Giovanni Marchi

Summary: DNA methylation (DNAm) of neighbouring CpG sites is highly correlated, making DNAm function in terms of blocks. DNAm patterns and functionality are linked to both chromatin structure of DNA and gene regulation. Defining biologically meaningful DNA methylation blocks from whole-genome bisulfite sequencing (WGBS) data remains challenging, as most existing methods rely on fixed genomic windows rather than the observed methylation pattern. We present FUSE, a data-driven segmentation method that captures intrinsic methylation segments directly from WGBS data by jointly analyzing multiple samples. FUSE identifies spatially homogeneous methylation blocks shared across the input cohort while allowing different methylation states across samples. Applied to 61 WGBS samples from the ENCODE database, FUSE identified segments which overlap significantly with promoters, enhancers, and repetitive elements. FUSE was able to recover the true segment breakpoints in synthetic data with high sensitivity under increased levels of noise. As such, FUSE facilitates post hoc methylation analyses by aggregating coherent CpG sites into candidate segments for downstream differential methylation testing or other comparative studies. Availability and implementation: FUSE is implemented as an R-package methFuse, available at https://github.com/holmsusa/methFuse and https://cran.r-project.org/package=methFuse. A GenomeSpy visualization of the data is available at https://csbi.ltdk.helsinki.fi/p/fuse_encode_gs/

Identifying new therapeutic approaches in high-grade serous ovarian cancer (HGSC) requires the development of more accurate preclinical models that replicate the patient-specific tumor and its microenvironment. To address this, we established immunocompetent patient-derived cultures (iPDC) for HGSC, cultured on a physiologically relevant human omentum gel matrix. We developed a high-throughput platform that combines drug testing, histologic analysis, genomic profiling, single-cell studies, and spatial biomarker discovery. Our results from 47 tumors showed that iPDCs recapitulated the tumor genomic and histologic characteristics while also retaining the intratumoral immune cells. The iPDC treatment responses correlated significantly with the patients’ clinical treatment responses. Using iPDCs and single-cell RNA sequencing, we identified potentially effective therapeutic options for patients with recurrent HGSC linked to distinct tumor cell states and mechanisms of resistance. High-throughput drug response profiling with single-cell imaging identified ataxia telangiectasia and Rad3-related inhibitor (ATRi) combined with an immunotherapy targeting autotaxin as a promising new combination treatment for HGSC. Using hyperplexed imaging and spatial analysis, we discovered that ATRi responses were associated with significant increases in both intra- and peritumoral T-cell infiltration, particularly in PD-1+ CD8+ T cells. Additionally, the ATRi-induced reactivation of CD8+ T cells was linked to spatial interactions with replication stress–positive tumor cells. Thus, our iPDC platform presents a representative high-throughput ex vivo model to advance precision oncology in HGSC, uncovering the ATRi-immunotherapy combination as a potentially effective therapeutic option for clinical translation.

Understanding how epigenetic mechanisms shape metastatic dissemination and therapy response in ovarian high-grade serous carcinoma (HGSC) has been hindered by the absence of large, anatomically diverse, and longitudinal datasets. Here, we present a comprehensive resource of the HGSC methylome, comprising 404 whole-genome bisulfite sequenced samples from 125 patients belonging to the real-world DECIDER trial. Tumor-specific methylomes were integrated with matched RNA sequencing, whole-genome sequencing and clinical outcomes, which enabled identification of > 8,500 regulatory promoters. Our results show that the regulatory methylome is largely established early in tumor progression and remains stable through chemotherapy. Tumors with poor response to treatment are marked by widespread promoter hypermethylation, particularly cancer cells in ascites, leading to epigenetic silencing of pathways targeted by chemotherapy agents. Our proof-of-principle analysis shows that these epigenetic signatures are detected in plasma-derived DNA, highlighting the value of DNA methylation in liquid biopsy-based disease monitoring. This study provides a foundational resource for dissecting the spatial and temporal dynamics of cancer methylomes and establishes a framework for integrating epigenetic states with clinical trajectories in HGSC.

Identification and functional validation of inactivating mutation on a source RC-L1