Abstract A024: High-throughput characterization of inhibitor impacts on chromatin dysregulation induced by oncofusions

Abstract Oncofusions, cancer-causing chimeric proteins formed by chromosomal rearrangements, are key drivers in approximately 16% of all cancer cases. These fusion proteins are associated with a wide range of malignancies, including varieties of leukemia, lung cancer, and sarcoma. Thousands of unique oncofusions have been identified in recent years, yet most of these fusion proteins lack mechanistic annotation. Many oncofusions dysregulate chromatin architecture in complex ways that are not yet fully elucidated. Investigating these chromatin alterations is both labor-intensive and costly, limiting the feasibility of large-scale studies. This lack of mechanistic insight has hindered the development of targeted therapies, with only a handful of efficacious small-molecule inhibitors directed toward oncofusions currently in clinical trials or approved for patient use. To address this critical gap, we adapted our recently developed PROD-ATAC method into a high-throughput, scalable platform to systematically elucidate the effects of dozens of candidate inhibitory small molecules on the chromatin dysregulation profiles induced by a pooled library of diverse oncofusions. In the PROD-ATAC scheme, a pooled protein-coding variant library is expressed in a single disease-agnostic cell line, and scATAC-seq (single-cell assay for transposase accessible chromatin) catalogs each variant’s effect on the chromatin landscape. We applied PROD-ATAC to simultaneously assay the chromatin regulatory impact of >40 small molecule inhibitors against a panel of tens of oncofusions (including the majority of patient-identified fusion sequences causal for Ewing sarcoma) in a single assay, generating genome-wide chromatin accessibility datasets representative of >1,000 unique drug - fusion interactions. In this manner, we characterized several small molecules as previously unidentified inhibitors of multiple oncofusions. Certain inhibitors were found to abrogate the chromatin dysregulation caused by an oncofusion either globally or in a targeted cell signaling pathway-specific manner. By profiling multiple rare fusions causal for the same cancers, we identified separate instances of homogenous and heterogenous modes of chromatin dysregulation associated with distinct fusion proteins. In this work, PROD-ATAC chromatin accessibility readouts coupled the identification of novel oncofusion inhibitors with the characterization of each inhibitor’s impact on the epigenetic landscape of oncofusion-expressing cells. Citation Format: James E. Corban, Max Frenkel, Srivatsan Raman. High-throughput characterization of inhibitor impacts on chromatin dysregulation induced by oncofusions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A024.