Hans Grossniklaus - US grants

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the United States. When the neovascular form of AMD, choroidal neovascularization (CNV), occurs under the fovea (subfoveal CNV), it leads to central vision loss and functional blindness. Subfoveal CNV may also occur in the ocular histoplasmosis syndrome (OHS), leading to disability in young adults. Fluorescein angiography has shown that CNV may have classic and occult patterns, each of which correlates with a different clinical outcome. Histologic studies have shown type l and type 2 topographies of CNV with theoretically differing clinical outcomes after surgery. The Submacular Surgery Trials is a phase III randomized clinical trials in which surgeons remove CNV specimens from patients with AMD and OHS. In this proposal, CNV specimens obtained from the SST are studied using light and electron microscopy and immunohistochemical stains to determine the structural and biochemical counterparts to classic and occult fluorescein angiographic patterns. The hypothesis that surgically removed type 1 CNV correlates with different clinical outcome than surgically removed type2 CNV compared with unoperated patients will be tested, and if there is a difference in outcome, fundus features of type 1 versus type2 CNV will be determined. Additionally, eyes obtained post-mortem from patients enrolled in the SST will be studied for pathologic correlation with clinical findings and outcomes.

DESCRIPTION (provided by applicant): The long-term goals of this research are to identify compounds that may be used in human trials to prevent and control eye melanoma metastasis. Despite advances in the treatment of primary ocular melanoma, there been no decrease in the mortality rate of this disease. In humans, ocular melanoma often spreads to the liver as micrometastases that have the potential to grow into vascularized metastases and lead to death. There is evidence that downregulation of the natural killer (NK) response is associated with an increase in micrometastases, and upregulation with immunotherapeutic agents such as interferon (IFN) decrease the number of micrometastases. Additionally, there is evidence that primary ocular melanoma produces angiostatin, an anti-angiogenic compound that suppresses growth of micrometastases into vascularized metastases. The objective of this proposal is to test the mechanisms of immunotherapeutic and anti-angiogenic/anti-melanoma invasion agents in a murine model of ocular melanoma that spreads to the liver and causes micrometastases that potentially grow into metastases. Intramuscular (IM) injections of IFN alpha-2b wil1 be given prior to enucleation and subcutaneous (SC) injections of angiostatin will be given just after enucleation of the eyes that contain melanoma. The IFN will eliminate micrometastases by enhancing the host NK response. Both the IFN and angiostatin will prevent the progression of micrometastases into metastases by anti-angiogenesis. Angiostatin will prevent metastases by an anti-melanoma invasion effect.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this project is to define the pathobiology of metastatic uveal melanoma to the liver. This knowledge will be used to control the metastatic process thus decreasing mortality. Uveal melanoma is the most common primary intraocular tumor and has an associated 30% mortality. This mortality rate has not decreased, despite improvements in treating the primary tumor. Uveal melanoma forms micrometastases in the liver and metastatic disease of the liver is the leading cause of death in patients with uveal melanoma. These micrometastases have the potential to grow, remain dormant, or involute, depending on intrinsic properties of the micrometastases and extrinsic properties of the host. Angiostatin promotes melanoma apoptosis and maintenance of dormancy by manipulating the intrinsic vascular endothelial growth factor (VEGF) to pigment epithelium derived factor (PEDF) ratio. Interferon alpha-2b (IFN) promotes micrometastatic melanoma apoptosis by boosting intrinsic hepatic NK cells. Angiostatin and IFN act synergistically via VEGFR1 which is expressed by melanoma cells and NK cells. The first aim is to manipulate the intrinsic PEDF/VEGF ratio in melanoma cell lines and within the micrometastatic environment in the liver. This is done by transfection for overexpression of VEGF or PEDF and siRNA blocking of VEGF or PEDF in melanoma cell lines, determining VEGF and PEDF mRNA levels in the micrometastases with laser capture microdissection, testing for VEGF/PEDF in co-cultures of melanoma/hepatocytes, and correlating hepatic micrometastases with hypoxic zones in the liver. The second aim determines the role of VEGFR1 stimulation of NK cells with adherence to hepatic endothelium in a coculture system and the contribution of FAS/FAS ligand, IFNgamma, and/or perforin to NK killing in knockout mice. The third aim determines the mechanism of combined angiostatin/IFN control of the micrometastases using methods in the first two aims. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There is an urgent need to develop novel therapies for patients with highly malignant uveal melanomas in the eye. Patients with uveal melanoma die within 1-2 years of diagnosis despite current conventional therapies, including eye enucleation, brachotherapy and chemotherapy. Hypoxia drives tumor progression by activating angiogenesis, cell motility and metastasis, as well as metabolic adaptation to growth under a hypoxic environment and is a major factor in the resistance of cancer cells to radio- and chemotherapies. Hypoxia activates transcription factors of the Hypoxia-Inducible Factor (HIF) family that induce the expression of genes that encode pro-angiogenic factors and glycolytic enzymes essential for tumor growth and favor tumor invasion. Based on these findings, we formulated the central hypothesis that development of hypoxia and activation of the HIF pathway play a critical role in ocular cancer growth and spread, and that therapeutic targeting of this pathway using small molecule inhibitors will inhibit ocular tumor growth and metastasis. We have generated substantial preliminary data validating this concept. We show that our lead probe (KCN1) is a potent inhibitor of the in vivo growth of uveal melanoma in the eye (70% tumor size reduction) and its metastasis to the liver (50% reduction in number of metastases), while being extremely well tolerated. The overall goals of this proposal are to refine the structur of the novel HIF pathway inhibitor (HPI) chemical probes we developed, optimize their potency and pharmacological properties, leading to the identification of 1- 2 clinical lead probes that wil be ready to undergo IND-directed pharmacology and toxicology towards phase 1 clinical testing in patients with malignant uveal melanoma through the NCI NExT program. Our multi- disciplinary team has expertise in major aspects of chemical probe development and will divide the project tasks into the following aims: screening analogs of the parent compound in primary and secondary assays to identify and confirm chemical probes with improved potency and solubility (Aim 1); screening analogs of the optimized probes for improved pharmacology and formulation development (Aim 2); and determine the anti- tumor efficacy of the optimized lead probe(s) in orthotopic uveal melanoma models in mice (Aim 3).

