SUN-343 Expression of Bromodomain and Extra-Terminal (BET) Proteins in Pancreatic Neuroendocrine Tumors and Mechanistic Implications of BET BD1- And BD2-Selective Inhibition

Abstract O.A. Shariq: None. K.E. Lines: None. K.A. English: None. I. Rioja: Employee; Self; GlaxoSmithKline. A. Selberherr: None. H. Ferry: None. A.L. Oberg: None. R. Prinjha: Employee; Self; GlaxoSmithKline. D. Richards: None. R.V. Thakker: None. Background: Effective treatments for metastatic pancreatic neuroendocrine tumors (PNETs) are scarce. We previously showed that inhibiting bromodomain and extra-terminal (BET) epigenetic reader proteins via non-selective targeting of their two bromodomains (BD1 and BD2) reduces proliferation in preclinical PNET models. However, the clinical relevance of BET proteins in PNETs and functional differences between BD1/BD2 are unclear. Here, we characterised BET expression in human PNETs and performed efficacy and mechanistic studies using next-generation BD1- (BD1i) and BD2-selective (BD2i) BET inhibitors. Methods: BET expression was evaluated by immunohistochemistry in functioning and non-functioning human PNETs (n=52) vs normal adjacent islets (n=18). RNA sequencing (RNA-seq) and gene set enrichment analyses (GSEA) were performed in an independent PNET cohort (n=46) to identify biological pathways upregulated in metastatic vs non-metastatic tumors. Effects of BD1i and BD2i on viability, apoptosis, and cell cycle progression were assessed in three PNET cell lines (BON1, QGP1, MIN6). Early (6h) and late (48h) transcriptional responses to BET inhibition were examined in vitro using nascent RNA-seq (SLAM-seq) and standard RNA-seq, respectively. Epigenomic responses were explored using chromatin immunoprecipitation sequencing (ChIP-seq) for the histone mark H3K27ac. Effects of BD1i and BD2i on viability were evaluated in ex vivo neoplastic pancreatic islets derived from Men1L/L/RIP2-Cre mice. Results: BET proteins (BRD2, BRD3 and BRD4) were overexpressed by 1.5-2.5-fold in human PNETs vs islets (p<0.01). GSEA showed enrichment of BET-regulated gene sets in metastatic tumors (normalized enrichment score 1.31, adjusted p=0.04). BD2i marginally affected cell viability (IC50 55-90 µM), apoptosis, and cell cycle profile, while BD1i had a moderate impact on all phenotypes (IC50 1-1.5 µM). Synergistic effects were observed when BD1i+BD2i were combined (IC50 0.2-0.5 µM). RNA-seq and SLAM-seq revealed transcriptional changes that were minimal with BD2i (n=73 and n=10 differentially expressed genes, respectively), modest with BD1i (n=354 and n=60 genes), and greatest with BD1i+BD2i combined (n=1739 and n=375 genes). ChIP-seq showed a reduction in H3K27ac enrichment at promoters and super-enhancers of genes involved in BET protein-regulated pathways (e.g. cell cycle progression), with the greatest decrease observed with BD1i+BD2i combined. In ex vivo cultured neoplastic islets, viability was reduced by <30% with BD2i (p<0.005), ∼40% with BD1i (p<0.0001), and ∼60% with BD1i+BD2i (p<0.0001). Conclusions: Our studies confirm BET proteins as clinically-relevant targets in human PNETs and show that dual (BD1+BD2) inhibition has synergistic efficacy. These findings have important implications for the clinical application of next-generation epigenetic modulators. Sunday, June 2, 2024