Farris, Natalie

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<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<0.0001), with ENDX and AT402Z showing superior potency. AT416E outperformed ENDX in wound-healing assays (p<0.0001). All NCEs induced comparable G1 cell cycle arrest in MCF7 (p<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<0.05). These NCEs also significantly induced apoptosis in MCF7 cells at 1-5 μM (p<0.0001), with AT416Z, AT402E, and AT402Z surpassing ENDX (p<0.0001). In T47D cells, apoptosis was significantly induced by ENDX, AT416E, and AT416Z (p<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.

Abstarct Introduction: (Z)-endoxifen (ENDX) is a potent orally bioavailable selective estrogen receptor modulator (SERM) and the most active metabolite of tamoxifen. Given its effective anti-estrogen activity and favorable safety profile, ENDX is being developed as a therapeutic solution in breast health. During its synthesis, two chemical biproducts (compound AT416 and AT402) with Z- and E- isomeric forms are produced. This study aimed to assess the anti-estrogenic activity of these four ENDX-related compounds with ENDX in ERα+ breast cancer. Methods: Compounds AT416E/Z are formed by acetylation of the final API with ethyl acetate during Step 3 of the manufacturing process of ENDX. Compounds AT402 E/Z are formed by coupling of impurity AT300 with propiophenone as a side reaction during Step 2 of the ENDX manufacturing process. The anti-cancer activities of the four ENDX-related compounds were tested across multiple ERα+ breast cancer cell lines (MCF7, T47D, UCD12), including those with ESR1 mutations (Y537S and D538G) known to confer endocrine resistance. Studies included cell proliferation, migration and invasion assays, cell cycle progression assessments, induction of apoptosis, activity of an estrogen response element (ERE) luciferase reporter construct and RT-qPCR analyses of ERα target gene expression. Results: In estrogen-containing media, ENDX exhibited strong anti-proliferative activity in MCF7 cells, regardless of ESR1 mutations. In MCF7 cells cultured in estrogen-deficient media, the four compounds demonstrated more pronounced anti-proliferative effects than ENDX. In T47D cells with the ESR1 Y537S mutation, AT416E showed superior anti-proliferative activity compared to ENDX, while in T47D cells with the D538G mutation, these two compounds exhibited comparable effects. In UCD12 cells, ENDX displayed the strongest anti-proliferative effects. Regarding the inhibition of migration and invasion, AT416E consistently outperformed ENDX in both T47D and MCF7 cells. ENDX was the most effective at inducing G1 phase cell cycle arrest followed. Both AT402E and AT402Z elicited pro-apoptotic effects comparable to ENDX in MCF7 cells, while in T47D cells, ENDX was the most effective at inducing apoptosis, followed by AT402E and AT402Z. In T47D and MCF7 cells, luciferase-based transactivation assays showed that ENDX was the most potent anti-estrogen followed by AT402Z. Finally, based on RT-qPCR analyses, ENDX was a more effective inhibitor of estrogen-induced ERα target gene expression, compared to the biproducts. Summary: These studies have revealed that four novel compounds derived from the manufacturing process of ENDX confer notable anti-tumor activity, with some performing comparably to or better than ENDX in several cancer-relevant cellular readouts. Future studies will validate these findings in additional in vitro/in vivo breast cancer models with the goal of identifying the basis for their varied anti-estrogenic and anti-cancer effects. Such discoveries should allow for the rational design of novel molecules with more potent therapeutic efficacy. Citation Format: Natalie Farris, Lena Batoon, Rajeev S. Muthyala, H. Lawrence Remmel, Sandra S. Hammer, John R. Hawse, Steven C. Quay. Anti-cancer activity of (Z)-endoxifen-related compounds in ERα+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A007.