DESCRIPTION (provided by applicant): Ocular melanoma is the most common primary eye cancer. Although the primary tumor in the eye can be controlled, frequent cancer spread to the liver results in significant mortality. There are currently no effective treatments for metastastic ocular melanoma in the liver. In the early/pre-metastatic stage of the disease, hypoxia induces the focal expression of chemokine/growth factor receptors in the eye tumor, rendering single melanoma cells responsive to activation by their respective paracrine ligands, stromal derived factor (SDF) and hepatocyte growth factor (HGF) produced in the liver. After extravasation into the circulation these cells home to the liver where they initially form micrometastatic foci that progress to dormant avascular colonies. In the late disease stage, an angiogenic switch leads to the formation of large hepatic macrometastases, which cause patient demise. We have discovered and characterized the anti-tumor properties of novel small molecule arylsulfonamides (ASAs), and obtained exciting preliminary data demonstrating that KCN1, our lead molecule, can potently decrease primary tumor growth, and the establishment and progression of hepatic metastases of uveal melanoma in an orthotopic mouse model we developed. We hypothesize that KCN1 alters the pro-tumorigenic signaling mediated by CXCR4/SDF and cMet/HGF that initiates metastasis, blocks STAT3 signaling involved in early progression in the liver and VEGF pro-angiogenic signaling that leads to macrometastasis, because Hypoxia Inducible Factor (HIF) can regulate these processes and KCN1 blocks HIF transcription. The goal of our proposal is to define the mechanism(s) underlying the anti-tumor effect of KCN1 at the different stages of disease progression. We will determine whether KCN1 inhibits i) the extravasation and survival of primary uveal melanoma cells into the circulation, and their homing to the liver (Aim 1), ii) the progression of micrometastatic foci to avascular melanoma cell colonies in the liver (Aim 2), and iii) the progression of avascular melanoma micrometastatic colonies to macrometastases in the liver by blocking micrometastases-induced angiogenesis (Aim 3). Our preliminary findings support our working hypothesis, as we demonstrate that KCN1 inhibits cMet cell surface receptor activation, STAT3 phosphorylation, and VEGF-mediated tumor angiogenesis in vivo. This work is important as it will better define the mechanisms of uveal melanoma metastases, identify therapeutic targeting points, and help the translation of the small molecules we identified towards becoming novel therapeutic agents for the control of metastasis of uveal melanoma.

The Structural Biology and Imaging (SBI) Core enhances vision research of AVRC members by assisting them in determining the relationship between normal and abnormal tissue structure in eye diseases, disease models and after experimental treatments. This SBI Core provides facilities and services for AVRC members to determine structural changes and how they relate to function through histology, electron microscopy, confocal scanning laser microscopy and experimental animal surgery imaging. The SBI Core also supports obtaining structural data and interpretation of this data for new initiatives of AVRC members with an emphasis on retinal function, retinal degeneration/injury, RPE morphometry, ocular oncology, and gene/drug delivery. The SBI Core enables localization of proteins involved with control of retinal function in health and disease; imaging of tissue changes in retinal and optic nerve degenerations; correlation of RPE morphometric changes with genotype and function; development of imaging techniques for primary and metastatic ocular tumors, including tissue correlations with MRI contrast agents and tele-imaging; and studying ocular tissue changes relative to gene and drug delivery to the posterior ocular compartment.

Summary: The retinal pigment epithelium (RPE) is the major barrier between the outer neural retina and the choroid, forming part of the blood retina barrier (BRB). Damage to the RPE cell and to the organization of the RPE sheet impairs the BRB, as seen in AMD, uveitis, retinal degenerations, and retinal detachment. Additionally, damage to the RPE can occur following subretinal surgery or subretinal injections. Extensive work by others with non-ocular epithelial monolayers indicates that such tissues respond to stress and restore their structure and function through a small number of protective mechanisms. Our preliminary experimental observations suggest that the RPE similarly responds to damage or disease, restoring the BRB via definable protective mechanisms. From these observations, we hypothesize: a) There are a limited number of responses that the RPE takes to restore the BRB; b) Each kind of response results in a unique pattern of RPE cell death; c) Each response type differs, thus modeling BRB repair must be flexible (Fig 1). For these reasons, we propose here to test for specific mechanisms that the RPE uses to respond to insult and to restore the BRB following chronic mild, moderate, and severe injury. While restoration of barrier functions has been investigated in Drosophila wings, lung alveoli, and throughout embryology, it has not been studied in the RPE sheet. Most research on epithelial monolayers, whether in RPE or in non-ocular tissue, uses 2- dimensional (2D) static or time-lapse motion photomicroscopy to study damage responses. In this project, we adapt clever software and mathematical tools from outside vision research that use quantitative spatiotemporal (4D: 3D in space & 1D in Time) dynamics to explore the damage responses of individual RPE cells and of the RPE sheet. Significance: Completion of these Aims will increase our understanding of spatiotemporal dynamics and biomechanics of repair of the BRB after short- or long-term, low- to high-level toxic insults to RPE cells. This new understanding of mechanisms will allow us to correct the loss of essential barrier function in blinding diseases by initiating early and inexpensive interventions.