Abstract 3882: YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma
Authors: Rashid Tooba , Burgess Breanne A. , Linardic Corinne M. , Deel Michael D. 
Published: September 2020

Abstract
Although more than 20 years have passed since the discovery that fusion-positive rhabdomyosarcoma (FP-RMS) is driven by the chimeric fusion oncogene PAX3-FOXO1 (P3F), therapeutically tractable components of the P3F tumorigenic program have yet to be uncovered and survival rates remain dismal (5-yr overall survival &lt;50%). YAP1 and TAZ, transcriptional co-activators of the Hippo pathway, are potent oncogenes known to mediate transcriptional addiction in malignancy. Here, we demonstrate that YAP1/TAZ complex with P3F and positively regulate P3F transcriptional activity in FP-RMS. Immunohistochemical (IHC) staining of human tissue microarrays were used to identify the relative abundance of YAP1 and TAZ in FP-RMS tumor samples. Functional interactions of YAP1/TAZ and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) was used to identify the YAP1/TAZ/P3F interactome. Experiments utilized gain- and loss-of-function vectors expressing control, wild-type YAP1/TAZ, constitutively active YAP1/TAZ (S89A/S127A), or shRNA knockdown. To identify P3F-mediated genes and pathways that are YAP1/TAZ-dependent, we used RNA-Seq and quantitative proteomics via tandem mass tag labeling. We demonstrate via IHC that YAP1/TAZ are highly abundant in FP-RMS, and through functional assays that YAP1/TAZ regulate many FP-RMS cancer phenotypes. Mechanistically, an interaction between YAP/TAZ and P3F was demonstrated via co-IPs for endogenous as well as epitope-tagged proteins. This was confirmed with IP-MS, which also revealed that YAP/TAZ and P3F share an enrichment for co-immunoprecipitated proteins involved in DNA binding and transcriptional regulation. IP-MS also demonstrated that chromatin remodeling complex proteins were enriched with TAZ immunoprecipitation. YAP1/TAZ functionally augment P3F transcriptional activity in reporter assays as well as expression of candidate P3F target genes. An unbiased approach using RNA-Seq and quantitative proteomics demonstrate that YAP1/TAZ positively regulate differential expression of P3F target genes, as well as proteins involved in cell cycle progression and pan-cancer related proteins that are typically up- or down-regulated across multiple malignancy types.In conclusion, we identify a novel complex between YAP1/TAZ and P3F, show YAP1/TAZ are positive regulators of P3F transcriptional activity, and identify the YAP1/TAZ axis as a vulnerability for P3F-transcriptional reprogramming in FP-RMS.
Citation Format: Tooba Rashid, Breanne A. Burgess, Corinne M. Linardic, Michael D. Deel. YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3882.