Abstract 2805: A lncRNA-specific CRISPRi screen identifies an essential role of <i>PDCD4-AS1</i> in colorectal cancer growth and survival

Colorectal cancer (CRC) ranks as the third most commonly diagnosed cancer worldwide and is the second leading cause of cancer-related mortality. Recent studies have highlighted the critical role of long non-coding RNAs (lncRNAs) in CRC development, influencing processes such as proliferation, invasion, and therapy resistance. However, the functional roles of most annotated lncRNAs in CRC remain poorly understood, underscoring the need to uncover novel lncRNAs that may impact CRC initiation, progression, and treatment response. To identify lncRNAs involved in CRC cell growth, we conducted a comprehensive lncRNA-specific CRISPR interference (CRISPRi) dropout screen targeting 675 lncRNAs highly expressed across various cancer entities. We utilized four CRC models with diverse molecular backgrounds, including two patient-derived 3D models. As a control, we included HEK293T cells to filter out common essential lncRNAs. This approach revealed 11 lncRNAs significantly depleted across all CRC models, indicating a shared regulatory role in CRC. To validate these findings, we inhibited expression of selected top-hit lncRNA candidates using CRISPRi and antisense oligonucleotide-mediated knockdown. In both approaches, we observed reduced cell growth compared to control conditions, closely aligning with the dropout screen findings. Among the candidates, PDCD4-AS1 was prioritized for further characterization, as it is not located near an oncogenic or common essential protein-coding gene, minimizing the likelihood of off-target effects. By generating loss-of-function patient-derived spheroid models, we showed that PDCD4-AS1 silencing significantly reduced cell growth, induced G2/M cell cycle arrest, and promoted apoptosis. To evaluate the therapeutic potential of targeting PDCD4-AS1, we performed knockdown experiments in a panel of patient-derived organoids (PDOs) both in vitro and in vivo. In a PDO xenograft mouse model, PDCD4-AS1 knockdown significantly suppressed tumor growth and prolonged overall survival. To explore the molecular mechanisms underlying the PDCD4-AS1 knockdown phenotype, we performed transcriptomic analysis by RNA sequencing. Here, we identified significant alterations in amino acid metabolism, suggesting a trans-regulatory role of PDCD4-AS1 in CRC. However, further analyses are required to identify the interaction partners of PDCD4-AS1 and gain deeper insights into its mechanism of action. Collectively, our findings highlight the potential of CRISPRi screens in uncovering novel molecular vulnerabilities in patient-derived CRC models. We present the previously uncharacterized lncRNA PDCD4-AS1 as a mediator of CRC progression and provide strong evidence for lncRNAs as promising therapeutic targets and/or biomarkers for personalized oncology. Citation Format: Vida Kufrin, Silvia Schäfer, Silke Brilloff, Maria Gabriela Pereira dos Santos, Elahe Rahimian, Sandra Küchler, Denis M. Schewe, Claudia R. Ball, Hanno Glimm, Marius Bill, Alexander A. Wurm. A lncRNA-specific CRISPRi screen identifies an essential role of PDCD4-AS1 in colorectal cancer growth and survival [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 2805.