MDB-28. High-content Imaging-based drug screen for selective targeting of Extrachromosomal DNA in Medulloblastoma

Extrachromosomal DNA (ecDNA) amplifications play a critical role in driving oncogene overexpression, contributing to the aggressive progression and treatment resistance of Group 3 Medulloblastoma. Developing targeted therapies against ecDNA remains a significant unmet clinical need. Therefore, we have now developed a high-throughput drug screening assay to identify inhibitors that selectively target ecDNA and can serve as potential therapeutic agents. First, we have optimized sample preparation in a 96-well plate format to enrich for metaphase spreads and to reliably detect ecDNA. High-content images are acquired using the Opera Phenix High Content Screening System, enabling automated detection of these metaphase spreads. Furthermore, we have devised an automated computational method to distinguish ecDNA from chromosomal DNA within cellular nuclei, specifically adapted for high-throughput detection in a 96-well plate format. We are now integrating these refined imaging and analysis workflows into a high-throughput format to systematically evaluate a collection of ∼500 small molecules in selected medulloblastoma cell lines with ecDNA-amplifications. Validation studies, including dose-response experiments and secondary assays, will confirm the specificity and efficacy of candidate drugs that significantly reduce ecDNA loads. By characterizing these compounds, we aim to gain insights into their effects on ecDNA dynamics and oncogene expression, ultimately contributing to the development of novel therapeutic strategies for ecDNA-positive medulloblastoma and other ecDNA-driven cancers