(Z)-endoxifen modulates estrogen receptor and cell-cycle signaling to induce synergistic antiproliferative effects with CDK4/6 inhibition in endometrial cancer models

Background: Endometrial cancer (EC) remains a major gynecologic malignancy driven in part by aberrant estrogen receptor (ER) signaling. The active tamoxifen metabolite (Z)-endoxifen exhibits dual SERM/SERD-like activity, modulating ER function while exerting additional noncanonical antiproliferative effects. Understanding (Z)-endoxifen's mechanisms of action and its interaction with cell-cycle regulators is critical to optimizing targeted therapy in EC. This study investigated the molecular and functional effects of (Z)-endoxifen, alone and in combination with the CDK4/6 inhibitor abemaciclib, across diverse EC models to define mechanistic activity and translational potential. Methods: ER+ (Ishikawa, HCI-EC23, patient derived organoid U1561.005) and ER-variable (ARK1, ARK2) EC models were treated with estrogen (0-10 nM), (Z)-endoxifen (125 nM-10 µM) or fulvestrant (0-2.5 μM), and abemaciclib (0-10 μM), as single agents or in combination. Cell viability was assessed using luminescence-based assays. Dose-response analyses and combinatorial synergy were evaluated to determine endocrine sensitivity, ER dependency, and downstream cell-cycle effects. Results: Estrogen induced biphasic proliferative responses in ER+ models, confirming ligand-driven mitogenic signaling. (Z)-Endoxifen monotherapy suppressed viability across all cell lines, demonstrating superior potency to fulvestrant and activity independent of ER expression status. Mechanistically, (Z)-endoxifen abrogated estrogen-induced proliferative peaks, consistent with ER antagonism and potential receptor degradation, while also exerting ER-independent effects suggestive of cell-cycle modulation. Abemaciclib monotherapy inhibited viability in both ER+ and ER− models, and its combination with (Z)-endoxifen yielded strong synergistic activity (Chou-Talalay combination index < 1), including in ER-negative ARK2 and the ER+ U1561.005 organoid. This synergy likely arises from concurrent disruption of ER signaling and CDK4/6-cyclin D regulatory pathways. Conclusions: (Z)-Endoxifen demonstrates dual ER-dependent and ER-independent activity in EC preclinical models. Synergy was observed in combination a CDK 4/6 inhibitor. These findings broaden the current understanding of (Z)-endoxifen's efficacy and support further exploration of its potential therapeutic role in ER-driven and mixed-phenotype EC, including evaluation of responses across ER expression levels.