title: Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13
creator: Friedrich, Mona Soledad
subject: ddc-570
subject: 570 Life sciences
description: Neuroblastoma (NB) is an extracranial childhood cancer and accounts for 15 % of all childhood cancer-related deaths. It is a remarkably heterogeneous disease and cases range from spontaneous regression to aggressive high risk disease. Amplification of the oncogene MYCN occurs in 20-30 % of cases and is an indicator of poor prognosis. As a transcription factor, MYCN drives several cellular programs favoring malignant behavior, for example proliferation, angiogenesis and metastasis. In order to gain a better understanding of direct and secondary transcriptional targets of MYCN, gene expression was analyzed in a time course experiment using cell cycle-synchronized cells with regulatable MYCN level. This approach revealed the continued upregulation of a number of cell cycle driver genes as well as genes involved in protein and DNA biosynthesis in MYCN high cells. Differences in the expression of other cell cycle and DNA replication genes occurred only in specific phases of the cell cycle. On the other hand, genes involved in alternative splicing and cell adhesion were constantly downregulated. The expression of snoRNAs strongly increased in the MYCN low condition towards the end of the observation period. An analysis of miRNA expression revealed that many differentially regulated miRNAs targeted genes involved in ribosome biogenesis, cancer processes and signaling pathways. Taken together, this data set indicates that MYCN directly regulates the expression of genes and miRNAs which contribute to accelerated proliferation, metabolism and metastatic growth in NB cells. As a consequence of the induced phenotype, a larger number of secondary targets are deregulated.  As MYCN heavily contributes to tumor malignancy, MYCN-amplified NB cells become addicted to high amounts of the protein. However, MYCN itself is notoriously difficult to target, therefore two siRNA screens were performed to detect synthetic lethal relationships with high MYCN levels. The second part of this thesis deals with the transcriptional kinase CDK13, which was identified as a potential candidate for novel targeted therapies. CDK13 and its highly homologous family member CDK12 were knocked down by several technical approaches. In a cellular MYCN overexpression model, CDK13 repression induced strong cell death only in MYCN high cells. CDK12 repression also elicited a small amount of cell death comparing MYCN high with low cells. Inducible CDK13 knockdown in a MYCN-amplified cell line caused modest reductions in cellular viability and colony formation capacity. CDK13, but not CDK12, knockout induced by the clustered regularly interspaced short palindromic repeats (CRISPR) technique reduced viability and caused a small increase in cells arrested in G1. However, analysis of CDK13 mRNA level revealed significant residual expression, suggesting that the majority of the polyclonal culture might be heterozygous for the induced mutation. A novel small compound inhibitor against CDK12 and CDK13 (BAY-587) was tested in a panel of eight NB cell lines and the effects were compared to that of a commercially available inhibitor, THZ531. BAY-587 was active at lower concentrations than THZ531. Both compounds strongly reduced viability, colony formation capacity and disrupted cell cycle distributions. BAY-587 treatment reduced the level of CDK12 protein and further induced apoptosis. Gene expression analysis revealed that CDK12/13 inhibition by BAY-587 caused downregulation of genes involved in alternative splicing and DNA damage repair, while transcription regulation genes were upregulated. In summary, CDK13 emerged as a promising new therapeutic candidate for the treatment of high risk NB patients.
date: 2021
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/26558/1/Dissertation_Friedrich.pdf
identifier: DOI:10.11588/heidok.00026558
identifier: urn:nbn:de:bsz:16-heidok-265586
identifier:   Friedrich, Mona Soledad  (2021) Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/26558/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng