Abstract Head and neck cancer is a debilitating malignancy, with the majority of cases arising in the oral cavity as oral squamous cell carcinoma (OSCC). A major driver of OSCC is the epidermal growth factor receptor (EGFR), whose activity is aberrantly upregulated in >90% of tumors. EGFR is highly modified with N-linked glycans; fucosylation of N-linked glycans interferes with EGFR dimerization and activation. Thus, post-transcriptional changes may govern EGFR activity. In OSCC, EGFR signaling converges on Wnt/ Β-catenin activity, known to play pivotal roles in the pathobiology of this malignancy through the interaction of nuclear Β-catenin with the histone acetyltransferase CREB-binding protein (CBP). We have shown that a small-molecule inhibitor of Β-catenin-CBP interaction, ICG-001, interferes with OSCC proliferation and aggressive features in cellular, zebrafish and murine models. Also, OSCC-cell line derived mouse tumor xenografts exhibit reduced EGFR abundance, and genomic analyses show a positive correlation between ICG-001 and EGFR inhibition. Given that modification of EGFR with N-glycans impacts its cell-surface localization and signaling, we hypothesized that ICG-001 affected EGFR N-glycosylation. We immunoprecipitated EGFR from indolent CAL27 and metastatic HSC-3 cells after treatment with ICG-001 or vehicle control and determined the effect of inhibition of Β-catenin/CBP activity on its N-glycosylation status. We subjected immunoprecipitated EGFR to proteolysis, performed glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC), analyzed glycopeptides with an Agilent 6550 Quadrupole Time-of-Flight (Q-TOF) MS using collision-induced dissociation, and compared site-specific glycoform patterns for the two cell types +/- ICG-001. At specific N-glycosylation sites, EGFR from indolent CAL27 cells had highly fucosylated N-glycans, while EGFR from metastatic HSC-3 cells displayed N-linked glycans with a paucity of fucose. Treatment of HSC-3 cells with ICG-001 revealed higher fucosylation at sites N151, N420, suggesting that ICG-001 promoted modification with terminal fucose, potentially inhibiting EGFR signaling. Parallel analyses of gene expression signatures in response to ICG-001 treatment in HSC-3 cells showed increased transcriptional expression of fucosyltransferases, FUT2 and FUT3 that fucosylate residues on the outer arms of N-linked glycans. Our studies suggest that the Β-catenin/CBP axis promotes EGFR signaling by inhibiting its fucosylation through downregulation of FUT2 and FUT3 expression and activity. Thus, inhibition of Β-catenin/CBP signaling with ICG-001 may serve as a therapeutic approach to downregulate EGFR protumorigenic activity in OSCC. Supported by NIH grants P41 GM104603 (CEC), F32 CA196157 (KBC), and by the Evans Center for Interdisciplinary Biomedical Research ARC #9950000118 (MAK). Citation Format: Kevin B. Chandler, Khalid Alamoud, Vinay K. Kartha, Khikmet Sadykov, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of Β-catenin/CBP signaling in oral cancer alters EGFR N-glycosylation and abundance [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 2516.