Abstract A022-PR005: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-Driven Alveolar Rhabdomyosarcoma

Abstract Functional and chemical genomic approaches, such as the Broad Institute’s Cancer Dependency Map and PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) chemical screening, have revolutionized the identification of context-specific genetic dependencies and therapeutic vulnerabilities across diverse cancer types. These datasets empower the identification of therapeutic strategies in cancers with limited actionable mutations. Alveolar rhabdomyosarcoma (aRMS), a pediatric cancer driven by a hard-to-target fusion oncoprotein, PAX3::FOXO1, typically expresses no immediately actionable mutations. Using aRMS as a disease model, we utilized the Broad Institute’s Cancer Dependency Map dataset, coupled with single-sample gene set enrichment analysis (ssGSEA), to systematically assess protein complex dependencies in aRMS. We nominated the Mediator complex and further identified CDK8, a member of the Mediator kinase module, as a clinically actionable target in aRMS. Other members of the Mediator kinase module, including CCNC, MED12, and MED13, are also essential to aRMS proliferation. In addition, in aRMS cell lines, there was a positive correlation (R=0.68) between the CDK8 gene-effect score and the effect of the CDK8 inhibitor, BI-1347, in the PRISM screening. We validated that both genetic loss of CDK8 and kinase inhibition by CDK8 small molecule inhibitors impaired aRMS cell line growth in vitro and induced evidence of myogenic differentiation. Our in vivo studies demonstrated that the clinical grade CDK8 inhibitor SEL-120-34A decreased aRMS cell line xenograft tumor growth. Functional genomic screens also serve as powerful tools to understand the mechanisms of small molecule inhibitors and to identify resistance and sensitizer mechanisms. We thus performed an unbiased genome-scale CRISPR-Cas9 screen with the CDK8 inhibitor BI-1347 in aRMS. We determined that the maximal anti-tumor activity of the CDK8 inhibitor requires the presence of the SAGA complex, particularly the SAGA HAT module. We further identified SIX4 as a key transcription factor mediating CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes. In addition, we revealed that the maximal activity of BI-1347 requires the presence of the Mediator kinase module, suggesting a trapping-related mechanism in addition to kinase enzymatic inhibition. Accordingly, we found that CDK8 inhibition increased the binding of components of the Mediator kinase module and SIX4 and TADA2B at enhancers of terminal muscle differentiation-related genes, such as VGLL2, SEMA3D, MYL1, leading to their transcriptional activation, as shown by PRO-seq analyses. These findings provide a framework for uncovering therapeutic targets using network-based analysis of functional genomic screens, and for studying the small molecular inhibitor mechanisms. In aRMS, CDK8 inhibition is a differentiation-inducing therapeutic strategy. Citation Format: Susu Zhang, Kathleen Engel, Assil Fahs, Clare Malone, Kenneth Ross, Marissa Just, Brian Guedes, Diyana Granum, Kristianne M Oristian, Alexander Kovach, Gabriela Alexe, Giulia Digiovanni, Leen Barbar, Rex Bentley, Christian Cerda-Smith, Ozgun Le Roux, Elizabeth Mendes, Seth P Zimmerman, Matthew Rees, Jennifer Roth, Jack F Shern, Kris C Wood, Christopher M Counter, Corinne M Linardic, Kimberly Stegmaier. CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-Driven Alveolar 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 A022-PR005.