Development of inhibitors to selectively target the CBX8 chromatin reader domain

Project Summary The mis-regulation of chromatin drives both cancer progression and chemotherapy resistance. For example, alterations in the trimethylation of histone 3 at lysine 27 (H3K27me3) is associated with numerous cancers; however, H3K27me3 is also important in numerous normal cellular processes as well. H3K27me3 marks are targeted by the chromodomains of the chromobox (CBX) subunits of Polycomb Repressive Complex 1 (PRC1), which compacts chromatin and represses transcription. There are five CBX paralogs (CBX2,4,6,7,8) that have differential expression in cancer. While CBX6 and CBX7 are often decreased in cancer, CBX2 and CBX8 are frequently increased in cancers. Chromodomains for several CBX paralogs (CBX4, 6, 7) have been successfully targeted with small molecules and peptides that display moderate potency and selectivity. To develop CBX2 and CBX8-selective inhibitors, our research team developed a robust and sensitive assay for affinity-based enrichment of ligands from DNA-encoded libraries and utilized these assays for selections of focused DNA- encoded libraries against a panel of chromodomains. The selective CBX8 inhibitors developed using this approach have validated the CBX8 chromodomain as a therapeutic target in MLL-AF9 leukemia; however, their low cell permeability limits their utility in targeting CBX8-specific function in these cancers. In this grant, we will develop improved CBX8 inhibitors using DNA-encoded libraries selected and designed for increased cellular activity compared to current probes. Hit molecules will be resynthesized off-DNA and tested for affinity and specificity in vitro, as well as in cells. Cellularly active CBX8 chromodomain inhibitors will be used to validate the selective inhibition of CBX8 as a viable, non-toxic therapeutic strategy in vivo.