Resident Physician Duke University Medical Center, Department of Pediatrics Duke University School of Medicine Durham, North Carolina, United States
Background: Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by the fusion oncogene PAX3-FOXO1 (P3F). P3F has been suggested to promote G2/M "checkpoint adaptation" – cell cycle progression despite unrepaired DNA. P3F is not currently pharmacologically targetable. We hypothesized YAP1, a transcriptional co-activator, is required for G2/M transit and mediates G2/M checkpoint adaptation.
Objective: To investigate the requirement for YAP1 on G2/M cell cycle progression; to evaluate whether YAP1 may contribute to resistance mechanisms for FP-RMS therapies targeting the G2/M checkpoint.
Design/Methods: In human FP-RMS cell lines, YAP1 gain- and loss-of-function was achieved using vectors expressing control, wild-type YAP1, constitutively active mutant YAP1 (S127A), and shRNA or CRISPR/Cas9 knockdown. mRNA and protein-level expression of YAP1 and P3F were evaluated with qRT-PCR and immunoblot. Cell cycle analysis using propidium iodide staining with flow cytometry was used to determine the functional requirement for YAP1 in promoting G2/M progression. Co-immunoprecipitation-coupled mass spectroscopy (IP-MS) was used to identify G2/M regulatory proteins that physically associate with YAP1. RNA-Seq and quantitative tandem-mass tag mass spectrometry (TMT-MS) were used to determine the impact of YAP1 suppression on the cyclins and kinases involved in cell cycle regulation. Pharmacologic inhibition of YAP1 activity was tested in murine xenograft studies using verteporfin (VP) and with VP in combination with vincristine (VCR).
Results: YAP1 promotes G2/M transit and is able to partially rescue FP-RMS cells from a G2 arrest caused by either VCR or siRNA suppression of P3F. IP-MS revealed that YAP1 physically associates with regulatory proteins involved in the G2/M checkpoint. Further, RNA-Seq and TMT-MS demonstrated that YAP1 is required for the differential expression of proteins involved in G2/M transit, and that YAP loss leads to upregulation of proteins involved in TP53 signaling. YAP1 mRNA expression is upregulated in VCR-resistant FP-RMS cells. Additionally, FP-RMS expressing S127A are more resistant to VCR. In xenografts, combining VP and VCR is more efficacious than either agent alone. Conclusion(s): YAP1 regulates the G2/M checkpoint and is required for G2/M progression in FP-RMS. YAP1 may contribute to checkpoint adaptation and acquired VCR resistance. Thus, YAP1 is a promising therapeutic target in FP-RMS. Although additional studies are required, combining YAP inhibition with current FP-RMS antitubulin agents may prevent therapy resistance.
Authors/Institutions: Vahakn S. Keskinyan, Duke University School of Medicine, Durham, North Carolina, United States; Tooba Rashid, Duke University School of Medicine, Durham, North Carolina, United States; Adam F. Pecoraro, Duke University School of Medicine, Durham, North Carolina, United States; Breanne Burgess, Indiana University School of Medicine, Indianapolis, Indiana, United States; Michael D. Deel, Duke University School of Medicine, Durham, North Carolina, United States
