Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS₀01, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS₀01 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS₀01-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0. 021-0. 94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS₀01 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS₀01 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS₀01 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS₀01 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS₀01 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS₀01 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS₀01 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS₀01 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS₀01, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y. W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4701.