Abstract 3151: Designing and utilizing a c-Rel specific bioassay in drug repurposing screen for HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer worldwide, with incidence anticipated to increase by 30% by 2030. HNSCC occurs most often in men in their 50-60s, although the incidence among women and younger individuals is rising. The current standard-of-care practices are surgical resection and chemoradiotherapy. HNSCC tumors exhibit frequent mutation of TP53 (72% of tumors). Two additional members of the TP53 gene family, TP63 and TP73, are frequently altered in HNSCC. TP63 encodes two major isoforms, ΔNp63 and TAp63 (containing an alternate or p53-like transactivation domain, respectively). Specifically, ΔNp63α is overexpressed in ∼80% of HNSCC tumors. Our lab has reported that ΔNp63α interacts with activated c-Rel (a nuclear factor-κB family member) in HNSCC, and this interaction is associated with uncontrolled proliferation, inflammation, and metastasis. Consistent with a role in growth regulation, ΔNp63α:c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human HNSCCs and normal keratinocytes overexpressing ΔNp63α. A robust nuclear co-localization in the proliferating compartment of primary HNSCC reflects the relationship between ΔNp63α and activated c-Rel. Stimulation of HNSCC cells with TNFα results in the activation and nuclear localization of c-Rel, which binds to ΔNp63α. This binding displaces and inactivates the tumor suppressor TAp73 from ΔNp63:TAp73 complexes. 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 evaluate compounds inhibiting c-Rel nuclear localization under TNFα stimulation. A 1536-well immunofluorescence assay was developed using high-content image analysis to quantify nuclear translocation. The specificity of the c-Rel antibody was validated using CRISPR/Cas9-generated c-Rel knockout cell lines. The optimized bioassay was used to screen the NCATS pharmaceutical collection of approved drugs. Of the 2, 807 compounds screened, we identified 168 inhibitors (6% hit rate). Interestingly, 17 compounds promoted/activated c-Rel nuclear translocation. These “hits” were then validated in dose-response to confirm activity. 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. Citation Format: Kristin A. Altwegg, Kathryn E. King, Natalia J. Martinez, Ty C. Voss, Dvir Blivis, Wendy C. Weinberg, Mark J. Henderson. Designing and utilizing a c-Rel specific bioassay in drug repurposing screen for HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3151.