Abstract B016: Biochemical mechanisms of PAX3::FOXO1 chromatin invasion in Rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer, with approximately 350 cases per year in the US. The highly aggressive tumor subtype, Fusion-Positive Rhabdomyosarcoma (FP-RMS) is characterized by chromosomal translocations yielding oncogenic fusion transcription factors (TFs). The most common RMS fusion (PAX3::FOXO1) forms an in-frame chimera of the DNA-binding domain of PAX3 with the activator domain of FOXO1. We hypothesize that understanding the precise mechanisms of PAX3::FOXO1’s chromatin engagement and nucleosome invasion would enable precision therapy to target these mechanisms to ultimately improve patient outcomes. Our studies are providing evidence that PAX3::FOXO1 has pioneer activity, including nucleosome engagement, and an ability to recognize multiple chromatin states including both open and repressive chromatin regions. We are investigating the biochemical regulatory functions of PAX3::FOXO1 binding to specific DNA motifs embedded within chromatin or nucleosomes, developing synthetic epigenetics to investigate motifs from our genomics-scale studies. We are interested in the preservation of naturally occurring post-translational modifications from the mammalian cellular context, and have sought to build a highly versatile system for investigation of TF/nucleosome targeting from cell extracts. In our studies, we are finding new evidence for PAX3::FOXO1’s targeting of homeodomain and paired domain DNA motifs in free DNA and also in a nucleosome context. We are validating these findings through parallel approaches including coupling electrophoretic mobility shift assays (EMSAs) with westerns and antibody-targeting of complexes within EMSA experiments. We are finding that PAX3::FOXO1’s binding to high-complexity motifs, conjoining paired domain and homeodomain sequences, occurs during nucleosome invasion. Through iterative comparisons, custom unmodified mono-nucleosomes containing three major classes of DNA binding motifs reveal PAX3::FOXO1’s binding to these nucleosomes without a strong motif preference within the categories examined in our series. These findings support a mechanistic hypothesis where PAX3::FOXO1 may invade nucleosomes, altering their local positioning prior to generation of chromatin accessibility. Additional investigations are underway to unravel these mechanisms further and probe the contexts for motif specificity or generality in nucleosome engagement, and to understand conformational changes in the target nucleosomes. The mechanisms we are uncovering will contribute to the search for new therapeutic vulnerabilities as our community progresses toward targeted therapies for FP-RMS. Citation Format: Chamithi Karunanayake, Hayden Statmore, Ehsan Akbari, Alexi Tallan, Frederic Barr, Michael G. Poirier, Benjamin Stanton. Biochemical mechanisms of PAX3::FOXO1 chromatin invasion in Rhabdomyosarcoma [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 B016.