Abstract 214: Chromatin profiling from formalin-fixed paraffin-embedded samples for precision oncology

Abstract As the oncology field moves towards a precision medicine model of patient care, understanding and profiling the epigenetic landscape is increasingly important. Gene expression and cell function are regulated by epigenomic features, including histone post-translational modifications (PTMs), transcription factors, and other chromatin proteins. Mapping the location of these features provides a powerful approach to study chromatin mechanisms driving disease and can be leveraged for biomarker and drug development. Formalin-fixed paraffin-embedded (FFPE) tissues banked from cancer clinical trials could be a rich resource for retrospective biomarker studies, due to their association with drug response and disease progression data. However, using FFPE tissues for chromatin mapping assays has been technically challenging for multiple reasons. For instance, the heavy cross-linking conditions make FFPE samples unsuitable for chromatin accessibility profiling (ATAC-seq), while the small amounts of chromatin extracted from FFPE tissues make standard epigenomic mapping assays (ChIP-seq) impractical. Combined, these difficulties have precluded widespread application of FFPE tissues for epigenomics research. To better leverage chromatin profiling for translational research, we are developing a modified CUT&Tag workflow that is compatible with FFPE samples (CUT&Tag-FFPE). CUT&Tag (Cleavage Under Targets and Tagmentation) is an immunotethering-based strategy that uses pAG-Tn5 to selectively cleave and ligate sequencing adapters at antibody-bound chromatin. The entire assay takes place in intact cells or nuclei bound to a solid support, enabling a highly streamlined, 2-day workflow. Compared to ChIP-seq, CUT&Tag requires 10-1,000X fewer cells, 5-10X reduced sequencing reads, and is scalable for large studies. The improved sensitivity of CUT&Tag makes it an attractive technique in fields limited by cell number, particularly preclinical research studies. Here, we will discuss optimization of upstream sample processing steps for efficient extraction of nuclei from heavily fixed cells and FFPE tissue. We have applied these modifications to successfully map RNA polymerase II (RNAPII) in heavily fixed cell lines, resulting in high quality chromatin profiles comparable to native controls. We are currently applying this workflow to map RNA Pol II in FFPE liver tissue samples. Future work will be done to validate additional chromatin targets using matched FFPE and fresh frozen tissue samples from various tissues. In parallel, we are optimizing workflows to pair with downstream single cell and spatial platforms to expand the impact of this technology. We envision that CUT&Tag-FFPE, and the associated automated assay service we are developing for large scale clinical trials, will accelerate the discovery of epigenetic drug targets and biomarkers to advance precision medicine. Citation Format: Ellen N. Weinzapfel, Alysha E. Simmons, Eva Brill, Vishnu U. Sunitha Kumary, Eva Oswald, Georg Kuales, Julia B. Schueler, Martis W. Cowles, Bryan J. Venters, Michael-Christopher Keogh. Chromatin profiling from formalin-fixed paraffin-embedded samples for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 214.