Abstract 289: Tracking APOBEC3A to overcome drug resistance in lung cancer.

Abstract Targeted therapies have transformed treatment for advanced non-small cell lung cancer (NSCLC), but acquired resistance remains a challenge. We recently showed that the cytidine deaminase APOBEC3A (A3A) is induced by lung cancer targeted therapies and plays a crucial role in promoting acquired drug resistance by facilitating the evolution of drug-tolerant persister (DTP) cells. While A3A mutational signatures are found in 70-80% of tumors post-TKI treatment versus 20-25% in untreated samples, some resistant tumors lack these mutations. Similarly, while many lung cancer cell lines upregulate A3A expression upon TKI treatment, others do not. Our understanding of intra- and inter-tumoral heterogeneity of therapy-induced A3A expression and activity is limited. Most available methods for studying A3A expression and activity are endpoint assays performed on bulk populations. Because A3A activation is episodic and occurs before detectable genomic changes can be quantified, resolving the activity of A3A in single cells over time has not been possible. To overcome these barriers we adapted the HiBiT nano-luciferase reporter system to develop a method for real-time single-cell monitoring of A3A activity in drug-treated tumor cells.Leveraging the preference of A3A for mRNA hairpin substrates, we incorporated optimized A3A-substrate sequences into the HiBiT tag. In the unedited configuration, HiBiT is unable to complement with LgBiT. However when A3A creates a CU edit within the mRNA hairpin loop, complementation occurs and the resulting luminescent signal reports that A3A is actively editing. We introduced the A3A-HiBiT reporter into patient-derived EGFR, ALK, and KRAS-mutant NSCLC cell lines. TKI treatment induced reporter editing, as detected by HiBiT luminescence and validated by allele-specific droplet digital PCR. No reporter activity was observed in the presence of catalytically inactive A3AE72A or in cell lines with deletion of A3A, confirming specificity for A3A-mediated editing. To investigate intra-tumoral heterogeneity of therapy-induced A3A activity in lung cancer cells treated with targeted therapies, we generated A3A-HiBiT cells stably expressing LgBiT. Experiments are underway to optimize ultra-sensitive bioluminescent microscopy to characterize and monitor the episodic nature of A3A editing in live single cells, as well as to correlate A3A-HiBiT editing with single cell transcriptional heterogeneity using single cell RNA-seq. In summary, our A3A-HiBiT reporter provides a sensitive, scalable, and quantifiable system for real-time monitoring of A3A activity, enabling new opportunities to study the role of A3A in tumor evolution and drug resistance. Citation Format: Maria Vittoria Di Marco, Christopher Eggers, Aaron N. Hata. Tracking APOBEC3A to overcome drug resistance in lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 289.