Development of Sialic Acid-GFP Sensors, a Cancer Associated Sugar, For Tumorigenic Mechanistic Elucidation and Glycan Therapeutic Development

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Title: Development of Sialic Acid-GFP Sensors, a Cancer Associated Sugar, For Tumorigenic Mechanistic Elucidation and Glycan Therapeutic Development Project Summary: Cancers utilize the sugar Sialic Acid (Sia) to engage in immune evasion, multidrug resist, and alter pro-inflammatory pathways, all resulting in survival. Three non- canonical forms of Sia exist that are found on cancer’s cell surface: acetylated-Sia (Neu5,9OAc), 5-N-glycolylneuraminic acid (Neu5Gc), and deacetylated neuraminic acid (Neu5Ac). These forms of Sia contribute to increased metastasis and immune evasion. Our lab was the first to identify that lung and colon cancers overexpress deacetylated-Sias inhibiting NK mediated cytotoxicity yet the relevant amount of deacetylated-Sias needed to propagate the mechanism of action remains unknown. Despite the evidence concerning the importance of glycan modification, further mechanistic elucidation that underpins each process (immune evasion, multidrug resistance, and metastasis) are incomplete due to the lack of methods to detect and quantify each Sia form thus a critical need for the development of sensors that will target each form Sia exists. Our long-term goal is to develop a toolkit of sialic acid targeting sensors that will allow for facile quantification of each cancer associated form of Sia in which to use to elucidate tumorigenic mechanistic thresholds. Our overall objective is to optimize sialic acid-GFP sensors to target acetylated and deacetylated sialic acids on the surface of colon cancer cells. Our central hypothesis is that Sia targeting sensors can be constructed utilizing Sia binding proteins. The formation of this hypothesis is based on our previous work in which we developed sensors for unconjugated ubiquitin using IsoT and the ubiquitin-associated domain. The rationale (and positive impact) that underlies the proposal is that the development of sensors to quantify the cancer related forms of Sia will provide mechanistic elucidation and lead to the robust development of glycan targeted therapies to mitigate cancer’s ability to survive. Performing the following specific aims will test the central hypothesis and accomplish the overall objective: develop sensors of sialic acid and its variants via SiaP, NanS, and SubAB, which bind to Neu5Ac, Neu5,9OAc, and Neu5Gc, respectively (Aim 1). We will then quantify binding and detection of Neu5Gc, Neu5,9OAc and Neu5Gc-GFP sensors on the surface of colon cancer cells via our optimized and published sialic acid cell surface binding assay with CRISPR-Cas 9 cancer cell lines in which the presence of Neu5Gc, Neu5Ac, and Neu5,9Ac has been previously confirmed and characterized (Aim 2). The binding of each sensor sensors will be measured via flow cytometric analysis.