Abstract 3670: Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness
Authors: Burgess Breanne A. , Slemmons Katherine K. , Linardic Corinne M. , Deel Michael D. 
Published: August 2019

Introduction: Fusion Positive Rhabdomyosarcoma (FP-RMS), a soft tissue sarcoma of adolescents and young adults, is driven by the oncogenic transcription factor PAX3-FOXO1 (P3F). Although most patients with FP-RMS initially respond to therapy, tumors frequently become refractory or relapse, underscoring a need to target resistance mechanisms and cancer stem cells. Because P3F is inherently disordered with no viable drug binding sites, it is currently not amendable to direct inhibition. Our prior work demonstrated that transcriptional co-activators TAZ and YAP are highly abundant in human FP-RMS and mediate several cancer phenotypes including apoptosis, differentiation, proliferation, and xenograft tumor growth. TAZ/YAP are essential co-activators for TEADs and AP1, which are among the top enriched transcription factor motifs at P3F binding sites. Here, we show that TAZ/YAP complex with P3F to positively regulate P3F transcriptional activity and that TAZ/YAP mediate FP-RMS stemness.

Methods: Functional interactions of TAZ/YAP and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) to identify the TAZ/YAP/P3F interactome is currently underway. For reporter assays, gain- and loss-of-function vectors expressing control, wild-type TAZ/YAP (WT), constitutively active TAZ/YAP (S89A/S127A), or shRNA knockdown (KD) were used. Epitope-tagged TAZ, YAP, and P3F were used for co-IPs and IP-MS. To investigate the role of TAZ/YAP in stemness, we evaluated the expression of stem cell genes in FP-RMS 3D-spheres expressing vector, WT, S89A/S127A, or KD. We are now performing RNA-Seq as an unbiased approach to examine genes and pathways regulating FP-RMS stemness.

Results: TAZ/YAP functionally augment P3F transcriptional activity in reporter assays and also regulate expression of P3F targets. Immunoblotting of 293T cells co-expressing epitope-tagged proteins demonstrate an interaction between TAZ/YAP and P3F. A TAZ/YAP/P3F complex is also seen in co-IPs of endogenous proteins in FP-RMS cells. Serial passaging of spheres increases TAZ/YAP expression, suggesting these may be important for stem cell enrichment. Indeed, expression of SOX2, OCT4, and NANOG are TAZ/YAP-dependent, whereby their expression decreases with KD and increases with S89A/S127A. Functionally, TAZ/YAP are required for FP-RMS sphere formation in limiting dilution assays.

Conclusions: These studies expand on prior work showing TAZ/YAP are potent oncoproteins in FP-RMS. We identify a novel complex between TAZ/YAP and P3F, show TAZ/YAP are positive regulators of P3F transcriptional activity, and demonstrate that TAZ/YAP regulate FP-RMS cancer cell stemness. Thus, targeting TAZ/YAP could be a vulnerability of P3F and for inhibiting the cancer stem cell population.

Citation Format: Breanne A. Burgess, Katherine K. Slemmons, Corinne M. Linardic, Michael D. Deel. Transcriptional co-activators TAZ/YAP are novel regulators of PAX3-FOXO1 transcriptional programing and fusion-positive rhabdomyosarcoma cancer cell stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3670.