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N-Myc and Aurora A: From Protein Stability to Chromosome Topology N-Myc and Aurora A: From Protein Stability to Chromosome Topology Myc and Aurora A: From Protein Stability to Chromosome Topology

Periodic Reporting for period 3 - AUROMYC (N-Myc and Aurora A: From Protein Stability to Chromosome TopologyN-Myc and Aurora A: From Protein Stability to Chromosome TopologyMyc and Aurora A: From Protein Stability to Chromosome Topology)

Reporting period: 2018-08-01 to 2020-01-31

The question that is being addressed is to understand the function of a complex of the MYCN protein with the Aurora-A kinase. The MYCN protein is an oncoprotein that drives the development of many, mainly neuroendocrine tumours. For example, many pediatric cancers are driven by the MYCN oncoprotein. Understanding the function of this protein and finding ways to target it for the therapy of tumours is therefore of significant theoretical and also medical benefit. Our previous work had shown that MYCN forms a complex with the Aurora-A kinase that stabilises the MYCN protein in human tumours. We and others have also provided evidence that targeting the complex via small molecule ligands of Aurora-A achieves considerable therapeutic benefit in mouse models of MYCN-driven tumors and in several models of tumours driven by the closely related MYC oncoprotein. Initial data in human trials of one such ligand had shown some efficacy but also pointed very clearly to the need for further improvement. The overall objective of the project is therefore to provide a rational understanding of the interaction of MYCN with its partner proteins, identify how and why this interaction is shaped by the Aurora-A kinase, and use this understanding for the rational development of therapies that target MYCN-driven tumors.
The work program comprises a biochemical and structural analysis of complexes of the N-MYC protein; initially, the work started from a proteomic analysis of N-MYC complexes. Meanwhile, we have also contributed to the proteomic identification of complexes of the closely related MYC oncoprotein.

Originating from this observations, several major results have been achieved:

(1) The team in Leeds has resolved the structure of the Aurora-A/N-MYC complex, providing a firm basis for understanding its effects on N-MYC stability and the effects of different Aurora-A inhibitors.

(2) The team in Würzburg has shown that the association with Aurora-A regulates N-MYC´s global transcriptional function in a cell cycle-dependent manner and provided evidence that the complex co-ordinates transcriptional elongation with DNA replication. In subsequent mechanistic work, we found that a major function of MYCN is to terminate transcription and that the balance between elongation and termination is determined by the phosphorylation status of the MYCN protein at T58. This leads to a model in which the stabilization of MYCN by complex formation with Aurora-A shifts the balance at a MYCN-bound promoter towards termination, enabling even highly proliferating tumor cells to escape conflicts of the elongating RNA Polymerase with a replication fork.

(3) As a consequence, ligands that disrupt Aurora-A/MYCN complexes cause replication transcription conflicts and establish a dependence of MYCN-driven tumor cells on the ATR kinase, which stabilizes stalling replication forks. This leads to a therapeutic strategy, in which Aurora-A and ATR inhibitors are combined to provoke conflicts and cause double-strand breaks in a highly tumor cell-specific manner. We have shown that such strategies open a large therapeutic window for MYCN-driven tumors and are exploring multiple ways to translate this into clinical praxis. One particularly exiting aspect is that tumor eradication upon Aurora-A/ATR inhibition depends on the host immune system, since a signaling pathway (STING) is activated that signals to the immune system. This opens multiple avenues to exploit these conflicts for new cancer therapies.

(4) Work is currently underway that defines the molecular pathways by which MYCN terminates transcription. For this, we have completed the functional screen and identified several termination factors that are recruited by MYCN to the RNA Polymerase. Their functional analysis is currently underway.

(5) Based on the observation that shows that MYCN associates with multiple proteins involved in chromosome topology, we have completed the mapping of TFIIIC, condensin and cohesin complexes. We have established Hi-C and Hi-ChIP methods that now enable us to analyze the way that MYCN protein shape the three-dimensional structure of the genome.

(6) Finally, while work trying to recapitulate that PP1 is a major T58 phosphatase did not confirm this finding, it contributed to two major publications on the role of PP1 complexes in MYC biology. One of these complexes co-ordinates transcription elongation with spliceosome assembly, while the other co-oridnates translation with MYC-driven elongation; both will offer new avenues to target MYC and MYCN-driven tumors.
The project will yield a largely complete biochemical model and structural insights into how N-MYC co-ordinates transcription with DNA replication. How these two processes are co-ordinated is a fundamental biological question. It has particular relevance for transcription-driven tumors and therefore also opens a new strategy for targeting these tumors. The project will show that this is a valid, feasible and effective strategy for the therapy of MYCN-driven tumors in all model systems that currently be used and suggest rational strategies for further improvements and also a rational strategy for the - larger - group of MYC-driven tumors.sIt will also give a clear view how MYCN shapes the three-dimensional structure of the genome and how these two processes are linked.