Abstract B029: Oncogenic MIR17HG Expression Is Transcriptionally Regulated by PAX3::FOXO1 and MYCN in Fusion-Positive Rhabdomyosarcoma

Abstract Fusion-positive rhabdomyosarcoma (FP-RMS), an aggressive pediatric soft tissue cancer, is driven by the oncogenic fusion transcription factors PAX3::FOXO1 (P3F) or PAX7::FOXO1. In a subset of FP-RMS cases, amplification of the MIR17HG locus leads to overexpression of the oncogenic miR-17–92 cluster. However, miR-17–92 is also upregulated in a subset of FP-RMS tumors lacking this amplification, suggesting the involvement of alternative regulatory mechanisms. Here, we show that P3F and MYCN cooperatively activate MIR17HG transcription in FP-RMS. CRISPR/Cas9-mediated knockout of P3F or MYCN in FP-RMS cell lines significantly reduced miR-17–92 expression. In a human myoblast model with inducible P3F expression, MYCN alone induced modest MIR17HG transcription, whereas co-expression with P3F led to robust activation of the cluster and acquisition of oncogenic phenotypes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed a P3F binding site located 1.84 Mb upstream of the MIR17HG promoter. CRISPR-mediated deletion of this site markedly reduced MIR17HG expression and impaired transformation in the myoblast system. Functional inhibition of mature miR-17–92 members using miRNA sponges also suppressed proliferation and transformation in FP-RMS cells. In vitro studies showed that miR-17–92 is necessary but not sufficient for transformation, highlighting its role as a key downstream effector of P3F and MYCN. Together, these findings define a cooperative transcriptional axis where P3F and MYCN activate MIR17HG through a distal DNA binding element, thereby revealing a novel therapeutic vulnerability in FP-RMS. Citation Format: Shabir Zargar, Pawam Raut, Hana Kim, Rachel Hoffman, Benjamin Z. Stanton, Frederic G. Barr. Oncogenic MIR17HG Expression Is Transcriptionally Regulated by PAX3::FOXO1 and MYCN in Fusion-Positive 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 B029.