Abstract PS4-04-19: A nucleic acid sensing system for selective therapeutic protein expression in breast cancer

Abstract Background: The challenge of cancer heterogeneity and the limitations of protein-targeting therapeutics underscore an urgent need for innovative, patient-scalable strategies that can precisely eliminate tumor cells while sparing healthy tissues. Towards this aim, breast cancer is dominated by genomic structural variation-which generates hundreds of tumor-specific nucleic acid sequences per cancer. We have previously shown that up to one-third of metastatic breast cancers harbor highly expressed fusion RNAs as a result of these genomic aberrations (Priedigkeit, SABCS 2023). Although fusions have been successfully targeted at the protein-level such as NTRK and FGFR-family fusions, many remain undruggable with conventional therapies. We hypothesized that advances in nucleic acid technology could be applied to therapeutically exploit these cancer-specific biomolecules. Methods: To test this hypothesis, we applied a modular nucleic acid targeting approach (RADARS, Jiang, Koob et al 2023) that utilizes engineered nucleic acid "sensors" designed to selectively hybridize to target RNA sequences. Upon hybridization, the sensors create a double stranded RNA duplex and recruit an endogenous protein called ADAR, which deaminates an intentional A:C mismatch in the sensor-triggering translation of a linked protein cargo only in the presence of a target RNA. We designed and tested 91 sensors against cancer fusion RNA targets with two broad designs—linear sensors that hybridize directly across the fusion RNA breakpoint sequence and gap sensors that target distinct regions of the 5' and 3' fusion partners on the same guide. We tested the selective expression of protein payloads using both designs against two clinically relevant fusion RNAs: (1) an ESR1 fusion and (2) an endogenous fusion RNA in MCF7 cells. Sensor activity was assessed with microscopy for fluorescent protein payloads, and cell viability readouts including CellTiter-Glo, protein quantification of apoptosis markers, and live-cell imaging for cell death payloads. Results: We first confirmed that outlier-expressed fusion RNAs are prevalent across luminal, HER2+, and basal-like cell models—mirroring our prior data on human tumors. We then tested sensors targeting an ESR1::SOX9 fusion RNA. In cells co-transfected with the sensors and fusion RNA, we observed robust mNeon expression, whereas cells without the fusion showed only minimal background fluorescence (mean 25-fold increase vs. no target controls, p 0.0001), validating the sensors' specificity. Next, we targeted an endogenous ARFGEF2::SULF2 fusion RNA in the MCF7 breast cancer cell line, deploying sensors with iCaspase9; a pro-apoptotic cargo. Transfection of both linear and gap fusion-targeting sensors into MCF7 cells and subsequent treatment with iCaspase9 inducer drug AP1903 resulted in a significant reduction in cell viability (up to 84% 36-hours post sensor transfection vs. non-targeting controls, p 0.0001). Live-cell imaging and probing for markers of apoptosis confirmed robust sensor-induced cell death in MCF7 cells, while fusion-negative cells exhibited no toxicity when treated with the same fusion-targeting sensors, demonstrating high selectivity. Conclusions: This study demonstrates the successful design and in vitro proof-of-principle of a biomolecule sensing system that selectively triggers a therapeutic protein effector in the presence of a cancer-specific nucleic acid. These findings have the potential to establish a novel therapeutic paradigm-in which an RNA medicine can repurpose cancer-defining nucleic acid sequences into a targeted, modular, and precise cell-killing mechanism. Our ongoing efforts focus on optimizing sensor design, interrogating mechanisms of sensor-mediated cell killing with diverse protein payloads, and addressing challenges of in vivo delivery. Citation Format: N. Priedigkeit, J. Koob, A. Lebrón-Torres, J. Liao, Y. Wang, M. E. Hughes, D. L. Abravanel, S. M. Tolaney, N. U. Lin, S. Oesterreich, A. V. Lee, F. Chen, T. R. Golub. A nucleic acid sensing system for selective therapeutic protein expression in breast cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-04-19.