Molecular analysis of the cdk1/CCNB1 complex and characterization of cdk-inhibitors in neuroblastoma

Das Neuroblastom ist der häufigste solide extrakranielle Tumor bei Kindern. Er entwickelt sich aus Vorläuferzellen der Neuralleiste und kann sich entlang des sympathischen Grenzstranges entwickeln. Die Heilungschancen gerade bei Rezidivpatienten sind mit einer 20 % 5-Jahres-Überlebensrate ausgesprochen schlecht insbesondere bei Vorliegen einer MYCN Amplifikation. Deshalb ist es notwendig, neue Therapiemöglichkeiten zu entwickeln oder bestehende zu verbessern. Aus diesem Grunde wurde in dieser Arbeit nach möglichen Targets für weitere Therapieansätze gesucht. Dazu wurden vorhandene Exon-Array-Daten primärer NB reanalysiert und nach Genen gesucht, die mit einem schlechten Überleben der Patienten korrelieren. Bei MYCN handelt es sich um einen Transkriptionsfaktor, der mit einem sehr aggressiven Verlauf der Krankheit verknüpft ist. So wurden als mögliche Zielstrukturen die Zellzyklusregulatoren cdk1 und der dazugehörige Interaktionspartner Cyclin B1 identifiziert und standen hier im Fokus der Untersuchungen. Es konnte gezeigt werden, dass cdk1 und CCNB1 in den getesteten Neuroblastomzelllinien überexprimiert sind. Ein direkter Zusammenhang zwischen dem MYCN-Status und der cdk1 bzw. CCNB1 Expression konnte in den Zelllinien jedoch nicht bestätigt werden. Gerade in den letzten Jahrzehnten wurde massiv an neuen Inhibitoren geforscht, die sich gegen Enzyme des Zellzyklus richten. Zu diesen neu entwickelten Inhibitoren gehört auch der cdk1 spezifische Inhibitor (RO33 06) und der pan cdk-Inhibitor (JNJ-77706621). Die beiden Inhibitoren wurden in dieser Arbeit verwendet, um die cdk1 Aktivität zu hemmen und so cdk1 als potentielles Therapieziel zu validieren. Hierbei konnte ein Zusammenhang zwischen dem Effekt der Inhibitoren und dem p53 Status der Zellen festgestellt werden. Zellen mit einer p53 Mutation bzw. einem geringen p53 Level zeigten sich wesentlich resistenter gegenüber beiden Inhibitoren. Des Weiteren konnte eine Aktivierung des p53 Signalweges durch RO3306 in den p53 wt Zellen nachgewiesen werden. Ebenso konnte gezeigt werden, dass die durch RO3306 ausgelöste Apoptose durch Caspasen vermittelt wird. Weiterhin konnte in dieser Arbeit gezeigt werden, dass die duale Inhibition von cdk1 und dem NB-spezifischen Onkogen ALK unter definierten Bedingungen einen synergistischen Effekt auf die Zellviabilität haben kann. Abschließend lässt sich sagen, dass unter Berücksichtigung des p53 Status die cdk1-Inhibition als Therapieansatz im Neuroblastom denkbar wäre. Um eine endgültige Aussage darüber treffen zu können, müssten die generierten Ergebnisse in „in vivo“ Versuchen validiert werden.Neuroblastoma (NB) is the most solid extracranial tumor of childhood. It originates from precursors of the neural crest and normally arises along the sympathetic trunk in the abdomen or neck. The curing prospects of relapse patients are markedly bad with a 5-year-survival-rate of 20%. Disease recurrence frequently affects patients harboring specific MYCN gene amplification. Therefore it is necessary to develop new therapies or to optimize the old ones. To explore future therapy opportunities Exon Array data of primary NBs were reanalyzed for genes correlating with overall survival. By doing so, we identified master regulators of the cell cycle, cdk1 and the corresponding interaction partner Cyclin B1, as candidate genes. In this thesis it could be shown that cdk1 and CCNB1 were overexpressed in neuroblastoma cell lines. No correlation between cdk1 or CCNB1 expression and MYCN status could be confirmed in these cells. As novel cell cycle inhibitors were developed in recent years, both a cdk1 specific inhibitor (RO3306) as well as a pan cdk-inhibitor (JNJ-7706621) were used in this thesis to inhibit cdk1 activation and to evaluate cdk1 as potential target for NB therapy. Interestingly, sensitivity to inhibitor treatment correlated with the p53 status of the cells. Cells harboring p53 mutations or presenting with lower p53 levels were more resistant to the inhibitors than p53 wt cells. Additionally it was shown that RO3306 activated the p53 signaling pathway as well as caspase mediated apoptosis. Furthermore, it was shown that the dual inhibition of cdk1 and NB-specific oncogene ALK has synergistic effects on cell viability depending on inhibitor concentration and on the order of treatment administration. Taken together cdk1 inhibition warrants further investigations as therapeutic approach in neuroblastoma given that the p53 status is taken into account. These findings deserve validation in “in vivo” experiments

