e16154 Background: Immune checkpoint inhibitors targeting the PD-1 pathway have become the standard of care for hepatocellular carcinoma (HCC), yet many patients develop resistance. Combination strategies to overcome PD-1 resistance are needed. Bromodomain and extra-terminal (BET) family proteins are epigenetic readers that regulate transcription of immune and cancer-related gene programs in both tumor cells and immune cells. While preclinical studies suggest that targeting BET-mediated transcription may modulate the tumor immune microenvironment, its impact on PD-1 responsiveness in HCC remains poorly defined. Here, we test the hypothesis that combining BET inhibition with PD-1 blockade favorably remodels the HCC tumor microenvironment and enhances antitumor immune responses, thereby improving sensitivity to PD-1 directed therapy. Methods: Tumor-bearing syngeneic mouse model of HCC (Hep53.4 cell line, n = 100) were randomized and treated with mouse anti-PD-1 for 14 days to model early exposure to immune checkpoint blockade, followed by re-randomization of tumors into anti-PD-1, BET inhibition (molibresib), or combination therapy. Tumor samples were subsequently analyzed by Bulk RNA sequencing using Illumina TruSeq libraries and NovaSeq sequencing. Differential gene expression and pathway enrichment analyses (GSEA) were performed using DESeq2 and FGSEA. In parallel, tumors were harvested for immune profiling and tumor infiltrating lymphocytes were isolated and analyzed by Cytometry by Time-Of-Flight (CyTOF) using a 37-marker panel. Results: The combination of the BET inhibitor molibresib plus anti-PD-1 prolongs survival versus PD1 alone ( p = 0.047). RNA-sequencing analysis revealed marked upregulation of genes indicative of transcriptionally active B cells (CXCL13) and lymphoid-associated stromal features (BANK1, and LYVE1). In the GSEA ontology gene set, adaptive immune response, lymphocyte activation, T cell activation and immune response activation were enriched, suggesting enhancement of adaptive immunity. This immune-rich phenotype was confirmed by (CyTOF) profiling which similarly demonstrated a marked upregulation of B cells and M1-like macrophages. Conclusions: In this preclinical study, BET inhibition in combination with anti–PD-1 was associated with improved survival and may influence immune activation within the tumor microenvironment when combined with PD-1 blockade, possibly via upregulation of transcriptionally active B cells within the TME. Further studies are warranted to better define the underlying mechanisms and to further explore BET inhibition as a potential clinical target.
Pazzi et al. (Thu,) studied this question.