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Galpha and MEK1/2 inhibition in combination with Baf A1 and CQ in MP41 cells

Response of uveal melanoma cell lines to MEK1/2 inhibition

Trametinib in combination with macropinocytosis or mitophagy inhibition

Figure S4. Primary tumor incidence, growth, survival and metastasis in Ptenf/f vs. Arf6Q67L mice. (a) Increased tumor incidence (% = # of mice that developed a tumor / # TVA positive, injected mice) in Ptenf/f vs. Arf6Q67L cohorts. Fisher's exact test, two-tailed, α

Figure S7. Low expression of ARF GAP genes correlates with reduced overall survival. (a) Individual gene plots of four ARF6 GAPs (ACAP1, ADAP1, ARAP2, SMAP2), an ARF1/ARF5 GAP (AGAP2) and one predicted ARF GAP (AGFG2). n = 439 patients. (b) Individual gene plots in Stage III specific cohort, n=142 patients. Log-rank (Mantel-Cox) test.

Figure S6. ARF6-GDP inhibits PI3K-AKT activation and is necessary for AKT activation. (a-b) Quantification of western blots shown in Figure 5c-d. Reproducible reduction in pAKT by Myc-ARF6T27N over expression in A375 cells (n=4), A2058 cells (n=4), and HEY-T30 cells (n=5). Reduced pAKT with siRNA knockdown of ARF6 in SK-MEL-147 (n=5), A2058 (n=4), and CACL (n=4) and HEY-T30 (n=6). Graphs show individual data points normalized to control along with geometric means, 95% CI, ratio paired t test. (c) Phosphokinase array images (left) and quantification of signal by densitometry (right, histogram), n=1. Expression of ARF6T27N in A2058 cells preferentially reduced pAKT and the AKT substrate PRAS40 over most other proteins in the array.

Bioluminescent images of hepatic tumors

YAP requirement in MP41 cells

Supplementary Materials and Methods

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients.Significance:These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.

Figure S1. Detection of HA-tagged ARF6Q67L in BrafCA;Cdkn2af/f melanoma. a) Anti-HA immunostain of FFPE sections of primary tumors with variable levels of detection of ARF6Q67L -HA. The strongest staining was observed in tumors with the highest level of mRNA detected by qRT-PCR (b). Absent staining of FFPE tumors was observed in tumors with low (6456) and undetectable (6457) levels of mRNA by qRT-PCR of frozen tumors. Scale bars = 20mm, 400x magnification. Control = BrafCA;Cdkn2af/f (Cre-only injection). b) qRT-PCR of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see Figures 1g and S3). With rare exception, positive tumors demonstrated Arf6 Q67L-HA expression levels lower than Gapdh.

Table S1: ARF6 pathways and related genes.

Purpose:Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma.Experimental Design:We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.Results:Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.Conclusions:These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

YAP activation in OMM1 cells

Figure S5: ARF6-GTP induces morphologic changes in mouse melanoma cells. Bright field images (100x magnification) of primary mouse melanoma cell lines derived from control BrafCA;Cdkn2af/f (5588) or BrafCA;Cdkn2af/f + Arf6Q67L (6431, 6455) mice. ARF6-GTP induces elongation/spindling of tumor cells relative to the more polygonal-shaped controls. Scale bars = 400um.

Immunofluorescence analysis of autophagy in OMM2.5 cells with or without trametinib

Validation of hepatic tumor model

Figure S3. Arf6Q67L-HA expression in tumors from BrafCA;Cdkn2af/f;Ptenf/f mice. qRT-PCR detection of Arf6 Q67L-HA from fresh, frozen primary tumor fragments. Bars represent the absolute difference in crossing threshold (Ct) between Arf6 Q67L-HA and Gapdh. The Ct threshold that defines detection was established above the background signal for controls in both the BrafCA;Cdkn2af/f and the BrafCA;Cdkn2af/f ;Ptenf/f cohorts (see also Figure S1). All mouse tumor negative by qRT-PCR showed detectable HA-tagged ARF6Q67L by immunohistochemistry (not shown, see Figure S1 for example).