Sensitivity to cdk1-inhibition is modulated by p53 status in preclinical models of embryonal tumors

Dysregulation of the cell cycle and cyclin-dependent kinases (cdks) is a hallmark of cancer cells. Intervention with cdk function is currently evaluated as a therapeutic option in many cancer types including neuroblastoma (NB), a common solid tumor of childhood. Re-analyses of mRNA profiling data from primary NB revealed that high level mRNA expression of both cdk1 and its corresponding cyclin, CCNB1, were significantly associated with worse patient outcome independent of MYCN amplification, a strong indicator of adverse NB prognosis. Cdk1 as well as CCNB1 expression were readily detectable in all embryonal tumor cell lines investigated. Pharmacological inhibition or siRNA-mediated knockdown of cdk1/CCNB1 induced proliferation arrest independent of MYCN status in NB cells. Sensitivity to cdk1 inhibition was modulated by TP53, which was demonstrated using isogenic cells with wild-type TP53 expressing either dominant-negative p53 or a short hairpin RNA directed against TP53. Apoptosis induced by cdk1 inhibition was dependent on caspase activation and was concomitant with upregulation of transcriptional targets of TP53. Our results confirm an essential role for the cdk1/CCNB1 complex in tumor cell survival. As relapsing embryonal tumors often present with p53 pathway alterations, these findings have potential implications for therapy approaches targeting cdks

Robust selection of cancer survival signatures from high-throughput genomic data using two-fold subsampling

Identifying relevant signatures for clinical patient outcome is a fundamental task in high-throughput studies. Signatures, composed of features such as mRNAs, miRNAs, SNPs or other molecular variables, are often non-overlapping, even though they have been identified from similar experiments considering samples with the same type of disease. The lack of a consensus is mostly due to the fact that sample sizes are far smaller than the numbers of candidate features to be considered, and therefore signature selection suffers from large variation. We propose a robust signature selection method that enhances the selection stability of penalized regression algorithms for predicting survival risk. Our method is based on an aggregation of multiple, possibly unstable, signatures obtained with the preconditioned lasso algorithm applied to random (internal) subsamples of a given cohort data, where the aggregated signature is shrunken by a simple thresholding strategy. The resulting method, RS-PL, is conceptually simple and easy to apply, relying on parameters automatically tuned by cross validation. Robust signature selection using RS-PL operates within an (external) subsampling framework to estimate the selection probabilities of features in multiple trials of RS-PL. These probabilities are used for identifying reliable features to be included in a signature. Our method was evaluated on microarray data sets from neuroblastoma, lung adenocarcinoma, and breast cancer patients, extracting robust and relevant signatures for predicting survival risk. Signatures obtained by our method achieved high prediction performance and robustness, consistently over the three data sets. Genes with high selection probability in our robust signatures have been reported as cancer-relevant. The ordering of predictor coefficients associated with signatures was well-preserved across multiple trials of RS-PL, demonstrating the capability of our method for identifying a transferable consensus signature. The software is available as an R package rsig at CRAN (http://cran.r-project.org)

