Abstract Introduction: Rhabdomyosarcoma (RMS) is a highly malignant soft tissue sarcoma. An aggressive subtype, fusion-positive RMS (FP-RMS), which is driven by PAX3/7::FOXO1 translocations, has a dismal 5-year overall survival rate of 13% for patients with metastatic disease. We hypothesized that targeting the oncogenic driver PAX3::FOXO1 with small molecules would be an effective treatment. To test this, we developed cell lines with endogenous PAX3::FOXO1 tagged with HiBiT epitope and performed a drug screen to identify drugs that downregulate PAX3::FOXO1 protein. Study Design: Using CRISPR-Cas9, we endogenously tagged PAX3::FOXO1 with HiBiT in two RMS cell lines (RH4 and SCMC), enabling the monitoring of PAX3::FOXO1 protein levels. We performed a drug screen using the Mechanism Interrogation Plate (MIPE) library of 2,480 compounds, of which 53% are FDA-approved or in clinical trials. NanoGlo Luciferase assays monitored levels of HiBiT-tagged PAX3::FOXO1, while CellTiterGlo measured cell viability at 24 hours. We selected hits that showed a difference in area under the curve (AUC) between the two readouts of ≥ 90 for drugs that preferentially reduce the fusion protein level over general cytotoxicity. We investigated whether inhibitors led to nuclear accumulation and reduced total protein levels using Western blot and immunofluorescent imaging. Candidates were validated in the parental cells and in vivo studies. Results and Conclusions: The screen identified 183 hits, including Eltanexor, an XPO1 inhibitor. XPO1 exports over 200 proteins from the nucleus by recognizing their nuclear export sequences (NESs). Since the fusion gene retains the NES of FOXO1, a known XPO1 target, we tested whether PAX3::FOXO1 is a substrate. We observed that Eltanexor enhanced PAX3::FOXO1 nuclear accumulation at 6 hours in RH4, and at 2 hours in SCMC, followed by protein downregulation at 24 hours by Western blotting. Furthermore, Eltanexor induced p53 nuclear accumulation, detectable at 6 hours in SCMC, suggesting that early accumulation of PAX3::FOXO1 may drive cytotoxicity. At 24 hours, RNA-seq in Eltanexor-treated cell lines demonstrated downregulation of PAX3::FOXO1 and MYCN signatures, components of the core regulatory network in FP-RMS. Preliminary in vivo studies also showed Eltanexor induces delays in tumor progression in an RMS xenograft model. Furthermore, Eltanexor in combination with Mivebresib, a validated BRD4 inhibitor, demonstrated significant synergy against FP-RMS cells. We will perform site-directed mutagenesis studies to disrupt PAX3::FOXO1’s NES and validate combination with Mivebresib in vivo. In conclusion, we identified Eltanexor, an XPO1 inhibitor, as a novel therapeutic agent that suppressed PAX3::FOXO1 activity and levels, induced nuclear accumulation and led to cytotoxicity in incurable FP-RMS. Citation Format: Soumili Dey, Yong Y. Kim, Katrina Jia, Mehal Churiwal, Michele Ceribelli, Teresa S. Hawley, Raj Chari, David Milewski, Young K. Song, Xinyu Wen, Hsien-Chao Chou, Vineela Gangalapudi, Jun S. Wei, Craig Thomas, Robert G. Hawley, Javed Khan. Small-molecule screening of HiBiT-tagged PAX3::FOXO1 rhabdomyosarcoma cell lines identifies eltanexor as a potent therapeutic agent against fusion-positive rhabdomyosarcoma 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 6405.
Dey et al. (Fri,) studied this question.