It is necessary to quantitatively determine substituent effects to accurately elucidate reaction mechanisms in the field of organic chemistry. This paper reports that the molecular electrostatic potential (MESP) can be used as a general and versatile measure for the substituent effects in various chemical reactions by performing extensive density functional theory (DFT) calculations for more than 400 molecules, followed by statistical analyses. We observed a robust and linear correlation between the electrostatic potential and the substituent parameters for various cases of reactive systems, regardless of the DFT functionals, basis sets, and solvation models used. In addition, we statistically analysed the normality of the residuals from the linear regression to demonstrate that strong linear relationships hold universally, which indicates that the electrostatic potential can serve as a physically meaningful quantity for the predictive estimation of substituent effects. In contrast, conventionally used methods based on the charge deviation in the aromatic carbons, as computed using various charge analysis methods, (

Heterostructures of complex transition metal oxides are known to induce extraordinary emergent quantum states that arise from broken symmetry and other discontinuities at interfaces. Here we report the emergence of unusual, thickness-dependent properties in ultrathin CaRuO3 films by insertion of a single isovalent SrO layer (referred to as δ-doping). While bulk CaRuO3 is metallic and nonmagnetic, films thinner than or equal to ~15-unit cells (u.c.) are insulating though still nonmagnetic. However, δ-doping to middle of such CaRuO3 films induces an insulator-to-metal transition and unusual ferromagnetism with strong magnetoresistive behavior. Atomically resolved imaging and density-functional-theory calculations reveal that the whole δ-doped film preserves the bulk-CaRuO3 orthorhombic structure, while appreciable structural and electronic changes are highly localized near the SrO layer. The results highlight delicate nature of magnetic instability in CaRuO3 and subtle effects that can alter it, especially the role of A-site cation in electronic and magnetic structure additional to lattice distortion in ruthenates. It also provides a practical approach to engineer material systems via highly localized modifications in their structure and composition that may offer new routes to the design of oxide electronics.

Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma.

Heterostructures of complex transition metal oxides are known to induce extraordinary emergent quantum states that arise from broken symmetry and other discontinuities at interfaces. Here we report the emergence of unusual, thickness-dependent properties in ultrathin CaRuO3 films by unique insertion of a single isovalent SrO layer (referred to as “δ-doping”). While bulk CaRuO3 is metallic and nonmagnetic, films thinner than or equal to ~15-unit cells (u.c.) are insulating though still nonmagnetic. However, δ-doping to middle of such CaRuO3 films induces an insulator-to-metal transition and unusual ferromagnetism with strong magnetoresistive behavior. The ferromagnetic ground state does not show fully long-range order. Atomically resolved imaging and density-functional-theory calculations reveal that the whole δ-doped film preserves the bulk-CaRuO3 orthorhombic structure, while appreciable structural and electronic changes are highly localized near the SrO layer. The results highlight delicate nature of magnetic ordering in CaRuO3 and subtle effects that can alter it, especially the role of A-site cation in electronic and magnetic structure additional to lattice distortion in ruthenates. It also provides a practical approach to engineer material systems via highly localized modifications in their structure and composition that may offer new routes to the design of oxide electronics.

Purpose:Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma.Experimental Design:We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.Results:Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.Conclusions:These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.

Urocortin 3 (UCN3) is a peptide hormone expressed in pancreatic islets of Langerhans of both human alpha and human beta cells and solely in murine beta cells. UCN3 signaling acts locally within the islet to activate its cognate receptor, corticotropin releasing hormone receptor 2 (CRHR2), which is expressed by delta cells, to potentiate somatostatin (SST) negative feedback to reduce islet cell hormone output. The functional importance of UCN3 signaling in the islet is to modulate the amount of SST tone allowing for finely tuned regulation of insulin and glucagon secretion. UCN3 signaling is a hallmark of functional beta cell maturation, increasing the beta cell glucose threshold for insulin secretion. In doing so, UCN3 plays a relevant functional role in accurately maintaining blood glucose homeostasis. Additionally, UCN3 acts as an indicator of beta cell maturation and health, as UCN3 is not expressed in immature beta cells and is downregulated in dedifferentiated and dysfunctional beta cell states. Here, we review the mechanistic underpinnings of UCN3 signaling, its net effect on islet cell hormone output, as well as its value as a marker for beta cell maturation and functional status.

Uveal melanoma is the most common primary malignancy of the eye, and a number of discoveries in the last decade have led to a more thorough molecular characterization of this cancer. However, the prognosis remains dismal for patients with metastases, and there is an urgent need to identify treatments that are effective for this stage of disease. Animal models are important tools for preclinical studies of uveal melanoma. A variety of models exist, and they have specific advantages, disadvantages, and applications. In this review article, these differences are explored in detail, and ideas for new models that might overcome current challenges are proposed.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. Significance: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.

