UNC6852

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

Protein degradation via the use of bivalent chemical degraders provides an alternative strategy to block protein function and assess the biological roles of putative drug targets. This approach capitalizes on the advantages of small-molecule inhibitors while moving beyond the restrictions of traditional pharmacology. Here, we report a chemical degrader (UNC6852) that targets polycomb repressive complex 2 (PRC2). UNC6852 contains an EED226-derived ligand and a ligand for VHL which bind to the WD40 aromatic cage of EED and CRL2VHL, respectively, to induce proteasomal degradation of PRC2 components, EED, EZH2, and SUZ12. Degradation of PRC2 with UNC6852 blocks the histone methyltransferase activity of EZH2, decreasing H3K27me3 levels in HeLa cells and diffuse large B cell lymphoma (DLBCL) cells containing EZH2 gain-of-function mutations. UNC6852 degrades both wild-type and mutant EZH2, and additionally displays anti-proliferative effects in this cancer model system.