Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13

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

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

© 2017 British Journal of Anaesthesia Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32–0.77); P\u3c0.01], but no difference in complication rates [OR 1.02 (0.88–1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62–0.92); P\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London