Abstract 5945: CRISPR-Cas9 dissection of heparan sulfate

Abstract

Abstract Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and play important roles in tumor growth and metastasis. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with a large number of matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. Previous studies have shown that HSPGs can modulate tumor growth kinetics and are abundantly expressed on the cell surface of many types of cancer. Additionally, genetically reducing the sulfation of HS has been shown to selectively inhibit tumor angiogenesis and lymph node metastasis. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. The overall goal of this project is to uncover and characterize novel genes whose expression influences HS-mediated regulatory networks in tumor growth and metastasis. A genome-wide CRISPR/Cas9-mediated screen was developed to uncover and characterize novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, a high-throughput screening assay was adapted to identify lentiviral-encoded sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs. Parallel screens using alternative HS-dependent toxins or plant lectins that cause cytotoxicity dependent on other types of glycosylation were performed in order to sort genes that selectively affect HS biosynthesis. From the toxin screens, we identified previously studied genes essential for HS formation and factors involved in the intoxication of cells by diphtheria toxin, an HS-dependent exotoxin. Furthermore, we uncovered potential candidate genes whose function is unknown relative to HS biosynthesis. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their potential involvement in the regulation of HS biosynthesis. Overall, these studies will provide a better understanding of the genetic regulatory factors involved in HS biogenesis. Additionally, the factors we identify could reveal novel targets for anti-cancer therapies, as well as lead us to methods to manipulate HS and its activities in other cellular processes that go awry in human diseases. Citation Format: Ryan J. Weiss, Philipp N. Spahn, Nathan E. Lewis, Jeffrey D. Esko. CRISPR-Cas9 dissection of heparan sulfate [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 5945. doi:10.1158/1538-7445.AM2017-5945