A unique dense network of multi-member-ring round hole defects is formed in monolayer WSe${}_{2}$ from the evolution of multivacancies by suitable control of a scanning focused electron beam, whereas the same process leads predominantly to chalcogen-vacancy line defect array in other trigonal-prismatic transition metal dichalcogenide (TMDC) monolayers. Density functional theory (DFT) calculations track the formation of the observed complex multivacancy structures and find that the underlying atomic-scale processes are quasi-thermodynamic, which elaborates the formation mechanism of dense round hole defects in WSe${}_{2}$ monolayers. The high-density round holes in WSe${}_{2}$ hold promise for novel applications such as atomic and molecular sieving.

The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT. To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin’s but not resveratrol’s anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.

Using first-principles calculations we have previously shown that placing graphene on a (111)-oriented perovskite $\mathrm{SrTi}{\mathrm{O}}_{3}$ (STO) surface provides a possible doping mechanism [D. Shin and A. A. Demkov, Phys. Rev. B 97, 075423 (2018)]. Further theoretical analysis presented here suggests that coupling of electrons in graphene to interfacial hybrid plasmon/optical modes via remote-phonon scattering may result in an effective attractive electron-electron interaction that, in turn, could lead to electron pairing and superconductivity. Specifically, we consider top-gated graphene supported by STO. Using the full dynamic polarizability within the random phase approximation for the entire system (including the hybrid modes arising from the coupling of the graphene plasmons to the optical phonons of the STO substrate and gate insulator), we estimate the superconducting transition temperature in the strong-coupling limit.

GNAQ and GNA11 (GNAQ/11) mutations are found in less than 2% of all melanoma, but more than 80% of uveal melanoma. Mutations in these Gα proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF->MEK1/2->ERK1/2) and YAP signaling. Metastatic uveal melanoma is refractory to all forms of pharmacologic treatment, such as FDA-approved targeted therapies inhibiting MEK1/2 (i.e. trametinib and binimetinib). We show that combining MEK1/2 inhibitors with 4-aminoquinoline antimalarials, chloroquine or hydroxychloroquine, resulted in synergistic and apoptosis-mediated cytotoxicity in GNAQ/11 mutant uveal melanoma cell lines. Interestingly, in contrast to our previous work in pancreatic and other RAS-driven cancers, the lysosomotropic role of chloroquine was not sufficient to promote cytotoxicity with MEK1/2 inhibitors, as neither lysosome inhibition with Bafilomycin A1 nor autophagy-specific and macropinocytosis-specific inhibition yielded enhanced cell death in combination with MEK1/2 inhibition. We then found that chloroquine prevented nuclear localization of the transcriptional coactivator, YAP, suggesting a novel mechanism of chloroquine. YAP inhibition combined with MEK1/2 inhibition enhanced cell death only in the presence of Bafilomycin A1. Gα-specific inhibition (inhibiting YAP and MAPK) combined with Bafilomycin A1 yielded similar results. This implies that the ability of chloroquine to inhibit both YAP signaling and lysosome function is required for promoting cell death in the presence of MEK1/2 inhibition. For in vivostudies, we utilized a hepatic colonization model using luciferized human metastatic uveal melanoma cell lines, OMM2.5 and OMM1. Daily treatment of trametinib with hydroxychloroquine in combination resulted in delayed tumor growth and increased overall survival compared to either treatment as monotherapy or chemotherapy. These findings were also recapitulated in an immunocompetent mouse model in which immortalized mouse melanocytes (Melan-A) with either a GNAQ or GNA11 activating mutation were implanted into syngeneic C57BL/6 mice. Our findings identify a novel mechanism of chloroquine and suggest a potentially effective strategy combining two FDA-approved drugs for the treatment of metastatic uveal melanoma. Citation Format: Amanda Truong, Michael Scherzer, Conan Kinsey, John Michael Sanchez, Jae Hyuk Yoo, Jackson Richards, Donghan Shin, Phaedra Ghazi, Michael Onken, Kendall Blumer, Shannon Odelberg, Martin McMahon. Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma [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 1890.

The electronic structure of Eu sesquioxide (Eu2O3) presents a significant challenge to the electronic structure theory due to the presence of correlated Eu semicore 4f electrons. The bandgap values do not agree between computational methods, and even experimentally, there are discrepancies between reports. Eu2O3 was grown epitaxially in a thin film form on n-type GaN (0001) by molecular beam epitaxy. The film was analyzed using UV and x-ray photoemission spectroscopies as well as inverse photoelectron spectroscopy in order to characterize both occupied and unoccupied states. Signatures of Eu2+ are detected after annealing in UHV or after exposure to air, which can be removed by subsequent O2 annealing. The sample reduction is shown to strongly affect the electronic structure. The bandgap of 4.3 eV, electron affinity of 2.2 eV, and band alignment to the substrate with a valence band offset of 0.2 eV for a stoichiometric Eu2O3 film were extracted from the measurements of the occupied and unoccupied electronic states. The electronic structure is interpreted in view of recent theoretical models, and the energy band alignment across the Eu2O3/GaN interface is discussed.

