Abstract B013: Uncovering and targeting resistance mechanisms to BET inhibitors in pediatric Fusion-Positive Rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. The fusion-positive (FP) subtype, driven by the chimeric transcription factor (TF) PAX3-FOXO1 (P3F), carries a high risk of recurrence and is strongly dependent on P3F for survival. While TFs like P3F are typically considered “undruggable, ” their expression and function can be modulated epigenetically. The BET family protein BRD4, an epigenetic reader, binds super-enhancers (SEs) to drive oncogenic transcription in several cancers. We previously showed that P3F reprograms the enhancer landscape in FP-RMS by recruiting BRD4 to oncogenic SEs. As a result, the BET inhibitor (BETi) JQ1 disrupts P3F function, halting tumor growth both in vitro and in vivo—highlighting a subtype-specific vulnerability in FP-RMS. However, as with many targeted therapies, resistance can develop. To explore mechanisms of acquired resistance, we generated JQ1-resistant FP-RMS cell lines through chronic exposure to escalating JQ1 concentrations. These cells exhibited significantly elevated JQ1 IC50 values in both 2D and 3D cultures and showed cross-resistance to various monovalent and bivalent BET inhibitors and degraders. Functionally, resistant cells maintained clonogenic potential, anchorage-independent growth, stem-like traits, and invasiveness despite BET inhibition. Importantly, resistance and cross-resistance were retained in vivo. Integrated transcriptomic and proteomic analyses revealed differential regulation of subsets of P3F/MYCN targets, suggesting partial restoration of the core transcriptional circuitry. Targeted antibody-based proteomics further indicated activation of the MEK-ERK pathway. Since MYCN stability is regulated post-translationally, especially through ERK-mediated phosphorylation at Ser62—we assessed MEK-ERK activity and MYCN phosphorylation. Resistant cells displayed elevated levels of phosphorylated MEK, ERK, and MYCN, alongside a ∼3-fold increase in MYCN protein half-life, indicating enhanced stability. Functionally, MEK-ERK inhibition using Trametinib (MEKi) resensitized resistant cells to BETi and showed synergistic effects in treatment-naïve FP-RMS cells. These findings suggest that ERK pathway activation promotes BETi resistance via MYCN stabilization, revealing a targetable mechanism of resistance. Combined MEK-ERK and BET inhibition could represent a promising therapeutic strategy for both resistant and treatment-naïve FP-RMS. This study was supported by Italian Ministry of Health Ministero della Salute (to S. P. and M. C. ). Citation Format: Erika Ferraro, Elisa Macrí, Young K Song, Berkley E Gryder, Simone Sidoli, Francesco Marampon, Robert G Hawley, Biagio De Angelis, Concetta Quintarelli, Franco Locatelli, Rossella Rota, Javed Khan, Matteo Cassandri, Silvia Pomella. Uncovering and targeting resistance mechanisms to BET inhibitors in pediatric Fusion-Positive Rhabdomyosarcoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr B013.