Neuroblastoma (NB) is a highly aggressive embryonal malignancy of the sympathetic nervous system. Despite

intensive multi-modal therapy, only half of high-risk patients survive while suffering from severe and long-term

treatment related side effects. While our discovery of ALK activating mutations opened the way to precision

medicine in a subset of about 10% of patients, the options for targeted therapy for the remainder of patients is

still limited. Although anti-GD2 immunotherapy raised hope, this approach only modestly improves survival while

often associated with neuropathic pain. Also, CAR-T ceil therapy is being explored but success is currently

hampered by multiple barriers. To widen the options for novel therapies, the host lab has explored the highly

recurrent DNA copy number gains as entry points and discovered several novel (potentially) druggable targets

related to transcriptional addiction (TBX2, SOX11) and replicative stress resistance (BRIP1, BRCA1, RRM2).

MYCN amplification, observed in half of high-risk cases, is known to cause replicative stress. This results from

replication fork stalling due to transcription-replication conflicts through MYCN enhanced global transcription

levels and depletion of essential co-factors for efficient DNA replication. We hypothesized that in emerging NB

cells recurrent DNA copy number gain are selected mediating gene dosage effects for genes essential for coping

replication fork stalling. These include BRIP1 and BRCA1 implicated in homologous DNA repair during replication

fork restart while multiple other DNA damage response and homologous repair genes are also upregulated

through other mechanisms. To target this presumed 'addiction', I have extensively explored the novel CDK12

inhibitor THZ531 which was recently shown to mediate global down regulation of expression of multiple DNA

damage response genes. THZ531 indeed strongly affected NB cell viability in the nanomolar range with evidence

of strong induction of DNA damage as monitored by yHA2X immunostaining. Given that monotherapy in general

often suffers from the development of resistance mechanisms and subsequent relapse, I subsequently screened

for the identification of a powerful synergistic combination therapy with THZ531 in NB. By serendipity I found that

combination treatment of THZ531 and MDM2 inhibitor Nutlin-3 leads to a very strong synergistic drug response

in TP53 mutated NB cell lines. I hypothesize that this synergism is mediated through TP53 independent Nutlin-

3 effects, one of which could be drug efflux pump inhibition. As TP53 mutations are recurrent in relapsed NB (up

to 15%) and can be anticipated to be even further positively selected upon in the currently initiated idasanutlin

phase I ITCC coordinated clinical trials, I propose that the observed THZ531-Nutlin-3 synergism could be

exploited towards second line therapy for therapy refractory NB. To finalize this essential and exciting part of my

PhD, I aim to include the further last experiments focused on the unravelling of the molecular basis underlying

this promising synergistic effect as well as assessing the effects of THZ531 -Nutlin-3 in TP53 mutated NB tumor

organoids as a relevant pre-clinical model system.