Abstract 4742: Novel (Z)-endoxifen-related new chemical entities exhibit potent anti-cancer activity in ERα+ breast cancer
Authors: Lena Batoon, Rajeev S. Muthyala, Sandra S. Hammer, Natalie Farris, Steven C. Quay, John R. Hawse
Journal: Cancer Research (2025)

Abstract Approximately 80% of breast cancers express estrogen receptor alpha (ERα, ESR1), which drives cancer progression. Despite the array of targeted therapies, ERα+ breast cancer exhibits the highest recurrence and late distant relapse rates among subtypes. (Z)-Endoxifen (ENDX), an active metabolite of tamoxifen, is a potent, orally bioavailable selective estrogen receptor modulator with a favorable safety profile now under development for breast health applications. Its synthesis yields endoxifen-related byproducts that are new chemical entities (NCEs), including AT416E, AT416Z, AT402E and AT402Z. This study compared the anti-estrogenic effects of these compounds to ENDX in ERα+ breast cancer cell lines (MCF7, T47D, UCD12), including those with ESR1 mutations (Y537S, D538G) linked to endocrine resistance. In 2D cultures with estrogen (E2), ENDX and all NCEs showed significant anti-proliferative effects across all lines, achieving 20-90% inhibition at 0.02-5 μM (p&amp;lt;0.0001). ENDX was the most effective in MCF7; ENDX, AT416Z, and AT402Z in T47D; and ENDX, AT416E, and AT416Z in UCD12. Under E2-deficient conditions, all NCEs had lower IC50 values than ENDX in MCF7 cells, while AT416Z and AT402Z outperformed ENDX in UCD12. In T47D D538G mutants, ENDX, AT416E, and AT402Z exhibited the highest potency, while all NCEs showed comparable efficacy in Y537S mutants. In MCF7 cells, ENDX, AT402E, and AT402Z demonstrated similar activity against Y537S mutants, while AT402Z was the most potent in D538G mutants. In 3D cultures at a clinically relevant concentration (∼1 μM), all compounds significantly reduced proliferation (∼75-90%; p&amp;lt;0.0001), with ENDX and AT402Z showing superior potency. AT416E outperformed ENDX in wound-healing assays (p&amp;lt;0.0001). All NCEs induced comparable G1 cell cycle arrest in MCF7 (p&amp;lt;0.0001), while AT402E and AT402Z showed stronger promotion of G1-to-S phase arrest and S/G2/M inhibition compared to ENDX in T47D cells (p&amp;lt;0.05). These NCEs also significantly induced apoptosis in MCF7 cells at 1-5 μM (p&amp;lt;0.0001), with AT416Z, AT402E, and AT402Z surpassing ENDX (p&amp;lt;0.0001). In T47D cells, apoptosis was significantly induced by ENDX, AT416E, and AT416Z (p&amp;lt;0.0001 vs. vehicle), with ENDX showing superior activity. RT-qPCR analysis at 0.1 μM revealed ENDX and AT416Z as the most potent inhibitors of E2-induced ERα target gene expression in MCF7 cells, with ENDX surpassing all NCEs in T47D cells. Synergy assays indicated that the NCEs do not synergize with ENDX, supporting their potential use as stand-alone agents. These findings highlight four NCEs with notable anti-cancer activity, with some matching or surpassing ENDX in several cancer-relevant cellular readouts, marking them as novel candidates for ERα+ breast cancer therapy. Further studies will advance one or more of these NCEs into in vivo models to confirm their therapeutic efficacy and potential for clinical application. Citation Format: Lena Batoon, Rajeev S. Muthyala, Sandra S. Hammer, Natalie Farris, Steven C. Quay, John R. Hawse. Novel (Z)-endoxifen-related new chemical entities exhibit potent anti-cancer activity in ERα+ breast cancer [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 4742.