Introduction: Rhabdomyosarcoma (RMS), a tumor characterized by skeletal muscle features, is the most common soft tissue sarcoma in children and adolescents. The standard of care treatment has not changed in over 40 years, resulting in a stagnant survival rate of less than 30% for high risk groups. Previous studies have identified the developmental pathways Notch and YAP as potent oncogenic signals in the embryonal subtype of RMS (eRMS). Understanding the signaling between Notch and YAP in cancers driven by these pathways, such as eRMS, will be crucial to designing rational combination therapies.

Experimental Procedures: For in vitro assays, we utilized 3D sphere cell culture methods that permit the study of stem cell behavior. To analyze signaling, we performed genetic gain and loss-of-function (dox-inducible shRNAs and constitutively active constructs), and pharmacologic inhibition. Target validation was assessed by qPCR and immunoblot, and mechanism was determined by ChIP and limiting dilution assays. For in vivo experiments, we performed subcutaneous xenografts in immunocompromised mice. Immunohistochemistry was used to analyze protein expression in human and murine tumors. Statistical analysis was performed to assess significant differences.

Results: We have discovered a novel Notch-YAP circuit that drives cancer cell stemness and tumorigenesis. In eRMS 3D spheres, Notch transcriptionally upregulates YAP and increases its activity, while YAP transcriptionally upregulates the Notch ligands JAG1 and DLL1 and the core Notch transcription factor RBPJ to provide positive feedback. In vivo, YAP suppression decreases Notch signaling, stemness, and proliferation. This circuit boosts expression of the stem cell gene SOX2, which is functionally required for eRMS cell stemness. Inhibiting one node of the circuit, for example through Notch pharmacologic blockade, is not sufficient to shut down the circuit, as stemness and cell growth can be rescued by ectopic YAP. Rather, dual inhibition of Notch and YAP is necessary to silence the circuit’s signaling and sphere growth. Importantly, nuclear Notch and YAP protein expression correlate in eRMS patient tumor samples, suggesting this circuit is intact in human eRMS tumors.

Conclusions: While unidirectional signaling between these two pathways has been reported in metazoan development and rarely in human malignancy, this is the first finding of an intact bidirectional circuit between Notch and YAP that supports cancer cell stemness and tumorigenesis. This study provides mechanistic insight into the long-standing question of how eRMS cancer stem cells maintain plasticity. Additionally, while this signaling is identified here in RMS, it could be applicable to many cancer types that are Notch and YAP-dependent. Lastly, this work provides evidence and rationale for research into combination therapies targeting this Notch-YAP signaling circuit.

Citation Format: Katherine K. Slemmons, Lisa E.S. Crose, Stefan Riedel, Manuela Sushnitha, Brian Belyea, Corinne M. Linardic. A novel Notch-YAP circuit drives stemness and tumorigenesis in embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5830. doi:10.1158/1538-7445.AM2017-5830