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 Fos-related antigen 1(Fra-1) in ICB resistance of HCC, RIL175-PD1-R cells with Fra-1 knockdown were constructed using the lenti-virus infection. To explore the phase separation ability of Fra-1, Fra-1 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 Fra-1 as the top transcription factor enriched for the open chromatin regions. CUT&Tag shows that Fra-1 binds to those accessible regions and alters chromatin looping in ICB-resistant cells. Modulating Fra-1 expression in HCC tumor cells significantly increases the therapeutic efficacy of ICB treatment. We also found that Fra-1 undergoes phase separation, which may mediate the changes in chromatin architecture between ICB-sensitive and resistant cells.
Discussion and Conclusion:
Fra-1 undergoes phase separation in HCC, particularly in ICB resistant cells. This characteristic of Fra-1 may shape specific three-dimensional chromatin architecture in HCC cells, contributing to their resistance to ICB treatment. Targeting Fra-1 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.
 

chromatin architecture,phase separation,hepatocellular carcinoma,immune checkpoint blockade (ICB) therapies