This paper addresses a novel technique of 3D clay printing using a robotic arm along with the architectural applicability of the proposed fabrication technique. In the initial phase of research, we selected the clay material to be used for printing in consideration of its viscosity and developed the clay extruder customized for the material. In the late phase, to verify how effective the robotic fabrication technique we propose is, we manufactured the freeform foam mold and tested multi-axis clay 3D printing on it by using three end-effectors consecutively: hotwire cutter, spindle, and clay extruder. Also, applying the proposed technique to a larger-scale ceramic structure consisting of nineteen panels, we checked the feasibility of fabricating actual ceramic building components with this technique. If this robotic clay 3D printing technique is improved more and fulfills the demands of the industry in future, it will enable cost-effective ceramic modular fabrication for a customized design in architecture.

Doping graphene layers presents a difficult practical and fundamental problem. We consider theoretically, the possibility of electrostatic doping of graphene by the intrinsic field of a polar substrate. By way of example, we perform density functional theory calculations for a graphene sheet placed on the (111)-oriented perovskite $\mathrm{SrTi}{\mathrm{O}}_{3}$ surface. We find that the Fermi surface moves well below the Dirac point of graphene, resulting simultaneously in a fast conducting channel in graphene, and a slow (large-effective-mass) channel at the oxide surface. Additionally, electrostatic gating may open a way to explore peculiar states that, through the ``no crossing,'' represent a hybrid carrier that exists simultaneously in both materials.

Introduction: Uveal melanoma is the most common primary ocular malignancy and there is currently no effective treatment for metastatic uveal melanoma largely because of the lack of understanding of the molecular mechanisms underlying this cancer. Although activating oncogenic mutations in GNAQ and GNA11 are present in 95% of uveal melanoma tumors, virtually nothing is known about the molecular mechanisms that drive oncogenesis in the remaining 5% of uveal melanomas that possess only wild type Gαq alleles and the molecular and cellular mechanisms that promote metastasis have not yet been discovered. This latter deficit in knowledge is extremely vexing, given that metastatic disease causes death in most uveal melanoma patients. We have recently shown that ADP-ribosylation factor 6 (ARF6), a small GTPase, is an immediate downstream effector of oncogenic Gαq and controls the entire major signaling pathways known to drive Gαq-mediated tumor formation and growth in uveal melanoma cells that have an activating mutation in Gαq. Moreover, in cutaneous melanoma, stimulation of the receptor by WNT5A activates ARF6 and controls metastasis via the release of β-catenin from N-cadherin and β-catenin's subsequent nuclear transport. Previous studies have also demonstrated that ARF6 plays a role in the internalization of various classes of membrane receptors including receptor tyrosine kinase (RTK) and G protein coupled receptor (GPCR) to control signal transduction, and WNT5A and its receptor promote tumor invasion and metastasis via receptor internalization in human cancers. Based on these intriguing findings, we postulate that ARF6 might be functioning to control tumor invasion and metastasis in uveal melanomas. Experimental Procedures: To test our hypothesis, we conducted cell invasion assays on uveal melanoma cell lines while targeting ARF6 expression by siRNA or ARF6 activity by pharmacological inhibition. We then proceeded with cell fractionation to look for any differences in the properties of subcellular components in treated cells compared to untreated cells. Unpublished findings: Here we show that WNT5A is expressed at high levels in Gαq wild type uveal melanoma cells and that WNT5A activates ARF6 through ROR2 receptor. When we block ARF6 expression by knockdown or ARF6 activity with a small molecule inhibitor, uveal melanoma invasion is significantly reduced. Notably, ARF6 is necessary for WNT5A-mediated ROR2 internalization. Conclusions: This work indicates that ARF6 functions in the internalization of WNT5A receptor to activate the signaling pathways that drive invasion and metastasis in uveal melanoma. These results would suggest that targeting ARF6 activation might be an effective therapy for the treatment of all uveal melanomas, regardless of their mutational status for Gαq. Citation Format: Donghan Shin, Coulson P. Rich, Lehi Acosta, Jackson R. Richards, Jae Hyuk Yoo, Allie H. Grossmann, Zongzhong Tong, Alan L. Mueller, Weiquan Zhu, Dean Y. Li, Shannon J. Odelberg. ARF6 controls WNT5A receptor internalization to promote uveal melanoma invasion and metastasis [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 5157.

