Determining the Mechanism of Rhabdomyosarcoma Transformation from a Non-Myogenic Cell of Origin

Project Summary/Abstract Rhabdomyosarcoma (RMS) is an aggressive solid tumor and the most common pediatric soft tissue sarcoma. RMS treatment consists of chemotherapy, radiation, and surgery with no FDA approved targeted therapies to date. Despite decades of research and clinical trials, high risk patients face an overall survival rate of less than 30%. Fusion-negative RMS (FN-RMS), the most common subtype, is genetically heterogeneous with cells resembling an arrested state of skeletal muscle differentiation. FN-RMS can arise anywhere in the body but predominately develops in the head and neck. Tumors commonly develop in regions completely devoid of skeletal muscle such as the urinary bladder, prostate, and biliary tree. This challenges the long-held conception that RMS originates from a skeletal muscle progenitor that escapes terminal muscle differentiation. However, it is currently unknown how a non-myogenic progenitor undergoes myogenic transformation in FN-RMS. The objective of this proposal is to determine the mechanism of FN-RMS transformation from a non-myogenic cell of origin. This will also identify the critical determinants of RMS cell fate which have yet to be elucidated. The overall hypothesis of this proposal is that RMS transformation from a non-myogenic cell of origin requires access to and expression of lineage specification factors upstream of myogenic regulatory factors such as MYOD1 and MYF5. I will address this hypothesis with the following specific aims: (1) define chromatin landscape changes during FN-RMS transformation, and (2) determine the transcription factors (TFs) required for rhabdomyosarcomagenesis. We characterized a mouse model of FN-RMS arising from a non-myogenic cell of origin where Cre-recombination driven by the adipose protein 2 (aP2) promoter activates oncogenic Sonic hedgehog (SmoM2) signaling (aP2-Cre;R26SmoM2). These tumors arise exclusively in the head and neck and resemble FN-RMS patient tumors by histology and gene expression profiles. Using this model, I will assess how chromatin accessibility, epigenetic marks, and interactions are altered upon myogenic transformation. I will then use a combination of conditional knockout mouse models and an in vitro directed differentiation CRISPR screen to determine the RMS-associated TFs required for transformation. To improve clinical outcomes, a better understanding of the biology driving RMS is critical. This project will provide novel insights into the key mechanistic determinants of FN-RMS cell fate allowing for myogenic tumors to arise in sites completely devoid of skeletal muscle. The critical drivers of RMS cell fate can then be leveraged to generate new targeted therapies.