocular 2-DG administration decreased tumor microvasculature, and importantly, induced EC apoptosis, as demonstrated by TUNEL+/CD 31 co-localization. CONCLUSIONS: Our data indicate that in angiogenic ECs, 2-DG's inhibition of N-linked glycosylation targets critical proliferation and survival pathways. 2-DG mediated Inhibition of AKT and ERK pathways may explain the significant endothelial sensitivity cytotoxic and pro-apoptotic effects of 2-DG in vitro and in vivo, and further support targeting EC glucose metabolism as a viable strategy for angiogenesis inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2332. doi:1538-7445.AM2012-2332</jats:p

Abstract 4348: Methylation analysis of uveal melanoma reveals definitive patterns in tumors harboring BAP1 mutations

Abstract Introduction: Uveal melanoma (UM) is the most common primary intraocular malignancy and can be classified by gene expression profiling into two distinct molecular classes that correspond to metastatic risk: Class 1 (low risk) and Class 2 (high risk). The less aggressive Class 1 UMs express a more differentiated phenotype and exhibit numerous characteristics of normal uveal melanocytes, while the metastasizing Class 2 UMs display loss of melanocytic differentiation and have acquired a primitive, stem-like phenotype. The majority of Class 2 UMs harbor loss of chromosome 3 in addition to inactivating mutations of the tumor suppressor BAP1, which also catalyzes ubiquitin removal from histone H2A and modulates gene expression. Histone modifications have been shown to be associated with DNA methylation and this study investigates the global methylation and gene expression changes in BAP1 mutant tumors. Methods: RNA-Sequencing data and Illumina Human Methyl 450K BeadChip Array data obtained from 92 primary uveal melanoma tumors was analyzed using optimized pipelines. Results: This analysis revealed that the more aggressive Class 2 tumors exhibit a distinct pattern of hypermethylation and silencing of developmental genes involved in neural crest migration and differentiation. Chromosomal regions that were significantly enriched for hypermethylated genes with decreased gene expression in Class 2 tumors included chromosome 3p14-26, 3q12-29 and 8p12-22, whereas the only significantly enriched region for genes that were hypomethylated with increased gene expression in Class 2 tumors was chromosome 8q22-24. Additionally, BAP1 itself was differentially hypermethylated in Class 2 UMs, suggesting that it may regulate its own transcription. Conclusions: Class 2 UMs harboring BAP1 mutations displayed distinct regions of differential hypermethylation in comparison to Class 1 UMs. In these tumors, the most significantly hypermethylated regions occurred on chromosome 3 and in regions that were enriched for genes encoding neural crest guidance cue proteins that regulate homing, migration and invasion. These findings suggest that BAP1 mutations are associated with marked reorganization of the DNA methylome, providing new insights into the pathophysiology of UM and suggesting that methylation may serve as a diagnostic to further stratify metastatic risk. Furthermore, these findings may open new opportunities for targeted therapy of Class 2 UMs. Citation Format: Michael Durante, Matthew Field, Stefan Kurtenbach, Parker Bussies, Christina Decatur, J. William Harbour. Methylation analysis of uveal melanoma reveals definitive patterns in tumors harboring BAP1 mutations [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 4348. doi:10.1158/1538-7445.AM2017-4348</jats:p

Abstract 2359: Using the glycolytic inhibitor 2-fluorodeoxy-D-glucose, a novel glycolytic inhibitor approach to target the chemoresistant, hypoxic cell population in advanced LHBETATAG retinal tumors

