Mechanisms of aggressive Rhabdomyosarcoma. - ABSTRACT Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma in the United States and displays features of skeletal muscle arrested at early stages of development. The aggressive MYOD1L122R mutated spindle/sclerosing RMS subtype accounts for 10% of pediatric diagnoses and have an extremely poor prognosis, even despite multi-modal treatment with surgery, radiation, and chemotherapy. The L122R mutation is predicted to modulate the DNA binding specificity of the Myogenic Differentiation 1 (MYOD1) transcription factor and regulates as of yet unknown transcriptional gene programs to elevate aggression and treatment resistance. To date, a detailed molecular understanding of how MYOD1L122R affects the genesis of RMS, its progression and drives therapy resistance is unknown. No cellular or molecular mechanistic studies of MYOD1L122R in RMS have been reported, no tractable genetically-engineered animal models have been developed, nor is it known if MYOD1L122R is required for continued human tumor growth or if it is rather a modifier of disease aggression, therapy resistance, or cancer stem cells (CSCs). The long-term goal and objective of our studies is to identify new molecular mechanisms and drug targets in MYOD1L122R mutant RMS that regulate aggression, therapy resistance, and CSCs. Our central hypothesis is that MYOD1L122R is a neo- morphic transcription factor that regulates pathways required for tumor maintenance and the production of therapy-resistant CSCs. The rationale and feasibility of our approach lies in our group’s recent discovery of molecularly distinct CSCs in MYOD1L122R mutant RMS, robust data showing that CSCs pathways and numbers are increased in isogenic knock-in and doxycycline-inducible MYOD1L122R RMS human cell models, and development of a new zebrafish model of MYOD1L122R-induced RMS that has elevated numbers of tumor- propagating cells. Aim 1 will assess roles for MYOD1L122R in enhancing tumor onset, growth, CSCs, and therapy resistance. Aim 1a will innovate new MYOD1L122R mutant RMS zebrafish models, testing the hypothesis that MYOD1L122R is itself not oncogenic, but drives elevated aggression only when complexed with other oncogenic drivers including activating mutations in RAS and PIK3CA. We also hypothesize that MYOD1L122R acts in part by increasing the overall fraction of tumor-propagating cells. Aim 1b will extend these findings to human RMS, testing the hypothesis that MYOD1L122R is required for continued tumor maintenance and regulates cell states including the production of therapy-resistance CSCs. Aim 2 will uncover MYOD1L122R regulated transcriptional targets, pathways, and mechanisms in RMS, testing the hypothesis that MYOD1L122R alters the DNA binding site specificity and transcriptionally regulates a novel set of genes to promote elevated RMS aggression, therapy resistance, and cancer stem cell fate. This work will have a positive translational impact by defining new pathways to kill MYOD1L122R mutant RMS, including those that target therapy-resistant CSCs, and identifying potential therapeutic targets that are likely shared across RMS subtypes.
