Abstract 3151: Designing and utilizing a c-Rel specific bioassay in drug repurposing screen for HNSCC
Authors: Kristin A. Altwegg, Kathryn E. King, Natalia J. Martinez, Ty C. Voss, Dvir Blivis, Wendy C. Weinberg, Mark J. Henderson
Journal: Cancer Research (2025)

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.