P1: The impact of epigenetic dysregulation associated with mutational crosstalk on driving early bladder cancer progression

在膀胱癌的发生过程中，很少有关于尿路上皮从原位癌（CIS）向肌层浸润性膀胱癌（MIBC）克隆性扩张的表观遗传景观的研究。因此，我们的目标是破译负责基因表达的DNA甲基化（DNAme）的患者内和患者间特异性模式，这些模式可能是肿瘤演变中侵袭性克隆的CIS（in）依赖性膀胱癌进展的原因。计划研究的一个特别优势是使用来自15名患者的组织学映射的膀胱癌标本的匹配组织样本的独特资源，每个患者包括正常尿路上皮（NU），空间上不同的CIS病变和MIBC。外显子组数据可通过DFG资助的另一个项目获得，并将在第一步中通过总体n=87个样本的DNA甲基化和RNA表达的全基因组数据进行补充。将与P7数据一起进行生物统计学分析，以推断个体和患者中肿瘤的遗传学、克隆性和遗传学表观遗传事件。为了指定克隆扩增背景下识别的DNAme模式，将在生物统计管道中实施P2小鼠研究的系统发育信息。在此阶段，将考虑项目P3和P4的已建立的人细胞培养模型的DNAme谱，以提供在编码表观遗传调节因子（如KDM 6A）的基因的遗传改变背景下推定的组蛋白-DNAme串扰的证据。在第二步中，将通过反映肿瘤进展的整个谱（CIS、MIBC和淋巴结和/或远处转移）的患者匹配样品的独立队列（总体n=175）来验证DNAme模式。根据Börries组P7公布的工作流程，将采用留一交叉验证方法提取一组与患者生存相关的候选基因。空间转录组学方法将有助于在体外研究的第三阶段进一步优先考虑功能验证的候选人。这些选定基因的研究将涉及在细胞培养系统（良性和/或恶性膀胱癌细胞系）中引入适当的遗传修饰（过表达或敲低/敲除），然后进行测定，以确定例如细胞增殖、迁移或侵袭。计划基于3D类器官（P6）的瞬时siRNA实验以确认在更生理系统中的结果。最后，我们将提供一个多维的，综合的遗传和表观遗传改变的基础上的不同类型的肿瘤侵袭性克隆的演变。

In bladder carcinogenesis little research on the epigenetic landscapes of urothelial clonal expansions from carcinoma in situ (CIS) towards muscle invasive bladder cancers (MIBC) has been done. Therefore, we aim to decipher intra- and interpatient specific patterns of DNA methylation (DNAme) responsible for gene expression which are potentially causative for CIS (in)dependent bladder cancer progression of invasive clones in tumor evolution. A particular strength of the planned study is the use of a unique resource of matched tissue samples from histologically mapped cystectomy specimens of 15 patients, each comprising normal urothelium (NU), spatially distinct CIS lesions and MIBC. The exome data are available through another DFG-funded project and will be complemented by the genome-wide data of DNA methylation and RNA expression of overall n=87 samples in a first step. Together with P7 data will be biostatistically analyzed allowing the inference of epigenetic events with genetics, clonality and phylogeny of the tumors in individuals as well as across patients. To specify the identified DNAme patterns in the context of clonal expansions, phylogenetic information of the mouse studies of P2 will be implemented into the biostatistical pipeline. At this stage DNAme profiles of established human cell culture models of projects P3 and P4 will be considered to provide evidence for a putative histone-DNAme crosstalk in the context of genetic alterations of genes encoding for epigenetic regulators such as KDM6A. In a second step, DNAme patterns will be validated by an independent cohort of patient-matched samples (overall n=175) reflecting the entire spectrum of tumor progression (CIS, MIBC and lymph-node and/or distant metastases). Following the published workflow of the Börries group P7, a leave-one-out-cross-validation approach will be applied to extract a robust panel of gene candidates associated with patient survival. A spatial transcriptomic approach will help to further prioritize candidates for functional validation upon a third phase of in vitro studies. These studies of selected genes will involve the introduction of appropriate genetic modifications (overexpression or knockdown/knockout) in cell culture systems (benign and/or malignant bladder cancer cell lines) followed by assays to determine, for instance, cell proliferation, migration or invasion. Transient siRNA experiments based on 3D organoids (P6) are planned to confirm results in a much more physiologically system. At the end we will provide a multi-dimensional, integrated view of the genetic and epigenetic alterations underlying the different types of tumor evolution of invasive clones.