Abstract 2803: Genome-wide CRISPR-Cas9 functional screens map long noncoding RNA dependencies in triple-negative breast cancer

Abstract Long noncoding RNAs (lncRNAs) are increasingly recognized as crucial regulators of gene expression, significantly influencing complex biological processes and disease progression, including cancer. Their transformative potential was underscored by the awarding of the 2024 Nobel Prize in noncoding RNA research, spotlighting lncRNAs as powerful modulators of biological regulation and therapeutic innovation. However, the precise functions of most lncRNAs remain largely unexplored. While GENCODE has cataloged over 20, 000 lncRNA genes, the true diversity of lncRNAs is likely much greater. To address this, we constructed an expanded lncRNA reference by integrating GENCODE, BIGTranscriptome, and MiTranscriptome databases. This enhanced reference enabled a comprehensive mapping of lncRNA dependencies in aggressive triple-negative breast cancer (TNBC) using a genome-wide CRISPR-Cas9 screen. Our screen specifically targeted the transcription start sites of over 1, 000 TNBC-associated lncRNAs, encompassing both annotated and novel transcripts. By employing a dual-excision CRISPR strategy in two TNBC cell models, we identified lncRNAs that are essential for TNBC cell growth and chemoresistance, validating several candidates using antisense oligonucleotides (ASOs). In total, 102 lncRNA hits were identified across both MDA-MB-231 and BT-549 TNBC models. Of these, three novel lncRNAs were found to be indispensable for TNBC survival in both two-dimensional and three-dimensional cultures. Analysis of a large TNBC cohort (n=360) further associated the expression of these lncRNAs with more aggressive TNBC subtypes. Loss-of-function studies with ASO Gapmers underscored the roles of these novel lncRNAs in regulating key cellular processes, including extracellular matrix organization, lipid metabolism, and GTPase signaling. Our study provides the first comprehensive map of lncRNA vulnerabilities in TNBC, highlighting their potential as therapeutic targets. This approach, while focused on TNBC, is adaptable for exploring lncRNA functions in other biological settings, paving the way for novel RNA-based therapies and clinical applications. Citation Format: Nehad M. Alajez. Genome-wide CRISPR-Cas9 functional screens map long noncoding RNA dependencies in triple-negative breast cancer [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 2803.