Investigating the role of MYCN in alternate splicing in MYCN-amplified neuroblastoma

Neuroblastoma (NB) is one of the deadliest childhood tumours, responsible for 15% of childhood cancer mortality. Children classified as high-risk for disease relapse have a long-term survival of less than 50%. The treatment for high-risk NB is very aggressive and it is associated with severe toxicities. There is a huge need to find efficient and less toxic ways to treat high-risk NB patients. The MYCN gene is amplified in half of the high-risk cases and as a transcription factor, it can regulate the expression of hundreds of genes affecting all hallmarks of cancer. Unfortunately, as many TFs, MYCN is considered undruggable. Preliminary data from the Halasz group indicated that MYCN might interact with the spliceosome and targeting splicing might be a vulnerability in MYCN-amplified NB. Therefore, during my PhD, I investigated a direct role for MYCN in alternative splicing. In the first results chapter, I explored if MYCN binds to RNA by investigating the composition of protein-crosslinked RNA from UV-crosslinked NB cells. In addition, in the second results chapter I investigated if MYCN directly binds to the splicing machinery by using chemical crosslinking and immunoprecipitation coupled to mass spectrometry. Lastly, in the third results chapter I evaluated if it is possible to combine a splicing inhibitor (isoginkgetin) with a currently used chemotherapy drug (vincristine) to reduce the toxic side effects associated with chemotherapy and to achieve better inhibition of NB growth. My results suggest that (i) MYCN do bind to RNA in NB cells; (ii) MYCN might directly interact with four spliceosome-related proteins in NB cells; and (iii) combining isoginkgetin with vincristine is synergistic in NB cells, and the combination treatment is better in arresting the cell cycle and inducing cell death. MYCN is known to regulate splicing molecules via transcriptional regulation. Here, I showed a new, non-transcriptional, direct role for MYCN in regulating alternative splicing. I also showed that targeting splicing might be a new way of targeting high-risk NB. This potential new role for MYCN may open new avenues for drug development for high-risk NB.