Complementary and alternative medicine in the (symptomatic) treatment of acute tonsillitis in children: A systematic review

Background: Acute tonsillitis (including tonsillopharyngitis) in childhood is a common disease with a peak of illness in school-age. Most of these cases have a viral origin and antibiotic therapy is not indicated, therefore, effective symptomatic therapy is required. For this reason, complementary, alternative and integrative medicine therapies might be a solution. Objective: The aim of this review is to demonstrate study status of such therapies. Methods: The databases PubMed, Cochrane Library, OVID, CAMbase, CAM-QUEST® and Anthromedics were systematically screened for studies investigating complementary, alternative and integrative therapy approaches in paediatric cohorts. Studies were analyzed by therapy approach, study design, cohort and outcome, using the PRISMA 2020 checklist. Results: The systematic literature search resulted in 321 articles. Five publications corresponded to the search criteria and were assigned to the following specific therapeutic categories: herbal medicine (3), homeopathy (1) and ayurvedic medicine (1). Clinical trials were found for the herbal compounds BNO 1030 (Impupret®) and EPs® 7630 (Umckaloabo), the homeopathic complex Tonzolyt® and the ayurvedic medicine Kanchnara-Guggulu and Pratisarana of Tankana‑Madhu. Antimicrobial effects of essential oils and carvacrol as single agents as well as in combination with erythromycin were analysed in an in vitro study. Conclusion: Clinical studies indicate an improvement of symptoms and a good tolerability of all investigated remedies of complementary, alternative and integrative medicine in the treatment of tonsillitis in childhood. Nevertheless, quality and quantity of the studies were insufficient to make a reliable conclusion regarding effectiveness. Therefore, more clinical trials are urgently needed to achieve a meaningful result

Zebrafish as an Orthotopic Tumor Model for Retinoblastoma Mimicking Routes of Human Metastasis

Background: Retinoblastoma (RB) is the most common eye cancer in children that has a high mortality rate when left untreated. Mouse models for retinoblastoma have been established but are time- and cost-intensive. The aim of this work was to evaluate an orthotopic transplantation model of retinoblastoma in zebrafish that also allows for tracking migratory routes and to explore advantages and disadvantages with respect to drug testing. Methods: Three fluorescence-labeled retinoblastoma cell lines (RB355, WERI-RB-1, Y79) were injected into the left eye of two-day-old zebrafish, while the un-injected right eye served as control. The migratory trajectories of injected retinoblastoma cells were observed until 8 days post injection (dpi), both in lateral and dorsal view, and measuring fluorescence intensity of injected cells was done for RB355 cells. Results: Time until the onset of migration and routes for all three retinoblastoma cell lines were comparable and resulted in migration into the brain and ventricles of the forebrain, midbrain and hindbrain. Involvement of the optic nerve was observed in 10% of injections with the RB355 cell line, 15% with Y79 cells and 5% with WERI-RB-1 cells. Fluorescence intensity of injected RB355 cells showed an initial increase until five dpi, but then decreased with high variability until the end of observation. Conclusion: The zebrafish eye is well suited for the analysis of migratory routes in retinoblastoma and closely mirrors patterns of retinoblastoma metastases in humans

Expert consensus-based clinical recommendation for an integrative anthroposophic treatment of acute bronchitis in children: A Delphi survey