Abstract Purpose: The aim of the current study is to assess the impact of 2-fluorodeoxy-D-glucose on tumor burden and hypoxia in the LHBETATAG retinal tumors. Methods: The study protocol was approved by the University of Miami Institutional Animal Care and Use Review Board Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 17-week-old (n=54) LHBETATAG transgenic mice were treated with 2-fluorodeoxy-D-glucose or saline control. These animals received three different treatments. They were treated: (1) with one injection and sacrificed at one day post-treatment, (2) with one injection and sacrificed at one week post-treatment, or (3) twice a week for three weeks and sacrificed at one day post-last injection. At the time of enucleation, all eye samples were snap frozen and analyzed for tumor burden and hypoxia using immunohistochemical techniques. Average densities of the different groups were statistically analyzed using ANOVA. Results were considered significant if p≤ 0.05. Results: There was no apparent toxicity associated with 2-fluorodeoxy-D-glucose treatment. There was a significant reduction in tumor burden following treatment with 2-fluorodeoxy-D-glucose at 1 day (86%) and 3 weeks (63%) post-treatment (p≤0.05). There was no reduction of tumor burden observed when mice were treated with 1 injection and eyes harvested at 1 week post-treament (2%, p=0.0640). There was a significant reduction of hypoxia areas following treatment with 2-fluorodeoxy-D-glucose at 1 day (100%) and 3 weeks (75%) post-treatment (p≤0.05). There was an increase in hypoxia of 12% following treatment at 1 week post-injection, but this increase was not statistically significant. Conclusions: 2-FDG significantly reduces tumor burden and tumor hypoxia following a single injection, with continued efficacy following repeated injections for 3 weeks. 2-FDG treatment is efficacious in murine retinoblastoma tumors and may enhance tumor control when combined with other therapies. 2-FDG appears to target hypoxic cells, a population that has been resistant to chemotherapy and radiation. Additionally, 2-FDG is commonly used in medical imaging and does not pose significant toxicities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2359. doi:1538-7445.AM2012-2359</jats:p

Abstract 794: Potential role of DLL4 in uveal melanoma vascular mimicry

Abstract Uveal melanoma is the most common malignancy of the eye. Thanks to gene array analysis it is possible to classify uveal melanoma in Class 1 (low metastasis risk) and Class 2 (high metastasis risk) tumor. This classification will ultimately determine the tumor treatment, risk of metastasis and patient surveillance. Progression to metastasis remains by far the greatest problem in uveal melanoma and is associated with loss of BAP1 tumor suppressor. Bioinformatic analyses of RNA-Seq indicated that pro-angiogenic genes such as DLL4, VEGFA, VEGFC and HIF1a are overexpressed in Class 2 compared to Class 1 uveal melanoma while angiogenic inhibitors such as ZFP36L1, HIF1AN, VEGFB, VHL and HIF3A are downregulated. Further, we found that DLL4 is among the 5 most highly overexpressed genes associated with BAP1 loss in clinical specimens and in uveal melanoma cell lines induced to deplete BAP1. DLL4 is a Notch ligand known to regulate endothelial cells, bone marrow endothelial cell progenitors and angiogenesis. We hypothesize that DLL4 contributes to vascular mimicry in uveal melanoma. To test this hypothesis, we will test uveal melanoma cell lines induced to deplete BAP1 using shRNA in cell culture-based and in vivo models. The results of this research have the potential to elucidate the mechanism by which vascular mimicry occurs in uveal melanoma. Citation Format: Julia Escandon, Matthew G. Field, Stefan Kurtenbach, Jeffim Kuznetzov, Christina L. Decatur, J William Harbour. Potential role of DLL4 in uveal melanoma vascular mimicry [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 794. doi:10.1158/1538-7445.AM2017-794</jats:p

PieParty: visualizing cells from scRNA-seq data as pie charts

Single-cell RNA sequencing (scRNA-seq) has been a transformative technology in many research fields. Dimensional reduction techniques such as UMAP and tSNE are used to visualize scRNA-seq data in two or three dimensions for cells to be clustered in biologically meaningful ways. Subsequently, gene expression is frequently mapped onto these plots to show the distribution of gene expression across the plots, for instance to distinguish cell types. However, plotting each cell with only a single color leads to repetitive and unintuitive representations. Here, we present PieParty, which allows scRNA-seq data to be plotted such that every cell is represented as a pie chart, and every slice in the pie charts corresponds to the gene expression of a single gene. This allows for the simultaneous visualization of the expression of multiple genes and gene networks. The resulting figures are information dense, space efficient, and highly intuitive. PieParty is publicly available on GitHub at https://github.com/harbourlab/PieParty