Development of a PROTAC targeting PFKFB4 in glioblastoma cells

Glioblastoma is one of the most aggressive brain tumors, with a median survival of only 12-15 months following diagnosis. It mainly affects adults between 65 and 75 years old, and despite the current standard-of-care therapies, including surgery combined with chemotherapy (temozolomide, TMZ) and radiotherapy, the prognosis remains poor due to the tumor’s specific anatomical localization, the blood-brain barrier, and its high intratumoral heterogeneity. In recent years, PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4), a glycolytic enzyme overexpressed in several malignancies such as breast, liver, prostate, lung, and renal cancers, has been shown to play a crucial role in tumor aggressiveness and growth. Tumor-promoting effects of PFKFB4 are mediated not only by its canonical metabolic role – namely the activation of PFK1 via fructose-2,6-bisphosphate production – but also through a non-metabolic function. In particular, PFKFB4 stabilizes HIF-1α by inhibiting the ubiquitin ligase activity of FBXO28, an enzyme that normally promotes HIF-1α degradation. To exploit this vulnerability, this study focuses on Targeted Protein Degradation (TPD) using PROTACs (Proteolysis Targeting Chimeras), bifunctional molecules that hijack the cell's ubiquitin-proteasome system (UPS) to selectively degrade specific targets. Therefore, the aim of this thesis was to biologically evaluate a panel of novel PFKFB4-targeting PROTACs, synthesized at the University of Salerno (Italy). We tested seven PROTACs (one VHL-based and six CRBN-based) across two different glioblastoma models: an immortalized cell line (U87) and a patient-derived cell line (NCH421k). We found that the PROTACs successfully induced target protein downregulation with varying efficiency and confirmed that this effect was mediated by the UPS. Subsequently, cell viability assays identified one specific PROTAC as the most potent compound in NCH421k, leading to a strong and significant decrease in cell survival. Finally, we evaluated the baseline expression of PFKFB4, VHL, and CRBN via Western blot across additional glioblastoma lines as well as other cancer entities to predict PROTAC sensitivity. In conclusion, these findings validate PFKFB4 as a druggable target via TPD and highlight the potential of these novel PROTACs as a promising therapeutic approach against glioblastoma, paving the way for further molecular optimization to achieve higher efficacy at lower concentrations.