Abstract 2854: The role of NONO in genome stability: Implications for RNA-DNA hybrid accumulation, genomic instability, and inflammatory signaling

Genomic instability, often resulting from replication stress and defects in DNA damage repair pathways, is a hallmark of cancer. The NONO protein, a multifunctional RNA-binding factor, plays a key role in DNA damage repair, transcriptional regulation and genome stability. Emerging evidence has linked NONO to cancer progression, though its mechanistic roles in maintaining genomic stability remain unclear. In our study, we demonstrate that NONO localizes at replication forks, as revealed by iPOND and SIRF assays, and interacts with replication polymerases. Loss of NONO in knockout (KO) cells leads to increased replication speed, significant RNA-DNA hybrid (R-loop) accumulation, and heightened genomic instability. These cells exhibit elevated micronuclei formation, abnormal chromosome segregation during metaphase, and increased 53BP1 foci, a marker of DNA double-strand breaks. NONO KO cells also show higher RPA loading under replication stress, highlighting their vulnerability to DNA damage. Additionally, the loss of NONO activates inflammatory response genes, including phospho-NFκB, p-IKKα, cGAS, and IRF3, suggesting that DNA-RNA hybrid accumulation and DNA damage may drive inflammatory signaling pathways. These findings position NONO as a critical regulator of genome stability, replication dynamics, and inflammation suppression. Our study highlights the essential role of NONO in maintaining genome integrity, interacting with replication machinery, and resolving R-loops. We will present ongoing work to further elucidate the mechanisms by which NONO contributes to genome stability and inflammatory responses in cancer cells. Citation Format: Vikas Gaur, Sara Knowles, Weihang Chai. The role of NONO in genome stability: Implications for RNA-DNA hybrid accumulation, genomic instability, and inflammatory signaling [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 2854.