A novel c-Rel bioassay for drug repurposing in head and neck squamous cell carcinomas

Summary: Abstract Body: The p63 isoform, ΔNp63α, is commonly overexpressed in human squamous cell carcinomas, including those arising in the head and neck (HNSCC). Our lab has reported that elevated levels of ΔNp63α activate and interact with NF-κB/c-Rel in primary murine epidermal keratinocytes. Furthermore, c-Rel is required for the continued proliferation seen in ΔNp63α-overexpressing primary keratinocytes under high Ca+2 conditions, and for orthotopic growth of lenti-ΔNp63α/v-RasHa carcinomas. Nuclear ΔNp63α and c-Rel co-localize in the proliferating compartment of primary HNSCC. Stimulation of HNSCC cells with TNFα results in the activation and nuclear localization of c-Rel, which binds to ΔNp63α, displacing and inactivating the tumor suppressor TAp73 from ΔNp63:TAp73 complexes. Consistent with a role in growth promotion, ΔNp63α:c-Rel complexes bind a promoter motif in both human HNSCC cell lines and lenti-ΔNp63α-expressing murine keratinocytes, repressing the expression of cyclin-dependent kinase inhibitor p21WAF1. By western blot, we confirmed that TNFα stimulation enhances nuclear c-Rel localization in human HNSCC cell lines. We then designed and optimized a high-throughput screening bioassay to measure c-Rel activation under TNFα stimulation. The specificity of the c-Rel antibody was validated using CRISPR/Cas9-generated c-Rel knockout cell lines. Both 1536-well and 384-well immunofluorescence assays were developed using high-content image analysis to quantify c-Rel nuclear localization and used to evaluate compounds from the NCATS pharmaceutical collection of FDA-approved drugs. Of 2,678 compounds screened, we identified 70 inhibitors (3% hit rate). Of these “hits” 27 compounds were confirmed as inhibitors in dose-response in 384 and 1536 well assays. Other NCATS compound libraries will be screened to discover new candidate inhibitor compounds. Top candidates will undergo further investigation for drug synergy with current standard-of-care therapeutics in vitro, ex vivo, and in vivo to explore the possibility of repurposing drugs for HNSCC therapeutics. Kristin A. Altwegg^{1, 2}, Kathryn E. King², Dvir Blivis¹, Ty Voss¹, Natalia Martinez¹, Mark Henderson¹, Wendy C. Weinberg² 1. NCATS/NIH, Rockville, MD, United States. 2. US FDA, Silver Spring, MD, United States. Translational Studies: Cell and Molecular Biology