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

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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.