LAUSR

Background: STING is an innate immune sensor critical for the development of immunity. Tumor cells can inactivate the STING pathway to avoid immune recognition, emphasizing its importance in generating tumor-specific immunity. Broad activation of STING in advanced cancers may be required to initiate CD8+ T cell priming against unique antigenic repertoires among distinct metastases and to reverse an immune-suppressive TME. Due its ubiquitous expression, systemic delivery of untargeted STING agonists may not achieve a therapeutic index. In contrast, expression of the cytosolic DNAse 3’ repair exonuclease (TREX1) is upregulated in tumor cells in response to genomic instability, inflammatory stimuli, and DNA replication, providing an opportunity for selective activation of the STING pathway. In addition to modulating cGAS/STING signaling, interaction with DNA replication enzymes that generate immunogenic DNA waste highlight a facet of TREX1 biology that may inform clinical development of targeted inhibitors. Methods: Using a structure-based drug design strategy, we designed and optimized small-molecule inhibitors of TREX1 with drug-like physicochemical properties that were profiled in biochemical and cell-based assays. X-ray crystallography studies, thermal shift, and biochemical assays were employed to determine mechanism of action. We evaluated the in vivo profile of select compounds. Results: Inhibitors of TREX1 with < 100 µM potency were optimized into a series with nanomolar potency against purified, recombinant murine and human TREX1 protein in biochemical assays. Inhibitors had similar IC50 values against TREX1 nuclease in an intact cell-based assay. Finally, TSA results demonstrated that compound interaction required magnesium. To our knowledge, we produced the first high-resolution co-crystal structures of inhibitor-bound human TREX1. We used this to dissect mouse- and human-specific interactions, confirming MOA predicted by earlier modeling efforts. Lead compounds demonstrated good bioavailability and achieved exposures necessary for target engagement in mouse models and resulted in tumor growth inhibition when combined with sub-therapeutic doses of doxorubicin. Conclusions: We present the identification and characterization of a potential first-in-class TREX1 inhibitor with nanomolar potency against human and mouse TREX1. Treatment with TREX1 inhibitors conferred profound anti-tumor activity when combined with DNA-damaging agents. Here we demonstrate that targeting TREX1 can specifically and locally engage the STING pathway in the tumor microenvironment, enhance tumor-specific immunity, and provide therapeutic benefit. Citation Format: Valerie Chen, Brian Francica, David Hsieh, Dave Freund, Anja Holtz, Saheli Samanta, Jamie Cope, Ryan Clark, Peppi Prasit, Anne Moon, Henry Johnson, Thomas Dubensky, Dara Burdette. Generation of novel potent human TREX1 inhibitors facilitated by crystallography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1636.