Abstract PS2-12-09: Global expression of small DNA damage-associated RNAs (sdRNAs) in breast cancers

Abstract Background: BRCA1 is a large protein that regulates many cellular tumor suppressive processes and maintains genome integrity, at least in part through DNA repair and euploidy maintenance. Interestingly, multiple studies suggest that the function of BRCA1 and/or the DNA damage repair mechanisms it regulates are altered in a substantial subset of breast cancers (BCs). Recently, we deciphered the molecular basis for a new process of BRCA1-driven, small RNA-mediated repair of R-loop-associated DNA damage. These previously undescribed, 30- 35 nt long small RNAs, called sdRNA (single-stranded, DNA damage-associated small RNA), are synthesized by BRCA1/RNAi-containing complexes. sdRNAs catalyze the repair of ssDNA breaks by promoting the formation of an atypical DNA repair complex composed of PALB2 (a bona fide BC suppressor gene product) and RAD52 (recently shown to be involved in R-loop processing at double-strand breaks, and also associated with BC). We sought to determine the levels and role of these sdRNAs in breast tumor formation using human specimens and patient-derived models of breast tissue. Methods: A total of 30 normal breast samples and 96 BC samples were collected. To ensure a sufficient amount of tissue available for RNA sequencing, some samples were expanded into organoids, which are three-dimensional tissue cultures that mimic the characteristics of the parent tissues. Whole-genome small RNA sequencing was used to determine the expression profiles of sdRNAs in BCs across all major subtypes. Abundance profiling and computational modeling methods were used to identify key BC-associated sdRNAs. Key sdRNAs, highly expressed in BCs, were overexpressed in organoid culture to determine the impact of sdRNAs on the growth of normal breast cells. Results: BC samples expressed very high levels of multiple sdRNAs compared to normal breast cells, as determined by focused RNA measurements (qRT-PCR) and by small RNA sequencing. sdRNAs were particularly increased in triple-negative BCs, compared to ER+ BCs, including sdRNAs associated with genes that play roles in DNA damage and repair, cell growth, survival, proliferation, and metastasis. We were able to identify a select few of these differentially expressed sdRNAs in an independent published study on circulating small RNAs in urine and blood in BC patients that underwent neoadjuvant therapy and surgery. The circulating sdRNAs were identified as differentially expressed throughout the study timeline. A dataset of small RNAs in BCs, specifically targeting the detection of sdRNAs, was generated across all major subtypes and linked to clinical annotations to identify sdRNAs for further evaluation in vitro. From this, 770 key BC-associated sdRNAs were identified, including for example sdRNAs associated with the AKT1 and SOD1 genes. Conditions were successfully optimized to transfect sdRNAs into breast organoids in 3-dimensional culture with high efficiency. After select sdRNAs were transfected in normal breast organoids, organoid growth rates were observed to be higher compared to non-specific control RNAs. Conclusions: BC samples have higher levels of multiple sdRNAs compared to normal breast cells. Our initial studies have identified specific sdRNAs associated with BC subtypes that increase the growth of normal breast tissue, consistent with a potential role in breast tumor development. Our data further suggest that sdRNAs highly expressed in BCs can be detected in liquid biopsies, and future studies will assess whether they can be used as a biomarker of therapy efficiency. Ongoing efforts are focused on further assessing the roles of sdRNAs in cancer initiation and exploring the expression of BC-associated sdRNAs in other cancer types. Citation Format: D. K. DeConti, J. Z. Yu, S. Warhadpande, F. Abderazzaq, A. Desbuleux, E. Hatchi, J. M. Rosenbluth. Global expression of small DNA damage-associated RNAs (sdRNAs) in breast cancers [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 PS2-12-09.