Identification of cisplatin resistance mechanisms and alternative treatment options for testicular germ cell tumors

Abstract

Type II testicular germ cell tumors (TGCTs) are the most prevalent tumors in young men aged 18 to 35 years. TGCTs are classified as seminomas and non-seminomas. Embryonal carcinomas (EC), representing the cancer stem cell population of non-seminomas, can differentiate into yolk sac tumors (YST), choriocarcinomas (Cc) and teratomas. There are curation rates of 95% due to high sensitivity towards cisplatin-based chemotherapy. However, several patients develop cisplatin resistant tumors or drug intolerance facing poor prognosis due to lacking treatment alternatives. With this study I aimed to identify novel cisplatin resistance mechanisms and investigated the potential of different compounds for TGCT treatment.<br /> Firstly, cyclin dependent kinase (CDK) inhibitors were investigated. While CDK inhibitors targeting the cell cycle have been studied extensively, no data were available describing transcriptional CDK (tCDK) inhibitors in TGCTs. tCDKs (CDK7, 8, 9 12, 13) are crucial for RNA polymerase II mediated mRNA transcription. Initially, the cytotoxic effects of Dinaciblib as well as Flavopiridol (both panCDK inhibitors), YKL-5-124 (CDK7), SY0351 (CDK7, 12, 13), THZ1 (CDK7, 12, 13), THZ531 (CDK12, 13), NVP2 (CDK9) and the CDK9 degrader THAL-SNS-032 were investigated on cisplatin resistant and cisplatin sensitive TGCT cell lines. Application of SY0351 and NVP2 showed very strong decrease in viability on the seminoma (TCam2) and EC (2102EP, NCCIT) cell lines as well as on the resistant sub cell lines (2102EP-R, NCCIT-R). YKL-5-124 treatment revealed a highly cytotoxic effect in 2102EP and 2102EP-R cells. The viability of the fibroblast cell line (MPAF) was not affected at all upon drug application. In 2102EP and TCam2 cells high levels of apoptosis induction as well as moderate cell cycle deregulation have been detected after NVP2, SY0351 and YKL-5-124 treatment. On mRNA level different cellular responses have been identified after NVP2 and SY0351 application while YKL-5-124 treated cells revealed a common response. NVP2 exposure of 2102EP and TCam2 cells resulted in deregulation of transcription and downregulation of mRNA processing, respectively. After YKL-5-124 application reduced mRNA processing and negative regulation of gene expression in 2102EP, TCam2 and MPAF cells was observable. The key findings after SY0351 exposure were upregulation of stress response and ubiquitin pathway deregulation. Thus, especially the cell line specific response after treatment with the highly potent inhibitors NVP2 and SY0351 suggests an opportunity for personalized therapy in seminomas and ECs.<br /> Next, I aimed to identify cisplatin resistance mechanisms in TGCTs using a genome scale CRISPR/Cas9 activation screen in 2102EP and JAR (Cc) cell lines. Aside from already known factors involved in DNA damage repair and cell cycle regulation, overexpression of the neddylation pathway core component NAE1 resulting in overactivated neddylation has been found. Neddylation is a posttranslational modification which regulates stability, function and localization of target proteins. The best characterized substrates of neddylation are cullins which are part of cullin-RING ligases (CRLs). Cullin neddylation activates the E3 ubiquitin ligase function of the CRL complex revealing polyubiquitination and subsequent proteasomal degradation of various substrates such as tumor suppressor proteins p21 and p27. Cisplatin resistance in TGCTs based on increased neddylation pathway activity could be validated by NAE1/GFP overexpression showing significantly decreased cisplatin sensitivity. Interestingly, NAE1 abundance is highest in TGCT tissues compared to other tumor entities and normal tissues. Thus, targeting the neddylation pathway seems to be a reasonable strategy. The covalent NAE1 inhibitor MLN4924 revealed strong cytotoxic effect in different TGCT cell lines. Most interestingly, MLN4924 application in combination with cisplatin significantly increased cytotoxicity compared to mono treatment in cisplatin sensitive and resistant cell lines. Further, combination treatment revealed G2/M-phase cell cycle arrest as well as strong apoptosis induction in 2102EP, JAR and TCam2 cells. Notably, MPAF fibroblast control cells were not affected all. Transcriptome analysis of 2102EP and JAR cells revealed elevated expression of apoptosis associated genes, deregulation of cell cycle and strong mesoderm/endoderm differentiation tendencies upon MLN4924 treatment in combination with cisplatin.<br /> To conclude, NVP2, SY0351 as well as MLN4924 revealed not only strong cytotoxic effects on parental TGCT cell lines but also on cisplatin resistant cell lines. Thus, these compounds might be promising alternative treatment options for TGCT patients independent of chemotherapy resistance.