AbstractRAS mutations drive approximately 30% of human cancers, representing a critical unmet clinical need. AN9025, an oral RAS (ON) inhibitor, with best-in-class potential, demonstrates picomolar potency against RAS-dependent tumor cell lines, showing greater potency than a benchmark molecule. AN9025 binds to cyclophilin A (CypA) to form a binary complex which then engages the RAS (ON) protein to create a tri-complex that effectively inhibits downstream signaling pathways such as MAPK and PI3K/AKT. Preclinical characterization shows that AN9025 binds to CypA with high affinity (KD=3.2 nM) and selectively engages with GMPPNP-bound RAS (ON), rather than RAS (OFF), to form a tri-complex with single-digit nanomolar affinity (KD=8.8 nM). Consistent with its high binding affinity, cell-based assays showed that AN9025 disrupts the KRAS-RAF1 interaction and downregulates phospho-ERK1/2 levels in KRASG12D- and KRASG12V-mutated cells, with IC50 values of 0.068 and 0.013 nM, respectively. AN9025 also exhibited potent antiproliferative effects across a range of RAS-dependent tumor cell lines with various mutations (KRASG12C, KRASG12D, KRASG12V, KRASG12R, KRASG12A, KRASG13C, NRASQ61H, NRASQ61L, NRASQ61K), achieving IC50 values between 0.001 and 0.1 nM. Additionally, AN9025 demonstrated cytotoxic activity in cell models resistant to KRAS(OFF) G12C inhibitors sotorasib and adagrasib, while showing no cytotoxicity in BRAF-mutant, RAS-independent cell lines. In the KRASG12D-bearing HPAC xenograft model, oral administration of AN9025 at a dose of 0.125 mg/kg QD achieved ∼95% intratumoral pharmacodynamic (PD) inhibition of DUSP6 and induced deep tumor regression compared to vehicle. Notably, AN9025 demonstrated sustained PD inhibition, with 60% inhibition persisting 14 days post-treatment cessation, suggesting extended therapeutic efficacy. In summary, AN9025 is a highly potent pan-RAS(ON) inhibitor that forms a tri-complex with CypA and RAS, effectively blocking downstream signaling and demonstrating robust antitumor efficacy, including in models that are resistant to mutant-selective RAS(OFF) inhibitors. Preclinical studies support its advancement into clinical development as a promising targeted therapy for RAS-driven cancers.Citation Format:Shuaishuai Liu, Meng Lv, Xiangyu Fu, Chenchen Zou, Hannah Yan, Tanya Yang, Feifan Li, Zhao Sun, Xiaoli Zhu, Hongyang Zhu, Meng Chen, Junying Li, Zhiyong Yu, Nanhai He. AN9025, an orally bioavailable pan-RAS(ON) inhibitor with potent, broad-spectrum anti-tumor activity [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 4377.