Mechanistic characterization of (Z)-endoxifen in ESR1-mutant and endocrine-resistant breast cancer

Background: ESR1 mutations (e.g.Y537S and D538G) drive ligand-independent ER activation and resistance in ER-positive breast cancer. (Z)-Endoxifen, the active tamoxifen metabolite, inhibits ER signaling, but its impact on ESR1-mutant signaling and therapy-resistant disease is not yet fully defined. Methods: HEK293T cells were transfected with ESR1-WT or mutant plasmids together with an ERE-luciferase reporter and Renilla control. After 24 h, cells were treated with endoxifen, elacestrant (10 µM-0.01 nM), or 0.1% DMSO for an additional 24 h. Firefly luciferase activity was normalized to Renilla, and mean ± SD values from three independent experiments were used to generate dose-response curves. Transcriptomic analyses of 166 ESR1 Y537S and 45 ESR1-WT MCF-7 samples from PandaOmics were performed, and differential expression after endoxifen treatment was assessed using 27 treated and 25 untreated samples processed with limma and gseapy. Plasma and tumor samples were extracted by methanol precipitation with deuterated endoxifen-d₅ and quantified by UPLC-MRM on a Poroshell 120 EC-C18 column using a six-point calibration curve. Results: (Z)-Endoxifen (ENDO) and elacestrant (ELAC) both produced dose-dependent inhibition of ER signaling in wild-type and mutant ESR1 constructs. At 100 nM, ENDO and ELAC showed comparable suppression of WT and three common ESR1 mutants, consistent with the ~80 nM steady-state ENDO plasma level in women taking 20 mg tamoxifen. At higher doses, ENDO more effectively inhibited Y537S and D538G than ELAC. Transcriptomic analyses revealed that ENDO reversed key mutant-associated transcriptional programs in MCF-7 cells. ENDO significantly downregulated estrogen response, apoptosis, E2F-target, and Myc-target pathways while restoring oxidative phosphorylation, xenobiotic and fatty acid metabolism, p53 signaling, and DNA repair (FDR < 0.05). Transcription factor enrichment showed suppression of resistance-associated POU5F1 and SOX11, and reactivation of FOXA1, ELF3, and SPDEF. ENDO also reduced expression of three genes (MTMR7, IL20, CA12) from a six-gene poor-prognosis ESR1-mutant signature. In the ongoing EVANGELINE phase 2 trial (NCT05607004), mean (Z)-endoxifen plasma levels at 40 mg/day reached 278.5 ng/mL (≈746 nM). Conclusion: In summary, (Z)-endoxifen and elacestrant both suppress ER-dependent transcription in ESR1-WT and mutant models, and clinically relevant (Z)-endoxifen concentrations are sufficient to inhibit mutant ESR1 activity. Endoxifen additionally reprograms key transcriptional and metabolic pathways associated with ESR1-mutant resistance and metastasis. These results support further evaluation of (Z)-endoxifen's pathway-level effects in ESR1-mutant breast cancer, and ongoing studies are testing its ability to block mutant ESR1-driven proliferation in preclinical models.