Margaretha A. Skowron

Background: The tight junction protein CLDN6 has been identified as a cancer‐associated cell surface marker that is rarely expressed in healthy tissues. In testicular germ cell tumors (GCT), CLDN6 is particularly detectable in seminomas, embryonal carcinomas, and choriocarcinomas. However, little is known about the biological function of CLDN6 in GCT. Objectives: This study aimed at characterizing the functional and molecular role of CLDN6 in GCT. Materials and Methods: Using CRISPR/Cas9 gene editing, CLDN6 ‐deficient GCT cell lines (TCam‐2, 2102EP, NCCIT, and JAR) were generated and validated on gene (PCR), mRNA (qRT‐PCR), and protein level (flow cytometry). The functional impact of CLDN6 ‐deficiency was assessed via proliferation, migration, adhesion, and aggregation assays in both 2D and 3D cell culture models and visualized by electron microscopy. Further, transcriptome‐ and proteome‐wide analyses were performed using RNA‐sequencing and mass spectrometry, respectively. Results: Compared to their respective parental controls, CLDN6 ‐deficient GCT cells showed diminished proliferative, migratory, and adhesive potential, as well as reduced capacity to form cellular aggregates, which could be attributed to disrupted cell‐cell contacts. The molecular analyses revealed only very few transcriptome‐ or proteome‐wide changes upon CLDN6 loss, suggesting a minor role in influencing gene expression. Discussion and conclusion: This study highlights the role of CLDN6 in cell adhesion, migration, and proliferation of GCT cells, suggesting that, in addition to its suitability as a therapeutic target, CLDN6 may also play a putative role during tumor progression.

Alternative therapeutic options targeting urologic malignancies, such as germ cell tumours, as well as urothelial, renal and prostate carcinomas, are still urgently needed. The membrane protein CD24 represents a promising immunotherapeutical approach. The present study aimed to decipher the molecular function of CD24 in vitro and evaluate the cytotoxic capacity of a third‐generation natural killer (NK) cell chimeric antigen receptor (CAR) against CD24 in urologic tumour cell lines. Up to 20 urologic tumour cell lines and several non‐malignant control cells were included. XTT viability assays and annexin V/propidium iodide flow cytometry analyses were performed to measure cell viability and apoptosis rates, respectively. Co‐immunoprecipitation followed by mass spectrometry analyses identified direct interaction partners of CD24. Luciferase reporter assays were used to functionally validate transactivation of CD24 expression by SOX2. N‐ and O‐glycosylation of CD24 were evaluated by enzymatic digestion and mass spectrometry. The study demonstrates that SOX2 transactivates CD24 expression in embryonal carcinoma cells. In cells of different urological origins, CD24 interacted with proteins involved in cell adhesion, ATP binding, phosphoprotein binding and post‐translational modifications, such as histone acetylation and ubiquitination. Treatment of urological tumour cells with NK‐CD24‐CAR cells resulted in a decreased cell viability and apoptosis induction specifically in CD24⁺ tumour cells. Limitations of the study include the in vitro setting, which still has to be confirmed in vivo. In conclusion, we show that CD24 is a promising novel target for immune therapeutic approaches targeting urologic malignancies.

Urological malignancies represent major challenges for clinicians, with annually rising incidences. In addition, 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 to understand 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 HDAC inhibitor 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.

Urological malignancies represent major challenges for clinicians, with annually rising incidences. In addition, 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 to understand 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 HDAC inhibitor 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.

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

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

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

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