Background: Acute bronchitis is one of the most common pediatric diseases. In addition to conventional therapies, a frequent use of complementary and alternative medicine (CAM) has been stated. Anthroposophic medicine (AM) is one of the most practiced complementary and integrative medicine (CIM) approaches in Central Europe but hitherto no consensus-based clinical recommendations or guidelines are available.Therefore, a consensus-based recommendation leading to an informed and reasonable use of AM in the treatment of acute bronchitis in pediatrics was developed. Methods: A total of 61 physicians in Germany with expertise in the field of anthroposophic pediatrics was invited to complete an online multistep Delphi process. Two independent reviewers quantitatively and qualitatively evaluated the results. The survey was completed when >75 % consensus was achieved. Results: The clinical recommendation comprises 15 subitems related to treatment as well as clinical and psychosocial aspects. All items reached strong consensus (>90 %; N = 9) or consensus (75–90 %; N = 6). Conclusion: The comprehensive clinical recommendation creates a scientific base for the anthroposophic integrative treatment of acute bronchitis in children in Germany. It will make the anthroposophic approach more applicable, understandable and comparable to a wider public of physicians and other health professionals in Germany

Comparative analysis of resource utilization in integrative anthroposophic and all German pediatric inpatient departments

Background: Integrative medicine (IM) combines conventional and complementary therapies. It aims to address biological, psychological, social, spiritual and environmental aspects of patients' health. During the past 20 years, the use and request of IM in children and adults has grown. Anthroposophic Medicine (AM) is an IM approach frequently used in children in Germany. From both public health and health economic perspectives, it is relevant to investigate whether there are differences in the resource utilization between integrative pediatric departments (IPD) and the entirety of all pediatric departments. Methods: Standard ward documentation data from all German integrative anthroposophic pediatric departments (2005-2016; N=29,956) is investigated and systematically compared to data of the entirety of all pediatric departments in Germany derived from the Institute for the Hospital Reimbursement System (2005-2016, N=8,645,173). The analyses focused on: length of stay, Diagnosis Related Groups (DRG), Major Diagnosis Categories (MDC), and effective Case Mix Index (CMI). Results: The length of stay in the IPD (M=5.387.31) was significantly shorter than the DRG defined length of stay (M=5.8 ± 4.71; p<.001; d=-0.07) and did not exceed or undercut the DRG covered length of stay. Compared to the entirety of all pediatric departments (M=4.74<plus/minus>6.23) the length of stay was significantly longer in the in the IPD (p <. 001; d=0.12). The effective CMI in IPD and all pediatric departments were identical (M=0.76). The frequencies of DRG and MDC differed between IPD and all pediatric departments, with higher frequencies of DRGs and MDCs associated with chronic and severe illnesses in the IPD. Conclusions: Treatment within integrative anthroposophic pediatric departments fits well in terms of the DRG defined conditions concerning length of stay, even though integrative pediatric patients has an increased length of stay of averagely 1 day, which is most likely associated to time consuming, complex integrative treatment approaches and to a certain extend to higher amount of chronic and severe diseases

Zebrafish as an orthotopic tumor model for retinoblastoma mimicking routes of human metastasis

Background: Retinoblastoma (RB) is the most common eye cancer in children that has a high mortality rate when left untreated. Mouse models for retinoblastoma have been established but are time- and cost-intensive. The aim of this work was to evaluate an orthotopic transplantation model of retinoblastoma in zebrafish that also allows for tracking migratory routes and to explore advantages and disadvantages with respect to drug testing. Methods: Three fluorescence-labeled retinoblastoma cell lines (RB355, WERI-RB-1, Y79) were injected into the left eye of two-day-old zebrafish, while the un-injected right eye served as control. The migratory trajectories of injected retinoblastoma cells were observed until 8 days post injection (dpi), both in lateral and dorsal view, and measuring fluorescence intensity of injected cells was done for RB355 cells. Results: Time until the onset of migration and routes for all three retinoblastoma cell lines were comparable and resulted in migration into the brain and ventricles of the forebrain, midbrain and hindbrain. Involvement of the optic nerve was observed in 10% of injections with the RB355 cell line, 15% with Y79 cells and 5% with WERI-RB-1 cells. Fluorescence intensity of injected RB355 cells showed an initial increase until five dpi, but then decreased with high variability until the end of observation. Conclusion: The zebrafish eye is well suited for the analysis of migratory routes in retinoblastoma and closely mirrors patterns of retinoblastoma metastases in humans