Abstract PR003: DHX9 inhibition as a novel therapeutic for ovarian and breast cancer with loss-of-function mutations in the DNA damage repair genes BRCA1 or BRCA2
Authors: Castro Jennifer , Daniels Matthew H. , Pai Sunaina , Laidlaw Monique , Wu Jie , Brennan David , Johnston Brian T. , Raman Anugraha , Lu Chuang , Blakemore Stephen J. , Silver Serena J. , Boriack-Sjodin P. Ann , Duncan Kenneth W. , Sager Jason A. , Copeland Robert A. 
Published: January 2024

Abstract
DHX9 is a multifunctional DEAH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G-quadruplexes. Given the delicate balance of R-loop formation and resolution in maintaining efficient transcription and replication, the ability of DHX9 to unwind R-loops is important in helping to maintain genomic stability. In addition, DHX9 can interact and regulate a large variety of proteins, including key proteins in DNA damage repair pathways such as BRCA1, ATR, Ku86, and WRN. Previously we demonstrated that DHX9 inhibition was efficacious in microsatellite high (MSI-H) CRC xenograft tumor models. Here we report results of in vitro and in vivo preclinical studies in ovarian and breast cancer that indicate patients with Loss-of-Function (LOF) mutations in the DNA damage repair genes BRCA1 and/or BRCA2, may also benefit from DHX9 inhibitor treatment. DHX9 small molecule inhibitors were tested for anti-proliferative activity in a panel of ovarian (N = 15) and breast cancer (N = 41) cell lines, and bioinformatic analyses performed to identify molecular variants that co-associate with sensitivity or resistance cell proliferation outcomes. Notably, selective dependency on DHX9 was observed in both ovarian and breast cancer cell lines that exhibit BRCA1 and/or BRCA2 LOF, as defined by somatic mutations including single-nucleotide variants and/or copy number loss. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress. Cell lines that exhibit BRCA1 and/or BRCA2 LOF appear unable to resolve this replication stress and show S-G2 phase cell cycle arrest prior to onset of apoptosis. Furthermore, a potent and selective DHX9 inhibitor ATX666 was dosed orally in vivo to assess DHX9 dependency within multiple human xenografts representing triple negative breast cancer and high-grade serous ovarian cancer with BRCA1 and/or BRCA2 LOF. In all models, ATX666 was well tolerated for a period of up to 28 days, with robust and significant tumor growth inhibition and regression observed in multiple BRCA1 and/or BRCA2 LOF models with minimal tumor growth inhibition observed in BRCA1 and BRCA2 wild type xenograft models. These results extend the opportunity for DHX9 inhibition to provide therapeutic benefit in solid tumor patients beyond what was previously reported for MSI-H CRC. Together, this preclinical data package validates DHX9 as a tractable new target with potential utility as a novel treatment for patients with BRCA1 and/or BRCA2 LOF across multiple tumor types including breast and ovarian cancer.
Citation Format: Jennifer Castro, Matthew H. Daniels, Sunaina Pai, Monique Laidlaw, Jie Wu, David Brennan, Brian T. Johnston, Anugraha Raman, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. DHX9 inhibition as a novel therapeutic for ovarian and breast cancer with loss-of-function mutations in the DNA damage repair genes BRCA1 or BRCA2 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR003.