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 and we have also contributed to the proteomic identification of complexes of the closely related MYC oncoprotein. Originating from this observations, the following 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 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 cell cycle progression. 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 highly proliferating tumor cells to co-ordinate elongation by RNA Polymerase with cell cycle progression and DNA synthesis.
(3) As a consequence, ligands that disrupt Aurora-A/MYCN complexes establish a dependence of MYCN-driven tumor cells on the ATR kinase, which monitors problems of DNA replication. 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) We have defined the molecular pathways by which MYCN terminates transcription. For this, we have completed a functional screen and identified several termination factors that are recruited by MYCN to the RNA Polymerase.
(5) Most importantly (see figure below) we have found that MYCN and TFIIIC integrate genes transcribed by RNA Polymerase III and genes encoding proteins involved in translation into a three-dimensional network. We have found that this network is a hub for growth-promoting genes that are highly expressed in MYCN-driven neuroblastoma cells. Integration into the network co-ordinates the the chromatin structure with cell cycle progression, since it allows a reversible heterochromatinization during S-phase, which is required to prevent R-loop formation, but prevents the formation of stable heterochromatin at these growth-promoting genes. We believe that this model provides a fundamental understanding of how MYCN maintains the proliferative capacity of tumor cells.
(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-ordinates DNA repair with MYC-driven elongation; both will offer new avenues to target MYC and MYCN-driven tumors.
