Head and neck squamous cell carcinoma (HNSCC) is a devastating malignancy associated with severe morbidity, high mortality and limited treatment options. The main subsite of HNSCC is the oral cavity, where the disease presents primarily as tobacco- and alcohol-associated HPV(-) oral squamous cell carcinoma (OSCC). Despite great progress in the understanding of genomic alterations in OSCC, the molecular details underlying the progression of non-invasive oral lesions to advanced disease with lymph node metastasis remain poorly understood. To gain insights into the mechanisms that contribute to OSCC progression to metastasis we have studied the interaction between nuclear β-catenin and cAMP-response element-binding (CREB)-binding protein (CBP) in OSCC by applying our newly developed computational methodologies coupled with genomic, epigenetic, molecular, biochemical and functional analyses. Our published and preliminary studies show that inhibition of β-catenin/CBP activity with small molecule antagonists, ICG-001 and E7386, in a panel of OSCC cell lines inhibits cell proliferation and mesenchymal phenotype while inducing cellular differentiation. Similarly, inhibition of β-catenin/CBP signaling in human OSCC cell line-derived tumor xenografts in nude mice inhibits tumor growth and metastasis and abrogates rapid metastases driven by subpopulations of OSCC stem cell-like cells, or cancer stem cells (CSCs), in embryonic zebrafish. Our recent global chromatin immunoprecipitation followed by sequencing (ChIPseq) studies show that β-catenin/CBP collaborates with the histone methyltransferase, MLL1, to promote global H3K4 trimethylation (H3K4me3) at transcription start sites (TSS) of numerous CSC genes. This finding is supported by our recent genomic analyses based on RNAseq and scRNAseq data showing that β-catenin/CBP activity is associated with aggressive cell states, including CSCs. Preliminary analyses also suggest that β-catenin/CBP complexes include the Hippo pathway effectors YAP and TAZ (YAP/TAZ), which, like β-catenin, are associated with resistance to both chemotherapy and cetuximab in HNSCC (19,20). Using well characterized OSCC cell lines, we integrated gene expression signatures associated with the inhibition of the β-catenin/CBP axis with OSCC data from The Cancer Genome Atlas (TCGA) to show that β-catenin/CBP activity is associated with progressive disease and reduced patient survival. Building on these collective findings we hypothesize that aberrant activation of β-catenin/CBP signaling underlies the expansion CSCs during HNSCC progression to metastatic disease and that its antagonism may inhibit advanced disease. This hypothesis will be tested in two aims that will: 1) define the role of the β-catenin/CBP axis in HNSCC progression to advanced disease; and 2) determine the molecular mechanisms underlying β-catenin/CBP activity in the induction of CSC phenotypes. Our studies will generate a dynamic integrated map aligning β-catenin-CBP- activity with distinct aggressive cell states and their associated in gene signatures, signaling networks and protein assemblies and provide a rationale for the development of new treatment strategies to combat this malignancy.

PROJECT NARRATIVE The goal of this revised application is to investigate how the aberrantly increased nuclear β-catenin/CBP branch of the Wnt/β-catenin signaling pathway promotes progression of early head and neck cancer tumors to metastatic disease. Based on our collective published and preliminary studies focused on oral cancer, a major subsite of head and neck cancer, we hypothesize that β-catenin/CBP activity induces distinct aggressive cell subpopulations which can be abolished by pharmacological inhibition of β-catenin/CBP signaling leading to reduced metastasis and improved patient outcomes. We now propose to integrate genomic, epigenetic, molecular and structural approaches to characterize the mechanisms responsible for the generation of aggressive cells with associated gene signatures, pathway networks and protein assemblies, and to identify new strategies for improving treatments for head and neck cancer patients.