Abstract 6504: Combinatorial profiling of pidnarulex and other G-quadruplex ligands in 3D multicellular tumor spheroids

Abstract G-quadruplexes (G4s) are four-stranded nucleic acid structures that help regulate key cellular processes and are attractive therapeutic targets in oncology. To assess the therapeutic potential of three G4 ligands—pidnarulex, APTO-253, and BRACO-19—we performed a high-throughput drug-combination screen across thirty-one multicellular tumor spheroids derived from patient tumors and established cancer cell lines. These 3D spheroids model salient features of the tumor microenvironment, incorporating malignant, endothelial, and mesenchymal components. Combination partners were selected for mechanistic relevance to G4 biology, including inhibitors of DNA damage response (DDR), replication stress, and chromatin regulation, based on the proposed roles of G4s in replication and genome stability. Responses were quantified using viability assays complemented by longitudinal brightfield imaging to track spheroid morphology and growth. Drug interactions were evaluated by Bliss independence and volume under the viability surface, providing complementary metrics of synergy and global response. Among the ligands, pidnarulex showed the broadest single-agent activity, whereas APTO-253 and BRACO-19 produced more limited effects. Combination screening with PARP inhibitors, DDR-kinase inhibitors (ATM, ATR, DNA-PK), and cell-cycle regulators (WEE1, PIM1) revealed model-specific synergy. Notably, pidnarulex exhibited consistent synergy in one of eight pancreatic adenocarcinoma models (966289-007-R4-J1) across multiple DDR-targeted combinations. Additional interactions were observed with HDAC inhibitors in a subset of models. Brightfield imaging corroborated enhanced suppression of spheroid growth in synergistic combinations. These data highlight the context-dependent activity of G4 ligands and support integrated functional plus imaging-based approaches to define therapeutic combinations in physiologically relevant 3D cancer models. This project was funded with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024. Citation Format: Thomas S. Dexheimer, Nathan P. Coussens, Thomas Silvers, Poorva Juneja, Eric Jones, Steven D. Gore, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Combinatorial profiling of pidnarulex and other G-quadruplex ligands in 3D multicellular tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6504.