Intrinsic signaling pathways modulate targeted protein degradation.

Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.

Development of a new in vitro assay system for evaluating the effects of chemicals on DNA methylation.

Epigenetic toxicity, a phenomenon in which chemicals exert epigenetic effects and produce toxicity, has been attracting attention in recent years due to advances in toxicology accompanying the development of life sciences. However, it has been difficult to identify epigenetic toxicants due to the lack of a simple experimental system to evaluate epigenetic toxicity. In this study, we developed a prototype of an in vitro reporter assay system for assessing the effects of chemicals on DNA methylation using two promoters showing different degrees of DNA methylation, Agouti IAP and Daz1 promoters, and a luciferase reporter. The system successfully detected DNA demethylating activity using 5-azacytidine, a chemical having DNA demethylation activity, as a positive control chemical, and demethylation of cytosine of CpG in the promoter was confirmed by pyrosequencing analysis. Next, in order to improve the detection sensitivity of the DNA demethylating activity of this system, we tried to increase the basal level of methylation of the Daz1 promoter by pre-methylase treatment of the reporter vectors. As a result, the detection sensitivity of the system was successfully improved in cells where the basal level of methylation was indeed increased by methylase treatment. Thus, the developed assay system here is effective for the simple evaluation of chemicals that affect DNA methylation.