%0 Generic
%A Nortmeyer, Maike Christine
%D 2019
%F heidok:26656
%R 10.11588/heidok.00026656
%T MYCN dependency of MYCN amplified neuroblastoma cell lines analyzed in relation to their interaction with BET proteins and in a novel orthotopic mouse model
%U https://archiv.ub.uni-heidelberg.de/volltextserver/26656/
%X Neuroblastomas are the most common extracranial solid tumors in early childhood and account for 7% of all pediatric cancers. Amplification of the MYCN gene occurs in 20-30% of neuroblastoma cases and is associated with aggressive cancers and a poor outcome for the patients. Despite decades of research on MYCN and c-MYC and several known functions of these proteins, there are no therapeutic approaches targeting transcription factors of the MYC family.   BET inhibitors inhibit growth and induce apoptosis in several MYC-overexpressing cancer types. Based on these findings, BET inhibitors were tested as part of this thesis for MYCN amplified neuroblastoma in vitro and in vivo. A panel of 23 neuroblastoma cell lines was screened for sensitivity upon treatment with the BET inhibitor JQ1 by using viability assays, soft agar assays and FACS live/dead and cell cycle staining. Based on the viability screening, 55% of the MYCN amplified and 30% of the MYCN non-amplified cell lines were categorized as JQ1 sensitive. Reduced viability was mirrored by the absence of anchorage independent growth and an increase of G1 and sub-G1 fraction after JQ1 treatment. Protein expression data were estimated by western blot and Reverse Phase Protein Array, showing that MYCN protein levels were reduced in 70% of the MYCN amplified neuroblastoma cell lines and that further proteins related to the G1/S-transition of the cell cycle were lower expressed. A shRNA-mediated BRD4 knockdown in MYCN amplified IMR5/75 cells did not affect MYCN protein levels but reduced the expression of Cyclin D1. Global mRNA expression of four JQ1-treated cell lines was analyzed by RNA-sequencing, showing differential expression of genes related to cell cycle, damage response and chromatin methylation. In vivo, treatment with the BET inhibitor BAY1238097 prolonged the survival of mice harboring xenograft tumors deriving from the neuroblastoma cell line LS.  Common neuroblastoma mouse models either belong to the MYCN amplified or to the MYCN non-amplified group of neuroblastoma. No neuroblastoma mouse model is available harboring both a strong and a low MYCN gene expression, allowing the analysis of MYCN on and MYCN off conditions with the same genetic background. A MYCN high/low orthotopic neuroblastoma mouse model was established with a tumor growth rate of 80%. Tumor imaging was performed by Magnet-Resonance-Imaging and in vivo bioluminescence. The MYCN knockdown was analyzed and falcified by quantitative PCR and RNA-sequencing.    In conclusion, BET inhibitors are probably an efficient therapeutic option for a genetically preselected number of neuroblastoma cases, although amplified MYCN alone is not predisposing neuroblastoma tumors for a therapy with BET inhibitors. The MYCN high/low mouse model needs further research for the establishment of a stable MYCN knockdown.