VALIDATING GENOME-WIDE CRISPR-CAS9 SCREEN CANDIDATES FOR LONG NONCODING RNA THERAPEUTIC TARGETS IN CHEMOTHERAPY-RESISTANT TRIPLE NEGATIVE BREAST CANCER

Abstract

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by poor clinical outcomes and a lack of targeted therapies. Less than 2% of the human genome encodes proteins, while over 80% is transcribed into non-coding RNAs, including long non-coding RNAs (lncRNAs). These lncRNAs have been shown to regulate key cellular processes and contribute to tumor progression and therapeutic resistance, making them promising targets for TNBC treatment. Methods: A genome-wide CRISPR-Cas9 pooled screen was performed under three-dimensional (3D) culture conditions to identify lncRNAs essential for TNBC. Two of the top identified candidates were functionally validated using knockout (KO) and knockdown (KD) TNBC models. Functional assays included colony formation (CFU), doxorubicin sensitivity, 3D organotypic growth, viability staining, transcriptomics, pathway enrichment analysis, and correlation studies on patients’ transcriptomic data to validate their role in TNBC progression and chemotherapy response. Results: Candidate 251 (Cand.251) and Candidate 254 (Cand.254) were identified as novel lncRNAs with an essential role in TNBC progression. Their depletion led to reduced cell proliferation, increased cell death, suppression of organotypic growth, and enhanced doxorubicin efficacy. Correlation analysis on patients’ transcriptomic data and enrichment analysis on KD TNBC models further supported their functional role in promoting TNBC. Conclusion: This study presents a comprehensive functional characterization of two novel lncRNAs essential for TNBC tumorigenicity, highlighting their potential as therapeutic targets. These findings expand the understanding of lncRNA-mediated regulatory mechanisms in TNBC, emphasizing their role in tumor progression and therapy response, and paving the way for future RNA-based targeted therapies in TNBC treatment.

Date of Award	2025
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences