Abstract 6448: Determinants of chemotherapeutic resistance in fusion-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, and is classified as fusion-positive (FP) or fusion-negative (FN) based on the presence of the PAX3::FOXO1 or PAX7::FOXO1 fusions. These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. FP status is associated with worse event free survival for patients with RMS. Regardless of fusion status, most RMS patients receive a chemotherapy regimen consisting of some combination of vincristine, actinomycin D, and cyclophosphamide (VAC). Unfortunately, tumors may acquire resistance to these drugs over time, leading to relapse. Our work aimed to uncover genes that drive VAC resistance in FP-RMS using a two-pronged approach. To assess how FP-RMS cells gradually acquire resistance, we cultured FP-RMS cell lines (RH30, RH41, CW9019, U48484, U66788) in sublethal concentrations of vincristine, actinomycin D, or 4-hydroperoxy cyclophosphamide (4-HC, the active metabolite of cyclophosphamide) for multiple passages. We periodically assessed the IC50 of cells growing in drugs and increased drug doses as cells developed resistance. We generated four cell lines with a ∼5- to ∼20-fold increase in IC50. Using RNAseq, we identified differentially expressed genes between the parental and resistant cell lines that may act as drivers of chemotherapeutic resistance. To assess de novo resistance in FP-RMS, we used CRISPR inactivation and activation library screening. We generated FP-RMS cell lines (RH30, RH41, CW9019) stably expressing Cas9 or dCas9-VPR, an endonuclease-dead version of Cas9 fused to three transcriptional activators. We then transduced these cells with a CRISPR inactivation sgRNA library (18, 885 gene targets) or a CRISPR activation sgRNA library (19, 113 gene targets). Transduced cells were cultured in vincristine, actinomycin D, or 4-HC, and surviving cells were expanded and sequenced to determine which genes provided drug resistance when activated or deleted. We hope that the findings of these experiments will contribute to novel therapeutic targets for patients with VAC-resistant RMS tumors. Citation Format: Taryn E. Shaw, Foram Shah, Jeffrey Toretsky, Aykut Üren. Determinants of chemotherapeutic resistance in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6448.