Abstract 4839: Harnessing the cGAS-STING signaling and immune checkpoint pathways to enhance the tumor microenvironment for breast cancer immunotherapy

Abstract Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, but resistance remains common with only some patients showing lasting responses. This underscores the need to understand resistance mechanisms and develop strategies to enhance responsiveness. In this study, we focus on breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, which is generally poorly immunogenic and responds unfavorably to ICB. Upon analyzing the METABRIC cohort’s immunogenic biomarkers and tumor mutational burden (TMB), we found that relapsed ER+ patients treated with endocrine therapy (ET) exhibit activated immune pathways. However, high levels of TMB and interferon-stimulated gene (ISG) expression correlated with worse outcomes in these patients, suggesting that ET modulates an active but clinically ineffective immune response. To explore the ET-dependent immune resistance mechanisms, we developed chronically tamoxifen-treated T47D and MCF7 cells (cTMX-T47D and cTMX-MCF7) and found increased type I interferon (IFN-I) signaling and innate immunity. This was confirmed in vivo with T47D xenograft showing enhanced IFN-I signaling. Further analyses revealed that tamoxifen’s DNA-damaging ability modulates the TME through mitochondrial DNA (mtDNA) damage-induced STING upregulation. Depleting mtDNA in tamoxifen-treated cells reduced STING upregulation, IFN-I pathway activation, and ISG expression. Furthermore, we identified that the RACK7/KDM5C H3K4 demethylase complex is crucial for such regulation. Tamoxifen induces the reduction of RACK7/KDM5C protein stability via its ER inhibitory ability, leading to the upregulation of STING and ISG expression. Surprisingly, we discovered that tamoxifen can also induce CEACAM1 upregulation via a RACK7/KDM5C-USF1/IRF2-CEACAM1 transcriptional regulatory axis. These findings highlight the tamoxifen-RACK7/KDM5C-CEACAM1 axis’s role in creating a tumor-favorable TME conducive to immune evasion and robust tumor growth. Preclinical TS/A (ER+) syngeneic tumor mouse model showed that combining tamoxifen treatment with RACK7 knockdown enhances innate immunity effects compared to either treatment alone. Blocking CEACAM1-TIM3 interaction with an anti-TIM3 antibody significantly inhibited TS/A tumor growth upon the combined tamoxifen and RACK7-knockdown treatment, underscoring the CEACAM1-TIM3 pathway’s role in tamoxifen resistance. In conclusion, our study provides direct evidence for tamoxifen-mediated ICB resistance mechanisms. Prolonged tamoxifen treatment reduces RACK7/KDM5C levels, increasing STING and CEACAM1 expression, creating an immunosuppressive TME via TIM3-dependent T-cell exhaustion. This fosters an immunosuppressive yet ready-to-fire TME, augmenting the efficacy of anti-CEACAM1/TIM3 immunotherapy. Citation Format: Shu-Ping Wang, Marvin Angelo Aberin, Saurabh Singh, Soumya Chatterjee, Guang-Zhi Sui, Joy Khag, Weng-Lang Yang, Chen-Yang Shen, Yao-Ming Chang, Shih-Yu Chen, Chandan Guha. Harnessing the cGAS-STING signaling and immune checkpoint pathways to enhance the tumor microenvironment for breast cancer immunotherapy [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 4839.