Abstract 244: DHX9 inhibition is synthetically lethal with homologous recombination deficient and dMMR/MSI-H cancers and synergizes with PARP1 inhibition and chemotherapy.

Abstract BACKGROUND: DHX9 is an RNA/DNA helicase that unwinds various RNA/DNA secondary structures, including R-loops and G-quadruplexes, which increase replication stress and transcription-replication collisions if not properly resolved. Homologous recombination deficient (HRD) and mismatch repair deficient/microsatellite instability-high (dMMR/MSI-H) cancer cells exhibit dependence on DHX9 due to enhanced formation of RNA/DNA secondary structures. PARP inhibitors (PARPi) are a front-line standard of care (SOC) for BRCA-deficient and HRD cancers but exhibit limited efficacy and durability. Topoisomerase 1 (TOP1) inhibitors are commonly used in dMMR/MSI-H cancers post-immunotherapy and in PARP and chemo-refractory HRD cancers. Both PARP1 and TOP1 have roles in R-loop prevention and resolution. We hypothesized that DHX9 inhibition combined with PARP or TOP1 inhibition might elicit deeper responses in a greater fraction of HRD or dMMR/MSI-H tumors than SOC therapy alone. METHODS: Cell lines were categorized as HRD based on genomic scar scores or as dMMR/MSI-H based on MMR LOF or MSISensor scores. Sensitivity of cell lines to siDHX9, DHX9 inhibition, or WRN inhibition was assessed using viability assays. Cell cycle changes after DHX9 or WRN inhibition were monitored using the PIP-FUCCI sensor using live cell imaging. Combined inhibition of DHX9 and PARP or TOP1 was evaluated using a viability assay, and synergy was quantified using Bliss Synergy scores. RESULTS: siDHX9 decreased cell viability in HRD and dMMR/MSI-H cells with no effect on homologous recombination proficient (HRP), microsatellite stable (MSS), and non-tumorigenic cells. Similarly, DHX9 inhibition altered the cell cycle and decreased viability in HRD and dMMR/MSI-H cell lines while sparing HRP, MSS, and non-tumorigenic cells. DHX9 inhibition exhibited synergism with both saruparib (PARPi) in HRD cell lines and with SN38 (TOP1i) in HRD and dMMR/MSI-H cell lines. On the other hand, WRN inhibition had no effect on the cell cycle or viability of HRD cell lines. While WRN inhibition reduced viability in a subset of dMMR/MSI-H cell lines, a greater number of dMMR/MSI-H cell lines were sensitive to DHX9 inhibition. CONCLUSION: These data support that DHX9 inhibition is synthetically lethal in HRD and dMMR/MSI-H cancers and demonstrate that DHX9 inhibition synergizes with SOC therapies. For the first time, these data highlight that DHX9 inhibition synergizes with both PARPi or TOP1i to elicit robust tumor cell killing in HRD and dMMR/MSI-H cancers. Additionally, these data demonstrate that DHX9 inhibition is a superior synthetic lethal target compared to WRN inhibition in both HRD and dMMR/MSI-H cancers. Citation Format: Samantha S. Hodge, Natalie Hill, Jhansi L. Leslie, Umida Djakbrova, Logan Chinn, Saranya Chandrasekar, Susan Paprcka, Ester Fernández-Salas. DHX9 inhibition is synthetically lethal with homologous recombination deficient and dMMR/MSI-H cancers and synergizes with PARP1 inhibition and chemotherapy [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 244.