Targeting Aurora-A and N-Myc in childhood cancer models using targeted protein degradation technology

The mitotic kinase Aurora-A is a promising target for cancer therapy. Several inhibitors have been developed and clinically tested. Unfortunately, no inhibitor has been able to achieve a survival benefit for patients. This was partly due to dose-limiting side effects. Targeted protein degradation drugs such as PROTACs (Proteolysis Targeting Chimeras) can improve selectivity and target both catalytic and non-catalytic functions of Aurora-A. Therefore, PROTACs could be used to eliminate oncogenic functions of Aurora-A that are not related to its catalytic function. One of these non-catalytic functions is the stabilising effect on N-Myc. N-Myc is a member of the MYC family of transcription factors. High MYCN expression is a known risk factor for certain cancer types, in particular neuroblastoma. The first Aurora-A PROTAC, called JB170, was published in 2020. The aim of this study was to develop a highly potent, highly selective Aurora-A-degrading molecule with the ability to destabilise N-Myc in neuroblastoma. New compounds should be orally available for preclinical studies in mice and clinical studies in humans. A total of 340 degrader molecules were tested, varying in Aurora-A ligands, linker and E3-ligase ligands. Structure/activity relationship studies showed that MK-5108- and 40f-based degradation molecules were both highly potent and selective. Ligand 2, a derivative of the CDK4/6 inhibitor Ribociclib, showed strong off-target degradation of cyclin-dependent kinases and Aurora-B. A luciferase-based reporter assay showed that selective degrader molecules induced a slow, continuous depletion of N-Myc while non-selective degraders like ligand 2- based compounds induced a rapid depletion of N-Myc within a few hours, most likely due to off-target degradation of CDKs and other kinases. Interactome studies of N-Myc showed that most interacting proteins of N-Myc stayed in a complex with N-Myc upon Aurora-A degradation. However, a minor fraction of the interactome of N-Myc switched from an RNA processing state to a chromatin remodelling state. In vivo metabolic studies showed that JB170 is mainly metabolised within the PEG-linker. Switching to an aliphatic linker improved metabolic stability in vitro. New MK-5108-based degradation molecules were highly selective and increased the degradation efficacy of Aurora-A compared to JB170 and cancer cell toxicity in leukaemia cells.