Abstract 2695: TGF-β accelerates STING signaling termination in the tumor microenvironment by enhancing AP3D1-mediated STING trafficking

Background: Activation of stimulator of interferon genes (STING) has shown great potential to enhance anti-tumor effect and reshape the tumor microenvironment (TME). Instead, the impact of TME on STING signaling remains elusive. Leveraging patient-derived samples at single-cell resolution from our clinical trial (NCT04507841), we find that TGF-β significantly attenuates STING signaling and downstream type I interferons production, which is related to the accelerated STING trafficking and degradation. Mechanistically, we identify AP3D1, a subunit of the AP-3 complex, could response to TGF-β and accelerate STING signaling termination in the TME. Methods and results: Utilizing unique samples with HRD precision stratification and PARPi treatment information from our clinical trial (NCT04507841), and employing cutting-edge technologies such as single-cell sequencing and multiplex immunohistochemistry to reveal the complex immune microenvironment, we identified TGF-β suppresses the STING signaling pathway and the production of downstream type I IFN. Additionally, through RT-PCR, western blot, and immunofluorescence co-localization, we discovered that TGF-β promotes STING transport from Golgi to lysosome, accelerating STING degradation. To elucidate the specific mechanism by which TGF-β promotes STING transport, we employed techniques including the STING-APEX2 based proximity biotin labeling system, phosphorylation mass spectrometry and IP-MS, our analysis revealed and verified that TGF-β activates MEK to facilitate phosphorylation of adaptor related protein complex 3 (AP-3) subunit delta 1 (AP3D1) at the S9 site, thereby enhancing the interaction between AP3D1 and STING and accelerating the degradation of STING. Furthermore, we conducted validation experiments in multiple mouse models to confirm the effectiveness of peptides that target the inhibition of AP3D1-STING binding at the AP3D1 R26 site and the blockade of AP3D1 phosphorylation at the S9 site. When administered in combination with PARP inhibitors and STING agonists, these peptides demonstrated satisfactory therapeutic outcomes in upregulating STING signaling, reducing tumor burden, and inducing anti-tumor immune responses, all without evident toxicity. Conclusions: Our work identified TGF-β as an exogenous STING regulator that accelerates AP3D1-mediated STING trafficking and degradation, providing an unprecedented insight into crosstalk between the TME and the STING signaling. Furthermore, our in-depth dissection of mechanisms offered conceptual strategies for new drug discovery and combined pharmacotherapy in cases where STING agonists fail to provide anticipated efficacy currently. Citation Format: Xingzhe Liu, Yuanjia Wen, Jiahao Liu, Guannan Li, Huayi Li, Xiaofei Jiao, Mengshi Luo, Yuewen Zhang, Xuejiao Zhao, Gorden B. Mills, Ding Ma, Guangnian Zhao, Qinglei Gao, Yong Fang. TGF-β accelerates STING signaling termination in the tumor microenvironment by enhancing AP3D1-mediated STING trafficking [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 2695.