Abstract 1092: RUNX2 inhibition alters PAX3::FOXO1 driven fusion-positive rhabdomyosarcoma cell state and growth
Authors: Mendes Elizabeth A. , Chambers Audrey L. , Weitzel Samantha J. , Deel Michael , Munshi Aanandi , Counter Christopher M. , Zimmerman Seth , Bushweller John , Linardic Corinne M. 
Published: March 2024

Abstract
Investigation: Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric cancer of skeletal muscle lineage. While the incidence of RMS is ~4.5 patients per million individuals aged &lt;20 years, 5-year survival for high-risk children is &lt;30%, and &lt;8% when metastatic. The PAX3::FOXO1 (P3F) fusion gene, resulting from t(2:13), is the most prominent fusion oncogene that drives FP-RMS. P3F is a fusion transcription factor and supports FP-RMS by keeping it in a persistent myoblastic state. We found P3F functions in part by suppressing MST kinase activity in the Hippo tumor suppressor pathway, a developmental pathway involved in cell fate and organ size. To investigate how this may rewire gene expression to support FP-RMS, we performed ATAC-seq analysis of a myoblast-based model of FP-RMS in which MST was further inhibited and found the RUNX2 gene locus was more accessible. Since RUNX2 is a pioneer transcription factor known to interact with the “WW” domains of Hippo pathway members to support mesenchymal cell fate, we hypothesize that RUNX2 maintains these relationships to support FP-RMS cell state and survival.
Methods & Results: Inquiry of in silico databases and previously published literature unveiled that sarcomas including RMS have a high dependency on RUNX2 and that P3F directly binds a RUNX2 enhancer. Interrogation of a proximity labeling proteomics study from our collaborators in the Counter lab found that RUNX2 was part of the protein interactome of 7/7 FP-RMS fusions including P3F. Additionally, Co-IP experiments confirmed that there is a direct protein-protein relationship between RUNX2, P3F, and TAZ. Next, we performed in vitro genetic loss of function (LOF) of RUNX2 using RNAi, which showed that suppression of RUNX2 caused cell death, growth inhibition, increased TAZ expression, and induction of both myogenic and osteogenic markers. We also performed complementary pharmacologic LOF using two available small molecules, AI-14-91 (Bushweller lab) in vitro, and CADD522 (commercially available) in vitro and in vivo. We found that these partially phenocopied the genetic LOF and that CADD522 reduces tumor volume and growth.
Conclusions: RUNX2 is a vulnerability in RMS based on in silico, in vitro, and in vivo investigation, and may act to prevent osteogenic and myogenic differentiation via protein-protein relationships in the Hippo pathway. The next step will be assessing mechanistic relationships between the Hippo pathway and RUNX2, as well as the genome wide changes upon RUNX2 LOF by performing ChIP-seq, RNA-seq, and scRNA-seq experiments.
Citation Format: Elizabeth A. Mendes, Audrey L. Chambers, Samantha J. Weitzel, Michael Deel, Aanandi Munshi, Christopher M. Counter, Seth Zimmerman, John Bushweller, Corinne M. Linardic. RUNX2 inhibition alters PAX3::FOXO1 driven fusion-positive rhabdomyosarcoma cell state and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1092.