Abstract 334: ASAP1’s role in regulating myogenic differentiation in fusion negative rhabdomyosarcoma
Authors: Kim Angela , Hebron Katie E. , Perkins Olivia L. , Jian Xiaoying , Girald-Berlingeri Sofia A. , Randazzo Paul A. , Yohe Marielle E. 
Published: June 2023

Abstract
Rhabdomyosarcoma (RMS) accounts for 5% of all pediatric cancers. The overall survival rate for patients with advanced RMS has been less than 20% for the last 30 years. Investigating novel treatment strategies for RMS is necessary to improve prognosis. PAX/FOXO1 Fusion Negative RMS (FN-RMS) is commonly associated with alterations in the RAS/RAF/MEK/ERK pathway and arises due to a disruption of differentiation in skeletal muscle precursor cells. Trametinib, a MEK inhibitor, induces myogenic differentiation in FN-RMS cell lines, thus nominating trametinib as a potential differentiation therapy for FN-RMS. However, it is imperative to note that trametinib-induced RMS differentiation is incomplete. Therefore, exploring additional targets to drive differentiation is necessary. ASAP1, an Arf GTPase-activating protein (GAP), is a candidate due to its role in osteogenic and adipogenic differentiation. Additionally, ASAP1 has been shown to be overexpressed in several cancers. Furthermore, we see an overexpression of ASAP1 in FN-RMS and lower survival rates for sarcoma patients in which ASAP1 is overexpressed within the tumor. In this study, we tested the hypothesis that ASAP1 plays a role in the myogenic differentiation of FN-RMS. Upon knockdown (KD) of ASAP1, trametinib-induced differentiation was reduced, while over-expression of ASAP1 potentiated trametinib-induced differentiation. Interestingly, KD of ARF1, a substrate of ASAP1’s GAP activity, also blocked trametinib-induced differentiation. Due to these conflicting observations, we examined the relationship between ARF1 and ASAP1. Expression of exogenous ARF1 and ARF1[I46D], a mutation that renders ARF1 insensitive to ASAP1’s GAP activity, following KD of endogenous ARF1, rescued trametinib-induced differentiation. This indicates that the GAP activity of ASAP1 is not necessary for trametinib-differentiation in FN-RMS. To further examine ASAP1 and ARF1’s interaction, we investigated trametinib-induced differentiation upon KD of known effectors of ARF1 (GGA1, GGA2, and GGA3). Similar to the KD of ASAP1, trametinib-induced differentiation was reduced, indicating that this may be a phenotype between ARF1 and its effectors. Furthermore, to investigate the mechanism in which ASAP1 is regulating differentiation, we turned to the Hippo pathway’s effectors, YAP and TAZ. ASAP1 is a transcriptional target of YAP/TAZ, and YAP/TAZ activity affects myogenic differentiation. We observed that ASAP1 negatively regulates TAZ activity by affecting TAZ localization. In summary, ASAP1 functions in part as an ARF1 effector that regulates myogenic differentiation of FN-RMS by affecting TAZ localization. Future work will consist of investigating the downstream signaling effects of the novel ASAP1 and ARF1 interaction in FN-RMS and understanding the role of manipulating myogenic differentiation in advanced and metastatic FN-RMS.
Citation Format: Angela Kim, Katie E. Hebron, Olivia L. Perkins, Xiaoying Jian, Sofia A. Girald-Berlingeri, Paul A. Randazzo, Marielle E. Yohe. ASAP1’s role in regulating myogenic differentiation in fusion negative rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 334.