Establishment and Evaluation of Dual HDAC/BET Inhibitors as Therapeutic Options for Germ Cell Tumors and Other Urological Malignancies

Urological malignancies represent major challenges for clini-cians, with annually rising incidences. In addition, cisplatin treat-ment 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 to understand their molecular mode-of-action. Treat-ment 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 consider- able induction of gene expression, accompanied by site-specific changes of chromatin accessibility after HDAC inhibitor applica-tion. 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 treat-ment, 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 under-standing of the underlying modes-of-action and risk assessment for the patient.