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(18_Suppl_2):Abstract nr B013.