Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death

Abstract

ABSTRACTTargeted protein degradation has emerged as a powerful technology – both as a biological tool and for broadening the therapeutic proteome. As tools to probe this approach on historically intractable targets, we have previously advanced ‘biodegraders’ — targeted degradation fusion constructs composed of mini-proteins/peptides linked to modified E3 ligase receptors. Herein, we gain deeper insights into the utility and potential of biodegraders, through a detailed study on Con1-SPOP, a biodegrader which rapidly degrades the potential cancer target, proliferating cell nuclear antigen (PCNA). In a variety of settings, the active biodegrader (Con1-SPOP) proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut). Specifically, in addition to more potent anti-proliferative effects in both 2D cell culture and 3D spheroids, PCNA degradation uniquely induced DNA damage, cell apoptosis and necrosis. Global proteomic profiling of a stable cell-line expressing Con1-SPOP under doxycycline (Dox) induction revealed that impaired mitotic division and mitochondria dysfunction is a direct consequence of PCNA degradation, effects not seen with the stoichiometric inhibitor protein. To evaluate the therapeutic potential of biodegraders, we showed that Dox-induced Con1-SPOP achieved complete tumor-growth inhibition in a xenograft model. To explore application of biodegraders as a novel therapeutic modality, modified mRNA encoding Con1-SPOP was synthesized and encapsulated into lipid nanoparticles (LNPs). The approach successfully delivered mRNA in vitro to deplete endogenous PCNA within hours of application and with nanomolar potency. Overall, our results demonstrate the utility of biodegraders as biological tools and highlight target-degradation as a more efficacious approach versus stoichiometric inhibition. Finally, once in vivo delivery and expression are optimized, biodegraders may be leveraged as an exciting therapeutic modality.