Background Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs. Results We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers (MZ-1, LP99) and polycomb-repressive complexes (PRT4165, GSK343). Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT. Conclusion Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs.

Urologic malignancies represent major challenges for clinicians with annually rising incidences. Additionally, cisplatin treatment-induced long-term toxicities and the development of therapy resistance emphasize the need for novel therapeutics. In this study, we analyzed the effects of novel histone deacetylase (HDAC) and bromodomain and extraterminal domain-containing (BET) inhibitors to combine them into a potent HDAC-BET-fusion molecule and understanding their molecular mode-of-action. Treatment of (cisplatin-resistant) germ cell tumors (GCT), urothelial, renal and prostate carcinoma cells with the HDAC, BET, and dual inhibitors decreased cell viability, induced apoptosis and affected the cell cycle. Furthermore, a dual inhibitor considerably decreased tumor burden in GCT xenograft models. On a molecular level, correlating RNA- to ATAC-sequencing data indicated a considerable induction of gene expression, accompanied by site-specific changes of chromatin accessibility after HDACi application. Upregulated genes could be linked to intra- and extra-cellular trafficking, cellular organization and neuronal processes including neuroendocrine differentiation. Regarding chromatin accessibility on a global level, an equal distribution of active or repressed DNA accessibility has been detected after HDAC inhibitor treatment, questioning the current understanding of HDAC inhibitor function. In summary, our HDAC, BET, and dual inhibitors represent a new treatment alternative for urological malignancies. Furthermore, we shed light on new molecular and epigenetic mechanisms of the tested epi-drugs, allowing for a better understanding of the underlying modes-of-action and risk assessment for the patient.

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T‐lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high‐throughput approaches of three‐dimensional (3D) co‐cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin‐based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass‐spectrometry‐based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin‐like growth factor (IGF) transport. To evaluate direct cell–cell contacts, green fluorescent protein (GFP)‐expressing GCT cells and mCherry‐expressing TM cells were co‐cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re‐analyses of secretome data with regard to lysine‐ and proline‐hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV‐ or fibronectin‐coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro‐tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin‐based chemotherapy for GCT patients.