Abstract 1155: High-throughput screening identifies synergistic drug interactions between CDK8/19 and DHFR inhibitors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue sarcoma marked by substantial treatment-related morbidity and poor outcomes, particularly in metastatic or recurrent disease. Although diagnostic and therapeutic approaches have improved, survival remains low for patients with high-risk molecular features, including tumors harboring the PAX3::FOXO1 fusion. This fusion protein drives an aberrant transcriptional program that promotes tumor progression and resistance to therapy, underscoring the need for new treatment strategies. In this work, we investigated therapeutic vulnerabilities created by inhibiting the transcription-regulatory kinases CDK8/19. Using a high-throughput drug combination screen, we tested several CDK8/19 inhibitors against a library of 2,803 preclinical, investigational, and approved small molecules. The screen identified a strong synergistic interaction between CDK8/19 inhibitors and dihydrofolate reductase (DHFR) inhibitors. Unsupervised hierarchical clustering showed that multiple DHFR inhibitors formed a distinct cluster with highly similar patterns of synergy, supporting the robustness of this interaction. Follow-up viability assays confirmed synergy in both PAX3::FOXO1-positive and fusion-negative RMS cell lines. To define the biological basis of this synergy, we examined effects on transcriptional regulation, cell-cycle dynamics, and apoptosis. Annexin-V/DAPI staining demonstrated that combined treatment produced significantly higher apoptosis and G1 arrest than either drug alone. EdU incorporation assays showed a strong decrease in DNA replication, indicating impaired nucleotide synthesis. Transcriptomic profiling, including gene set enrichment analysis, revealed that CDK8/19 inhibitors upregulated PAX3::FOXO1 fusion targets, MYC-regulated genes, and oxidative phosphorylation pathways, whereas DHFR inhibitors primarily induced immune-related and KRAS signaling programs. The transcriptional responses to each drug class were largely distinct, and combination treatment reversed expression patterns driven by single agents. Importantly, DHFR inhibition triggered compensatory activation of pyrimidine biosynthesis genes, a response that was suppressed by CDK8/19 inhibition, providing a mechanistic basis for the observed synergy. ChIP-seq and proteomic data further supported these findings. Additionally, CDK8/19 inhibition amplified DHFR inhibitor-induced DNA damage, reflected by elevated γH2AX accumulation. Overall, these results highlight a therapeutic strategy that concurrently disrupts PAX3::FOXO1-driven transcriptional programs and nucleotide-dependent DNA repair. The combination of CDK8/19 and DHFR inhibitors may therefore overcome resistance mechanisms and enhance treatment efficacy in RMS. Citation Format: Ukhyun Jo, Ying Wu, Lisa M Jenkins, Tapan Kumar Maity, Seth P Zimmerman, Chris M. Counter, Assil Fahs, Corinne Linardic, Craig J. Thomas, John F. Shern. High-throughput screening identifies synergistic drug interactions between CDK8/19 and DHFR inhibitors in 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 1155.