Abstract 6986: Discovery of trihydroxyflavone from botanical sources targeting the epigenomic driver NSD3 in lung squamous cell carcinoma

Abstract Lung squamous cell carcinoma (LSCC) represents a highly aggressive subtype of non-small cell lung cancer, accounting for approximately 30% of all lung cancer cases. Despite its significant prevalence, effective targeted therapies for LSCC remain elusive, contributing to poor clinical outcomes. Recent molecular research has identified NSD3 as a critical oncogenic driver, with gene amplification observed in 60% of LSCC cases. NSD3 catalyzes the di-methylation of histone H3K36, leading to the epigenetic upregulation of key oncogenes such as MYC, CDK1, and CCNB1. Furthermore, the hyperactive NSD3-T1232A mutation has been implicated in accelerating tumor progression. This study aimed to develop and characterize novel small-molecule inhibitors targeting both wild-type NSD3 and the hyperactive NSD3-T1232A mutant in LSCC. Using AI-assisted molecular docking, kaempferol, an anticancer compound derived from medicinal plants, emerged as a promising lead. Comprehensive in silico drug design, cellular cytotoxic screening, surface plasmon resonance (SPR) assays, NSD3 chemiluminescent assays, and immunoblotting were employed to identify potent NSD3 inhibitors among kaempferol derivatives. Trihydroxyflavone (THF), one such derivative, demonstrated top DiffDock Confidence and SMANA Affinity scores, indicating a strong potential for NSD3 binding. SPR analysis confirmed the physical interaction between THF and NSD3, yielding favorable binding kinetics with a dissociation constant (KD) of 6.9 µM. Additionally, THF inhibited NSD3-catalyzed histone H3K36 methylation and disrupted critical oncogenic pathways involved in chromatin remodeling, cell cycle control, and mTOR signaling. Cell viability assays revealed that THF significantly reduced the proliferation of LSCC cell lines with NSD3 amplification or gain-of-function mutations, achieving an IC50 of 2.3 µM. In conclusion, our findings demonstrate the therapeutic potential of THF as a targeted inhibitor for NSD3-driven LSCC, laying the groundwork for future preclinical studies aimed at developing selective NSD3 inhibitors to improve outcomes for LSCC patients. Citation Format: Betty Yangari, Kelsey Zou, Kala Mignone, Huisheng Xie, Jianliang Zhang. Discovery of trihydroxyflavone from botanical sources targeting the epigenomic driver NSD3 in lung squamous cell carcinoma [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 6986.