Abstract 3500: Synergistic targeting of EP300/CBP and EYA co-activators collapses the rhabdomyosarcoma core regulatory circuit

Abstract Rhabdomyosarcoma (RMS) is a high-risk and lethal pediatric sarcoma that resembles developing skeletal muscle. RMS tumors have low mutation burdens, but these scant mutations often alter genes involved in transcriptional control. Transcriptional dysregulation is critical to RMS pathogenesis, supported by studies in both RMS tumors carrying mutationally derived chimeric transcription factors (“fusion positive (FP)”), or those without (“fusion negative” (FN)). However, mechanisms to selectively target dysregulated transcription in RMS remain outstanding. Here, we develop a novel approach targeting RMS transcription comprising simultaneous targeting of two distinctly acting transcriptional co-activators. We discover a common cell identity-controlling pan-RMS core regulatory circuit (CRC) composed of oncogenic and lineage-specific myogenic master transcription factors (mTFs). These mTFs are regulated by super-enhancers, and they co-bind genome-wide to control the malignant transcriptome of both FP- and FN-RMS. Using a super-enhancer-based reporter screen, we identify the EP300/CBP inhibitor A485 as a potent inhibitor of the pan-RMS CRC, though efficacy of this compound was limited by toxicity. To enhance on-target specificity, we identify the protein EYA2 as a co-factor that binds directly to SIX1, a member of the pan-RMS CRC and exploit a recently developed second-generation EYA1/2 tyrosine phosphatase inhibitor, LG1-34, to inactivate its function. While A485 and LG1-34 independently reduce mTF transcription and drive RMS cell death, in combination, these agents function synergistically to reduce RMS growth in vitro and in vivo. These results demonstrate that combined targeting of enhancer maintenance and CRC cofactors is a powerful strategy to suppress the RMS transcriptome and enforce RMS cell death. Citation Format: Annika Gustafson, Stephanie Nance, Berkley Gryder, Noha A. Shendy, Lars Wick, Grace McKay-Corkum, K. Elaine Ritter, Stephen Connor Purdy, Arthur R. Wolin, Sheera R. Rosenbaum, Sabateeshan Mathavarajah, Nickerson A. Demelfi, Yueyang Wang, Yang Zhang, Mark W. W. Zimmerman, Anoop M. Kavirayani, John Hardin, Alexander LaVeck, Xiang Wang, Neekesh V. Dharia, Andrew Hong, Guillaume Kugener, Jesse S. Boehm, Jennifer Roth, Javed Khan, Francisca Vasquez, Kristin B. Artinger, Rui Zhao, David M. Langenau, Jun Qi, Kimberly Stegmaier, Heide L. Ford, Adam D. Durbin, Brian J. Abraham. Synergistic targeting of EP300/CBP and EYA co-activators collapses the rhabdomyosarcoma core regulatory circuit abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3500.