Abstract A066: Enhancing breast cancer immunotherapy by modulating the cGAS–STING and CEACAM1–TIM3 pathways in the tumor microenvironment
Authors: Wang Lan-Hsin , Aberin Marvin Angelo E , Singh Saurabh , Wang Yi-Fu , Lu Ya-Ting , Khag Joy , Wang Weng-Lang , Chang Yao-Ming , Chen Shih-Yu , Guha Chandan , Wang Shu-Ping 
Published: February 2025

Abstract
Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, delivering impressive results across various cancers. However, resistance is common, and only a limited number of patients experience lasting responses. This underscores the urgent need to understand resistance mechanisms and develop new strategies to enhance responsiveness. Here we focus on breast cancer, particularly ER+ breast cancer, which is generally poorly immunogenic and responds poorly to ICB. To address this, we analyzed the METABRIC cohort's immunogenic biomarkers and tumor mutational burden (TMB). We found that relapsed ER+ patients treated with endocrine therapy exhibit activated immune pathways. However, high TMB correlates with lower overall survival (OS), suggesting an active but clinically ineffective immune response. To explore the immune-resistant mechanisms, we developed chronically tamoxifen-treated T47D and MCF7 cells (cTMX-T47D and cTMX-MCF7) by exposing them to the drug for over six months. RNA sequencing data revealed increased type I interferon (IFN-I) signaling and innate immunity in these cells, confirmed in vivo in T47D xenografts. Tracing tamoxifen’s effects, we found that IFN-I signaling and ISG induction depend on the cGAS/STING pathway. Short-term tamoxifen exposure induced only modest ISG expression, indicating additional prerequisites during long-term treatment. We identified that the RACK7/KDM5C H3K4 demethylase complex is crucial for tamoxifen-induced ISG upregulation, with both proteins decreasing over time. Knocking down RACK7 intensified tamoxifen's activation of the cGAS/STING signaling and ISG expression. Combined tamoxifen treatment and RACK7 knockdown significantly enhanced these innate immunity effects compared to either treatment alone. Furthermore, tamoxifen’s DNA-damaging ability modulates the tumor immune microenvironment, particularly through mitochondrial DNA (mtDNA)-induced STING upregulation. We discovered key tamoxifen-mediated mechanisms for cGAS/STING-IFN-I pathway activation via STING upregulation, involving the tamoxifen–mtDNA–STING and tamoxifen–RACK7/KDM5C–STING axes. While IFN-I signaling can promote anti-tumor immunity, our tumor/T-cell co-culture experiments revealed that tamoxifen inactivates T-cells through tumor-intrinsic mechanisms, likely involving T-cell exhaustion pathways. We investigated tamoxifen’s effects on immune checkpoint molecules, finding that CEACAM1, the primary ligand for TIM3, was upregulated in cTMX-T47D and cTMX-MCF7 cells. Further analysis of tamoxifen-treated T47D xenografts revealed elevated CEACAM1 levels, suggesting an immunosuppressive mechanism via the CEACAM1-TIM3 pathway. To investigate this axis in vivo, we used syngeneic tumor mouse models with TS/A (ER+) cells in BALB/c mice. Tamoxifen induced CEACAM1, increased CD45+ immune cell infiltration, and caused TIM3+ CD8 T-cell exhaustion, contributing to tumor progression. Blocking the CEACAM1-TIM3 interaction with an anti-TIM3 antibody significantly inhibited tumor growth, highlighting its role in tamoxifen resistance.
Citation Format: Lan-Hsin Wang, Marvin Angelo E Aberin, Saurabh Singh, Yi-Fu Wang, Ya-Ting Lu, Joy Khag, Weng-Lang Wang, Yao-Ming Chang, Shih-Yu Chen, Chandan Guha, Shu-Ping Wang. Enhancing breast cancer immunotherapy by modulating the cGAS–STING and CEACAM1–TIM3 pathways in the tumor microenvironment [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A066.