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 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.

泌尿生殖系统恶性肿瘤是临床医师面临的重大挑战，其发病率逐年攀升。此外，顺铂治疗可引发长期毒性反应，并催生治疗耐药性，这凸显了开发新型治疗手段的迫切需求。本研究针对新型组蛋白去乙酰化酶（HDAC）抑制剂与含溴结构域及额外末端结构域（BET）抑制剂展开效应分析，旨在将二者整合为强效HDAC-BET融合分子，并阐明其分子作用机制。将HDAC抑制剂、BET抑制剂以及双重抑制剂分别作用于（顺铂耐药型）生殖细胞肿瘤（GCT）、尿路上皮癌、肾癌与前列腺癌细胞后，可降低细胞活力、诱导细胞凋亡，并影响细胞周期进程。此外，双重抑制剂可显著降低生殖细胞肿瘤异种移植模型中的肿瘤负荷。在分子层面，通过关联RNA测序与ATAC测序数据发现，应用HDAC抑制剂后，基因表达得到显著诱导，同时染色质可及性出现位点特异性改变。上调基因可与细胞内外物质运输、细胞组织结构以及包括神经内分泌分化在内的神经元活动过程相关联。从全局层面分析染色质可及性时，HDAC抑制剂处理后，DNA可及性的激活与抑制状态呈现均匀分布，这对当前关于HDAC抑制剂作用机制的认知提出了质疑。综上，本研究开发的HDAC抑制剂、BET抑制剂以及双重抑制剂可为泌尿生殖系统恶性肿瘤提供全新的治疗选择。此外，本研究揭示了受试表观遗传药物（epi-drugs）的新型分子与表观遗传调控机制，有助于更深入理解其潜在作用模式，并为患者的风险评估提供依据。