Abstract This study elucidates ST6Gal-I, a sialyltransferase, as a functional driver of a cancer stem cell (CSC) phenotype regulated by the stem cell transcription factor Sox2. ST6Gal-I is upregulated in 98% of ovarian cancers (OC) and its high expression correlates with reduced overall and progression free survival in high-grade serous ovarian carcinoma. ST6Gal-I functions to add an α2-6 sialic acid, a large, negatively charged sugar, to N-glycosylated proteins bound for the cell surface. Normal differentiated epithelia have very low expression of ST6Gal-I, however, expression is turned on in stem cell compartments and transformed tissues. Furthermore, our work has shown that ST6Gal-I plays a causal role in conferring hallmark CSC properties including greater tumor-initiating capabilities, and resistance to tumor-associated stressors like chemotherapies, serum deprivation, and hypoxia. However, despite this stark upregulation and functional importance of ST6Gal-I in cancer, very little work has been done to identify the transcriptional drivers of ST6Gal-I expression. We identified that Sox2 and ST6Gal-I are both located on one of the most commonly enriched amplicons in human cancer, amplicon 3q26. We then examined the TCGA databases and found that these two genes are co-amplified in 48/73 cancer cohorts, including ovarian cancer. Furthermore, in those 48 cohorts with co-amplification, there is also co-occurrence per individual patient samples. In addition to being genetically co-amplified, we identified Sox2 response elements in the ST6Gal-I promoter and performed a chromatin immunoprecipitation assay (ChIP) to confirm Sox2 binding. These data implicated Sox2, a key stem-associated transcription factor in CSCs, as a transcriptional activator of ST6Gal-I expression. To confirm this hypothesis, Sox2 was overexpressed or knocked-down in ovarian cancer cells, and it was consistently found that high expression of Sox2 directly induces expression of ST6Gal-I mRNA and protein. Finally, given the relationship between Sox2 and ST6Gal-I in the promotion of a CSC phenotype, we profiled the reprogramming of the cell population into a more stem-like state by quantifying expression of stemness markers Oct4 and Nanog. Overexpression or knockdown of Sox2 resulted in up- or down- regulation of CSC markers Oct4 and Nanog, respectively. Importantly, forced ST6Gal-I knockdown in cells with high Sox2 inhibited the Sox2-induced ST6Gal-I upregulation and subsequently prevented the enhanced expression of Oct4 and Nanog. These data suggest that Sox2 requires ST6Gal-I expression to promote a CSC phenotype. Collectively, our results highlight a novel, glycosylation-dependent mechanism that drives a CSC phenotype. Citation Format: Kaitlyn A. Dorsett and Susan L. Bellis. SOX2 DRIVES ST6GAL-I EXPRESSION AND ACTIVITY TO PROMOTE A CSC PHENOTYPE IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-071.