Abstract 5631: ZSTK3744, a novel AhR agonist, demonstrates antitumor efficacy in TNBC cell lines <i>in vitro</i> and <i>in vivo</i>

Background: Breast cancer is the most commonly diagnosed cancer among women worldwide. Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and has limited treatment options. TNBC lacks key molecular targets, making it challenging to treat with conventional therapies and limiting the availability of effective treatment options. Herein, we report a novel small molecule, ZSTK3744, which exhibits potent inhibitory effects on the proliferation of TNBC-derived cell lines both in vitro and in vivo. Method: We found a compound, ZSTK3744, that exhibits potent antitumor effects against the TNBC-derived cell line MDA-MB-468. We employed DNA microarray analysis and CRISPR-Cas9 technology to determine the molecular targets of ZSTK3744. Mechanistic studies were performed to evaluate ROS generation, DNA damage, and intracellular signaling pathways in ZSTK3744-treated cells. In vivo, ZSTK3744 was administered at a dose of 2.5 mg/kg every four days (days 0, 4, and 8) via tail vein injection in an MDA-MB-468 xenograft model. Tumor size and body wight were measured until day 28. In addition, safety was evaluated in dogs with weekly dose escalation to assess tolerability. Results: ZSTK3744 exhibited potent antitumor activity (GI50: 0.69 nM) against MDA-MB-468 cells. Target analysis identified ZSTK3744 as an aryl hydrocarbon receptor (AhR) agonist and demonstrated that AhR is essential for its growth-inhibitory effects. ZSTK3744 induced ROS generation, DNA damage, and apoptosis in MDA-MB-468 cells in a dose-dependent manner. In vivo, ZSTK3744 demonstrated significant anti-tumor activity, reducing tumor size from baseline and maintaining this reduction throughout the 3-week post-administration observation period. Importantly, ZSTK3744 treatment did not adversely affect the body weight of treated mice. In contrast to aminoflavone, a previously developed AhR agonist associated with severe lung toxicity, ZSTK3744 showed no evidence of lung toxicity in dog studies. Conclusion: ZSTK3744 demonstrated potent antitumor effects on TNBC-derived cells in vitro and in vivo, as well as having a favorable safety profile. These results suggest that ZSTK3744 is suitable for development as a novel therapeutic agent for TNBC. Ongoing studies are needed to identify biomarkers that predict response to ZSTK3744. Citation Format: Kayoko Kawai-Asami, Takahiro Ohashi, Sayuri Terada, Kotomi Akatsuka, Yuko Nagata, Shinsuke Hiramoto, Tsubasa Nagasaki, Eika Higashi, Ryuichiro Ohshita, Keiko Fukushima. ZSTK3744, a novel AhR agonist, demonstrates antitumor efficacy in TNBC cell lines in vitro and in vivo [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 5631.