ARF6 is a member of the adenosine diphosphate (ADP)-ribosylation factor (ARF) family of small GTPases and is part of the RAS superfamily. We have shown that pharmacologic inhibition of ARF6 can inhibit proliferation of uveal melanoma and invasion/metastasis of cutaneous melanoma. Most cutaneous melanomas are defined by mutually-exclusive driver mutations in BRAF, NRAS, or NF1. These subtypes share similar mechanisms of oncogenesis because they align along a continuum of RAS signaling. Mutations in the PI3K pathway occur in a fraction of all melanoma subtypes, indicating a selective advantage for PI3K/AKT activation beyond BRAF or RAS activation. We and others have reported ARF6-dependent ERK activation occurring upon either extracellular ligand stimulation or mutant oncoprotein signaling. Recently, ARF6 has also been shown to be upstream of PI3K signaling. Based on these findings, we hypothesized that ARF6 is critical for both the RAF and PI3K arms of the RAS pathway and that loss of ARF6 will impede tumor growth driven by these oncogenes. We tested this hypothesis using human melanoma cell lines and with an inducible, genetically engineered mouse model (GEMM) of melanoma. Our data reveal that pharmacologic inhibition of ARF6 can reduce proliferation of BRAF, NRAS and NF1-mutated melanoma cells. Furthermore, loss of Arf6 causes a reduction in mean tumor size and improves survival in BRAFV600E/CDKN2ANULL/PTENNULL/AKTE17K and BRAFV600E/CDKN2ANULL/PTENNULL mice. These results indicate that ARF6 is necessary for melanomagenesis and for oncogenic BRAF and/or PI3K signaling. Our data suggest that ARF6 may be an actionable node in the RAS pathway and that therapeutic targeting of ARF6 has the potential to improve current treatment methods for progressive disease. Citation Format: Lehi Acosta, Aaron Rogers, Jingfu Peng, Alan Mueller, Zongzhong Tong, Donghan Shin, Jae Hyuk Yoo, Dean Y. Li, Shannon J. Odelberg, Sheri L. Holmen, Allie H. Grossmann. The small GTPase ARF6 is necessary for melanomagenesis [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 4367.

Existing methods for the production and installation of free-form ceiling structures were not suitable with respect to construction cost and time period that were assigned to a Shinsa-town project. Hence, we selected the Robotic-based Digital Fabrication Method that was being tested at that time. Considering the construction cost and period, we selected expandable polystyrene (EPS) as the material of the ceiling structure, and we developed and utilized BAT (a Grasshopper plug-in), to process the work as a free-form production method. We also invented a new cutting method to implement the specific types of components that were otherwise unlikely to be implemented due to the limitation of the straight hot-wire. This paper describes a transport system for the components of the framework, and a robotics-based on-site installation method that is required for the utilization of a robot in the fabrication of these structures.

The high-pressure hexagonal phase of Eu2O3 has been grown epitaxially on C-plane GaN (0001) by molecular beam epitaxy. A structural phase transition from the hexagonal to the monoclinic phase is observed with increasing film thickness by ex-situ X-ray diffraction. The critical thickness for the structural transition is between 2 and 6 nm. The observed epitaxial relationships between the substrate and the film are GaN (0001) ǁ Eu2O3 (0001), GaN ⟨11 2¯0⟩ ǁ Eu2O3 ⟨11 2¯0⟩ for the hexagonal phase, and GaN (0001) ǁ Eu2O3 (20 1¯), GaN ⟨11 2¯0⟩ ǁ Eu2O3 [020] with six rotational domains for the monoclinic phase. The (0.8 ± 0.2) eV conduction band offset and bulk dielectric constant of ∼14 makes Eu2O3 a possible gate dielectric for a GaN-based field effect transistor.

Using density functional theory and model Hamiltonian analysis, we investigate the localized states induced by an oxygen vacancy in rutile TiO2. We identify two classes of localized states—the hybrid and the polaron. The hybrid state is caused by the orbital overlap between three Ti atoms next to a vacancy and is mainly derived from the Ti eg orbitals. The polaron state is caused by the local lattice distortion and is mainly composed of one particular t2g orbital from a single Ti atom. The first principles calculation shows that the polaron state is energetically favored, and the tight-binding analysis reveals the underlying connection between the bulk band structure and the orbital character of the polaron. The magnetic coupling between two nearby polaron states is found to be ferromagnetic. Using this picture, we analyze the results of recent theoretical calculations and experiments and discuss the connection to vacancies in SrTiO3.