Abstract LB317: Liquid-liquid phase separation drives tumorigenesis by modulating the chromatin accessibility of paused and non-paused genes in hepatocellular carcinoma

Abstract Biomolecular condensates partition various cellular processes including transcription, DNA repair, and RNA metabolism. Pol II pausing occurs in more than 30% of all genes and a mechanism for cells to control and synchronize transcription. Pol II pausing is important in maintaining cellular homeostasis and response to stress, which is facilitated by Negative Elongation Factor (NELF) complex. NELF includes four proteins, NELFA, NELFB, NELFC/D, and NELFE. It is known that NELF requires interaction with nascent RNA (via NELFE) to initiate pausing and speculated that nucleosomal accessibility mediates this activity. Additionally, the current literature indicates that NELF facilitates transcriptional regulation distinct from Pol II pausing and that NELFA-induced LLPS is required for Pol II pausing to downregulate genes under stress. However multiple gaps in knowledge remain unknown. First, the role of NELF and Pol II pausing as an oncogenic mechanism remains elusive because current work has not been able to reconcile the vast transcriptomic changes it causes upon its depletion in cells. Our work fills these three gaps. Second, loss of NELF promotes both up and down-regulation of genes - why and how this is regulated remains unresolved. Third, how NELF facilitates transcriptional regulation distinct from Pol II Pausing is unknown. For the first time, we show NELFE, not NELFA, promotes LLPS to facilitate Pol II Pausing by modulating the chromatin to regulate transcription. This modulation is through SMARCB1, which acts distinctly from the SWI/SNF complex. Specifically, we show NELFE LLPS promotes increased Pol II pausing at pro-apoptotic Pol II paused genes, subsequently “pausing” induced cell death signals. Simultaneously, we show DNA regions of pro-survival genes are more accessible due to NELFE LLPS, allowing cancer cells to survive under stress conditions. Our work provides evidence that NELFE/SMARCB1 relationship is distinct from both NELF and SWI/SNF and provides a new theoretical framework whereby LLPS mediates the pro-oncogenic and inhibition of tumor suppressive mechanisms to drive tumorigenesis in hepatocellular carcinoma. Citation Format: Alavaro Lucci, Anna E. Barry, Victoria Johnson, Brittany Ruiz, Laura Reynolds, Adam Wojnar, Kasonde Chewe, Yotsawat Pomyen, Christoph Eckert, Kai Zhang, Jason Hill, Ashesh Shah, Adam Bodzin, Elda Grabocka, Matthias M. Gaida, Nicolas Fawzi, Hien Dang. Liquid-liquid phase separation drives tumorigenesis by modulating the chromatin accessibility of paused and non-paused genes in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB317.