Exploring the oncogenic role of the deubiquitinase USP7 in pancreatic cancer using PROTAC technology

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers worldwide, posing unique therapeutic challenges. Despite significant advances in scientific understanding of the biology and pathophysiology of PDAC, integrating this knowledge into clinical practice to enhance patient outcomes has proven to be challenging. In recent years, it has gradually become apparent that ubiquitin-mediated processes play a pivotal role in regulating protein functionality, localization, and turnover. Deubiquitinating enzymes (DUBs) function to maintain a state of relative homeostasis within the cellular ubiquitome by removing the post-translational modification ubiquitin moieties from substrates. The dysregulation of DUBs can disrupt the dynamic equilibrium of the ubiquitome, which can in turn lead to the development of various diseases, particularly cancer. A comprehensive understanding of the oncogenic function of DUBs at the molecular level remains elusive. In consequence, a focused library of DUB inhibitors was screened using a multiplexed in vitro live cell migration assay. At the highest non-toxic dose, all inhibitors targeting the ubiquitin-specific protease 7 (USP7) enhanced migration of cells. Based on this finding and to advance clinical drug discovery and provide a platform for investigating protein-specific functions in cancer, a valuable chemical toolbox was developed, comprising a novel USP7-inhibitor, a chemical chimera of two commercial USP7 inhibitors, and a highly potent and selective proteolysis-targeting chimera (PROTAC). Strikingly, in this thesis it could be observed that chemical degradation and pharmacological inhibition of the USP7 protein resulted in two distinct cellular phenotypes. Inhibition of USP7 triggered a shift towards a mesenchymal-like cell identity, accompanied by a reduction of in vitro cell proliferation and colony formation. Furthermore, cells reprogrammed their metabolism as inhibition of USP7 led to a disruption of mitochondrial respiration and the switch to a glycolytic cellular phenotype. In contrast, complete degradation of the USP7 protein still enhanced the migratory capacity of cells, yet did not elicit any of the described effects. This suggests that the developed PROTAC exhibits a higher degree of target specificity, while the USP7 inhibitor possesses minimal off-target activity. This conclusion was further substantiated by proteomic analysis, which revealed that the PROTAC is highly selective for its substrate, whereas the USP7 inhibitor regulates a broader range of proteins. Among the most significantly downregulated proteins in both treatment arms, members of the Polycomb complex, an essential chromatin regulatory machinery involved in repressing the transcription of various developmental and oncogenic genes, were identified. Given that proteins involved in the Polycomb complex have been previously described as regulated by USP7 and have also been associated with disease-driving characteristics in PDAC, it can concluded that the increased migration of cells is an USP7-specific phenomenon and dependent on the activity of Polycomb proteins. Moreover, PROTAC treatment caused a reduction in the expression of mitochondrial complex chain assembly proteins and an increase in the expression of proteins involved in the cholesterol pathway and remodeling of lipoproteins. These findings confirm the metabolic activity of USP7 in pancreatic cancer. In conclusion, a highly selective and potent chemical toolbox was developed, offering the possibility of investigating USP7-specific oncogenic functions in vitro and in vivo. Furthermore, this thesis uncovered novel USP7 functions in cancer, as it was observed that the rewiring of metabolism and enhancement of migratory capacity in pancreatic cancer cells are critical processes that are dependent on the activity of USP7. Further research is required to evaluate the potential efficacy of USP7-targeting therapies as treatment options for patients with PDAC.