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.