Abstract C087: DHX9 inhibition as a novel therapeutic modality in microsatellite instable colorectal cancer

Abstract

Abstract Background: DHX9 is a DEAH-box RNA helicase which has been reported to play important roles in replication, transcription, translation and RNA splicing/processing, all of which contribute to DHX9’s role in the maintenance of genomic stability. Functionally, DHX9 unwinds and/or resolves 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 (circRNA) and DNA/RNA G-quadraplexes. Overexpression of DHX9 is evident in multiple cancer types, including colorectal cancer (CRC) and lung cancer. In particular, microsatellite instable (MSI) tumors exhibiting defective mismatch repair (dMMR) show a strong dependence on DHX9, making this helicase an attractive target for oncology drug discovery. Here, we will demonstrate validation of DHX9 as a novel target for CRC-MSI, and the identification of potent and selective in vitro and in vivo small molecule inhibitors of DHX9. Materials and Methods: DHX9 targeted siRNA or DHX9 small molecule inhibitors were used to assess anti-proliferative activity in multiple different CRC-MSI and CRC-MSS cell lines through either CellTiter-Glo proliferation or colony formation assays. Downstream biological consequences of DHX9 knockdown or inhibition in vitro were determined using immuno-fluorescent imaging, western blot, flow cytometry to measure RNA/DNA secondary structure, cell cycle changes, apoptosis, and qPCR to measure circBRIP1 induction. CRC-MSI and CRC-MSS tumor xenografts were treated with DHX9 inhibitor ATX968 BID orally for 21-28 days. Tumor and plasma samples were collected for pharmacokinetics (PK) and pharmacodynamic (PD) (circBRIP1) measurement. Results: We demonstrate that DHX9 inhibition in CRC-MSI leads to an increase in RNA/DNA secondary structures such as R-loops, G-quadruplexes and Alu mediated circRNA such as circBRIP1, leads to subsequent DNA damage and increased replication stress. Cell lines that exhibit defective DNA repair pathways such as dMMR are unable to resolve this replication stress and demonstrate S-G2 phase cell cycle arrest prior to onset of apoptosis. We confirmed this selective dependency in a panel of cancer cell lines, where anti-proliferative effects mediated by DHX9 inhibition were associated with dMMR status. Furthermore, DHX9 tool compound ATX968 was well tolerated in vivo across a 28-day treatment period with robust and durable tumor regression observed in the MSI CRC tumor xenograft model. Following cessation of treatment, minimal tumor regrowth was observed in a 28-day post treatment window. Tumor and plasma concentrations of ATX968 and changes in PD markers of DHX9 inhibition were measured and resulting PK, PD and efficacy data were highly correlated. Conclusions: Together, these preclinical data validate DHX9 as a tractable new target with potential utility as a novel treatment for patients with CRC-MSI. Conflict of Interests: All authors are current or former employees and shareholders of Accent Therapeutics, Inc. Citation Format: Jennifer Castro, Matthew H Daniels, David Brennan, Brian T Johnston, Rishabh Bansal, Monique Laidlaw, Chuang Lu, Deepali Gotur, Young-Tae Lee, Kevin Knockenhauer, April Case, Jie Wu, Anugraha Raman, Jae Eun Cheong, Julie Liu, Shane M Buker, E. Allen Sickmier, Stephen J Blakemore, P. Ann Boriack-Sjodin, Kenneth W Duncan, Serena J Silver, Scott Ribich, Robert A Copeland. DHX9 inhibition as a novel therapeutic modality in microsatellite instable colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C087.