Abstract 1736: Novel fourth-generation ALK inhibitors targeting compound mutations in <i>ALK</i>-positive cancers
Authors: Yuhao Gao, Jinghan Wang, Kai Zhang, Chang Lu, Yun Bai, Tianxia Liu, Miao Zhang, Sanxu Chen, He Wang, Xijie Liu, Hong Luo, Yinghui Sun
Journal: Cancer Research (2025)

Abstract The emergence of compound mutations in the ALK tyrosine kinase domain presents a significant challenge in treating ALK-positive cancers, especially in patients who have received sequential therapy with second- and third-generation ALK tyrosine kinase inhibitors (TKIs). Key compound mutations, such as G1202R/L1196M, I1171N/D1203N, single mutations (G1202R and I1171N) induce conformational changes that hinder drug binding and severely limit the efficacy of existing therapies, highlighting the urgent need for advanced fourth-generation (4G) ALK TKIs with enhanced potency, selectivity, and increased central nervous system (CNS) penetration. In this study, we developed a series of novel 4G ALK TKIs targeting ALK compound mutations, demonstrating potent activity in vitro and in vivo. Enzymatic assays revealed robust inhibition of ALK compound mutations, with IC50 values below 1 nM and high selectivity against off-target kinase NTRK. Strong cellular antiproliferative effects were observed in series of Ba/F3 EML4-ALK compound-mutant cell lines with low-nanomolar IC50 values. These compounds significantly inhibited ALK autophosphorylation and downstream signaling pathway in a dose-dependent manner, resulting in G0/G1 cell cycle arrest and cell apoptosis. In vivo efficacy studies using subcutaneous xenograft models with EML4-ALK wild-type and compound-mutant cell lines (Ba/F3 and NCI-H3122) demonstrated dose-dependent tumor growth inhibition, with some cases achieving complete tumor regression. Notably, these 4G ALK TKIs exhibited effective transport across the blood-brain barrier (BBB) with a notable Kp, uu in rats. In intracranial xenograft models harbouring EML4-ALK (wild-type or compound-mutant), these 4G ALK compounds substantially inhibited intracranial tumor growth, underscoring the potent CNS antitumor activity. And the treatments were well tolerated across all animal models, with no significant body weight loss, supporting a favorable safety profile. Meanwhile, pharmacokinetic studies in animals also revealed favorable properties of these compounds. In conclusion, these selectivity 4G ALK compounds demonstrate significant in vitro and in vivo efficacy, effective CNS penetration, and a favorable safety profile. They represent a promising advancement in the treatment of ALK-driven cancers, providing potential benefits for refractory cases following second- and third-generation ALK TKI sequential therapy and as viable front-line options. Comprehensive preclinical evaluations are warranted to support further clinical investigation. Citation Format: Yuhao Gao, Jinghan Wang, Kai Zhang, Chang Lu, Yun Bai, Tianxia Liu, Miao Zhang, Sanxu Chen, He Wang, Xijie Liu, Hong Luo, Yinghui Sun. Novel fourth-generation ALK inhibitors targeting compound mutations in ALK-positive cancers [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 1736.