Abstract 2751: Role of transcriptional regulation in chromatin architecture and immunotherapy resistance of hepatocellular carcinoma

Abstract Introduction: Chromatin architecture is frequently altered in cancers, including one of the most prevalent cancers, hepatocellular carcinoma (HCC). Emerging evidence suggests that liquid-liquid phase separation plays a crucial role in shaping chromatin architecture and regulating gene transcription. Immune checkpoint blockade (ICB) therapy has become a new standard of care for HCC. However, the majority of HCC patients exhibit either primary or adaptive resistance to this immunotherapy, highlighting the urgent need for effective combination therapies. This study aims to uncover the differences in chromatin architecture between ICB-sensitive and resistant HCC tumors and dissect the underlying regulatory mechanisms. Methods: Hi-C, ATAC-seq, CUT&Tag, and RNA-seq were performed on RIL175 cells from a well-established adaptive ICB-resistant mouse model to generate the multi-omics dataset. To explore the functional role of F1 in ICB resistance of HCC, RIL175-PD1-R cells with F1 knockdown were constructed using the lenti-virus infection. To explore the phase separation ability of F1, F1 protein tagged with mCherry was expressed in E. coli and purified. Droplet formation assay and fluorescence recovery after photobleaching (FRAP) assay were applied. Results: Three-dimensional genomic analysis by Hi-C reveals comparable chromatin interactions between ICB-sensitive and resistant cells at both compartment and topologically associated domain (TAD) levels but an increase in loops within ICB-resistant cells. In accord with this, ATAC-seq results indicate upregulated chromatin accessibility in a subset of chromatin regions in ICB-resistant cells compared to ICB-sensitive cells. Motif analysis identifies F1 as the top transcription factor enriched for the open chromatin regions. CUT&Tag shows that F1 binds to those accessible regions and alters chromatin looping in ICB-resistant cells. Modulating F1 expression in HCC tumor cells significantly increases the therapeutic efficacy of ICB treatment. We also found that F1 undergoes phase separation, which may mediate the changes in chromatin architecture between ICB-sensitive and resistant cells. Discussion and Conclusion: F1 undergoes phase separation in HCC, particularly in ICB resistant cells. This characteristic of F1 may shape specific three-dimensional chromatin architecture in HCC cells, contributing to their resistance to ICB treatment. Targeting F1 condensates may provide a novel therapeutic strategy to reprogram the genome architecture to improve ICB efficacy for patients with HCC. Keywords: chromatin architecture, phase separation, hepatocellular carcinoma, immune checkpoint blockade (ICB) therapies Acknowledgement: This work is supported by NSFC/RGC (460795048) & Li Ka Shing Foundation. Citation Format: Siyun CHEN, Yingnan LIN, Chengpeng Zhong, Yalin TU, Haoran WU, Yiling ZHANG, Jia WANG, Alfred Sze Lok CHENG. Role of transcriptional regulation in chromatin architecture and immunotherapy resistance of hepatocellular carcinoma [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 2751.