Abstract B038: Discovery of an anticancer seed targeting cancer stem cells and analysis of its mode of action

Abstract Background: Cancer stem cells (CSCs) represent a highly malignant subpopulation of cancer cells characterized by enhanced tumorigenicity and resistance to both conventional anticancer and radiation therapies. Even after elimination of the bulk tumor cells, residual CSCs can regenerate tumors, making them a major cause of cancer recurrence. Recently, there has been increasing interest in targeting CSCs; however, effective agents have yet to be developed. In this context, we established a novel screening system using CSC spheroids derived from the human colorectal cancer HT-29 cell line. As a result of screening from microbial metabolites, we identified polyether cation ionophores as hit compounds, including lenoremycin, which selectively inhibited CSC spheroids formation via reactive oxygen species production (J. Antibiot. 2022, 75, 671). In this study, we further advanced our screening and discovered that compound X isolated from the actinomycete Streptomyces sp. KUSC-240 selectively inhibited CSC spheroids. Methods: HT-29 cells cultured in Tumorsphere Medium XF generate spheroids with enhanced CSC-like properties compared to those cultured in normal RPMI medium. By comparing the drug sensitivities of these spheroids, we screened for in-house microbial metabolite library and purified the active compound (compound X) through silica gel column chromatography, reversed-phase HPLC, and Sephadex LH-20 chromatography. The chemical structure of compound X was elucidated by HR-ESI-MS and NMR analysis. The inhibitory activities against CSCs were evaluated using ATP assay, propidium iodide staining, and DilC1(5) mitochondrial membrane potential (MMP) staining. Western blotting and transcriptomic profiling were performed to assess CSC markers expression and molecular mechanisms, respectively. Results: The culture extract of Streptomyces sp. KUSC-240 selectively inhibited the formation of CSC spheroids. Compound X, isolated as the active compound, was determined to possess a unique dimeric anthracennone-based scaffold and exhibited selective cytotoxicity against CSCs compared to non-CSCs. Compound X significantly reduced the MMP, furthermore inducing the upregulation of apoptotic markers and downregulation of CSC markers including Nanog, Oct3/4, and Sox2. Transcriptomic analysis revealed that compound X activated the unfolded protein response, in which we furthermore demonstrated that the IRE1–XBP1 axis was specifically involved in this activation. Conclusion: We identified compound X, a dimeric anthracenone-derived metabolite isolated from Streptomyces sp. KUSC-240, as a selective inhibitor of CSC spheroids. Treatment with compound X resulted in elevated levels of apoptotic markers, downregulation of CSC markers, and induction of ER stress response. Notably, compound X features a unique chemical scaffold distinct from previously reported CSC inhibitors, such as polyether ionophores, suggesting its potential to provide new insights into CSCs regulatory mechanisms and to serve as a promising lead for the development of novel CSCs-targeted therapeutics. Citation Format: Miyuki Matsui, Hiroaki Ikeda, Kazuko Sakai, Kazuto Nishio, Masaya Imoto, Hideaki Kakeya. Discovery of an anticancer seed targeting cancer stem cells and analysis of its mode of action [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B038.