Abstract 5685: Targeting the NR2F6-NACC1-NOTCH3 axis: a promising therapeutic strategy to combat tumor progression and resistance in endometrial cancer

Abstract Background: Uterine corpus endometrial carcinoma (UCEC) is the most common gynecologic malignancy, with poor prognoses in advanced, high-grade, or treatment-resistant cases. The transcription factor NR2F6 (nuclear receptor subfamily 2 group F member 6) has emerged as a key regulator of immune evasion, tumor progression, and therapy resistance, suppressing T-cell activation by regulating PD-L1 (programmed death-ligand 1) expression. Nucleus accumbens-associated protein 1 (NAC1), encoded by the NACC1 gene, is a transcriptional repressor critical for tumor growth, metastasis, and drug resistance. NOTCH3 (Notch homolog 3), a downstream regulator, contributes to therapy resistance and tumor aggressiveness. This study investigates the significant therapeutic potential of targeting this axis. Methods: Gene expression data from 529 UCEC patients were obtained from The Cancer Genome Atlas (TCGA), and survival analyses were performed using R Studio. Microsatellite instability (MSI) cell line HEC1A and microsatellite stable (MSS) cell line KLE were obtained from ATCC. Knockdowns of NR2F6 and NACC1 were performed using small interfering RNA (siRNA). Semi-quantitative real-time PCR (RT-qPCR) and Western blot were used to measure NR2F6, NACC1, NOTCH3, and PD-L1 expression. Cell proliferation was evaluated using Promega assays. NIC3, a small-molecule inhibitor that prevents NAC1 homodimerization and induces proteasomal degradation, was applied to validate findings from siRNA experiments. Statistical significance was determined using paired Student’s t-tests. Results: High expression of NR2F6 and NACC1 was associated with poor survival in UCEC patients (NR2F6: p = 0.0017; NACC1: p = 0.019). Correlation analyses showed a strong relationship between NR2F6 and NACC1 (Pearson r = 0.54, p < 0.0001) and a moderate relationship between NACC1 and NOTCH3 (r = 0.45, p < 0.0001). RT-qPCR and Western blot analyses revealed that NR2F6 knockdown significantly reduced NACC1, NOTCH3, and PD-L1 expression. NACC1 knockdown similarly decreased NOTCH3 and PD-L1 expression, confirming its regulatory role in the axis. Notably, siRNA-mediated knockdowns and NIC3 treatment significantly reduced cellular proliferation across all treated cell lines (HEC1A, KLE, and Colo 684). NIC3 similarly demonstrated the ability to degrade NAC1 protein and reduce downstream NOTCH3 and PD-L1 expression. Conclusions: This study highlights the therapeutic potential of targeting the NR2F6-NACC1-NOTCH3 axis in UCEC. Validation at both mRNA and protein levels, combined with the observed reduction in cellular proliferation, underscores the promise of this axis as a critical target in high-grade or treatment-resistant tumors. The small-molecule inhibitor NIC3 offers a novel strategy to disrupt NAC1-mediated signaling, and potentially target UCEC. Citation Format: Xiaoqi Sun, Xiaofang Guo, Tram Le, Vaagn Andikyan, Thomas J. Rutherford, Eduardo Sotomayor, Matthew L. Anderson, Adrian Kohut. Targeting the NR2F6-NACC1-NOTCH3 axis: a promising therapeutic strategy to combat tumor progression and resistance in endometrial 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 5685.