Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Protein-protein interaction inhibitors as an innovative approach to target the Aurora-A/N-Myc complex in neuroblastoma

Neuroblastoma is the most common extracranial solid tumour in childhood, accounting for ~10% of all infant cancers. Although several signalling pathways and genomic features associate with neuroblastoma progression, the amplification of the MYCN gene, coding for the N-Myc transcription factor, correlates with high aggressiveness and poor outcome. MYCN-amplified (MNA) neuroblastoma cells are addicted to the transcriptional profile ensured by N-Myc, which, due to its disordered conformation, is considered an undruggable target. The recent evidence that the Aurora-A kinase interacts with N-Myc, protecting it from proteasome mediated degradation in MNA neuroblastoma cells, disclosed a new strategy of intervention. Indeed, targeting the Aurora-A/N-Myc complex turned out to be a good strategy against high-risk neuroblastoma. In particular, a sub-class of ATP-competitive inhibitors of the kinase, i.e., conformational disrupting (CD) molecules, are able to induce a drastic rearrangement of the Aurora-A structure, which impairs N-Myc binding and causes the decrease of N-Myc protein levels. In this thesis, I investigated new approaches to inhibit the interaction between Aurora-A and N-Myc via a two-pronged strategy: i) identify, among already known Aurora-A kinase inhibitors, new compounds affecting the conformation of the kinase and ii) develop protein-protein interaction inhibitors of the complex, using a peptide-based scaffold. Using Bioinformatics approaches, we identified already known Aurora-A kinase inhibitors, which could act as CD molecules. Then, Surface Plasmon Resonance (SPR) experiments were carried out to assess the ability of such compounds to hamper the binding of Aurora-A and N-Myc in vitro. We then confirmed the ability of the most promising inhibitor, PHA-680626, to impair the Aurora-A/N-Myc interaction in MYCN overexpressing cell lines. Moreover, we observed a decrease in N-Myc protein levels and the appearance of cell death markers in a MNA neuroblastoma cell line. Regarding the second part of this study, we assessed the ability of Protein Kinase A Inhibitor (PKI)-based peptide inhibitors, in silico modified to allow the binding with Aurora-A, to impair the Aurora-A/N-Myc complex in MYCN overexpressing cells. Therefore, the molecules identified in this study expand the repertoire of in neuroblastoma.