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Neuroblastoma is the most abundant, solid, extracranial tumor in early childhood and the leading cause of cancer-related childhood deaths worldwide. Patients with high-risk neuroblastoma often show MYCN-amplification and elevated levels of Aurora-A. They have a low overall survival and despite multimodal therapy options a poor therapeutic prognosis. MYCN-amplified neuroblastoma cells depend on Aurora-A functionality. Aurora-A stabilizes MYCN and prevents it from proteasomal degradation by competing with the E3 ligase SCFFBXW7. Interaction between Aurora-A and MYCN can be observed only in S phase of the cell cycle and activation of Aurora-A can be induced by MYCN in vitro. These findings suggest the existence of a profound interconnection between Aurora-A and MYCN in S phase. Nevertheless, the details remain elusive and were investigated in this study.
Fractionation experiments show that Aurora-A is recruited to chromatin in S phase in a MYCN-dependent manner. Albeit being unphosphorylated on the activating T288 residue, Aurora-A kinase activity was still present in S phase and several putative, novel targets were identified by phosphoproteomic analysis. Particularly, eight phosphosites dependent on MYCN-activated Aurora-A were identified. Additionally, phosphorylation of serine 10 on histone 3 was verified as a target of this complex in S phase. ChIP-sequencing experiments reveal that Aurora-A regulates transcription elongation as well as histone H3.3 variant incorporation in S phase. 4sU-sequencing as well as immunoblotting demonstrated that Aurora-A activity impacts splicing. PLA measurements between the transcription and replication machinery revealed that Aurora-A prevents the formation of transcription-replication conflicts, which activate of kinase ATR.
Aurora-A inhibitors are already used to treat neuroblastoma but display dose-limiting toxicity. To further improve Aurora-A based therapies, we investigated whether low doses of Aurora-A inhibitor combined with ATR inhibitor could increase the efficacy of the treatment albeit reducing toxicity. The study shows that the combination of both drugs leads to a reduction in cell growth as well as an increase in apoptosis in MYCN-amplified neuroblastoma cells, which is not observable in MYCN non-amplified neuroblastoma cells. This new approach was also tested by a collaboration partner in vivo resulting in a decrease in tumor burden, an increase in overall survival and a cure of 25% of TH-MYCN mice. These findings indicate indeed a therapeutic window for targeting MYCN-amplified neuroblastoma.