A ROLE FOR NOTCH IN SELF RENEWAL IN EMBRYONAL RHABDOMYOSARCOMA

Self-renewing tumor-propagating cells drive continued tumor growth and are responsible for relapse. If the processby which tumor cells self-renew could be turned off, then tumors would regress and patients would remain relapsefree. The goal of this updated proposal is to define the cellular and molecular mechanisms by whichNotch regulates tumor-propagating potential and plasticity of the tumor propagating cell state inembryonal rhadomyosarcoma (ERMS), a devastating pediatric malignancy of the muscle. Relapse is the majorclinical problem facing patients with ERMS, with less than 40% of relapse patients surviving their disease. Progresson this project using a combination of in vivo experiments in the zebrafish ERMS model and in in vitro experimentsusing ERMS cell lines and primary tissues has validated the hypothesis that Notch pathway activation increases thepool of tumor- propagating cells (TPCs), but rather surprisingly in vivo cell transplantation experiments finds thatNotch enables the dedifferentiation of non-TPCs into TPCs. Using human patient samples, ERMS cell lines andcorrelative data in zebrafish has identified critical Notch regulated targets in human ERMS including SNAI1,MEF2C, PAX7 and MYF5. Preliminary data within my proposal shows that RAS-driven ERMS contain amolecularly distinct population of ERMS-propagating cells that express high levels of myf5 but lack differentiatedmuscle marker expression. These cells can be directly visualized in live, fluorescent- transgenic zebrafish, allowingunprecedented access to visualize self-renewal in live animals. Building on these observations, my proposal willdetermine the cellular and molecular mechanisms by which Notch alters tumor-propagating potential in both zebrafishand human ERMS. Specifically, Aim 1 will assess if Notch pathway activation alters symmetric vs. asymmetricdivisions in the ERMS-propagating cell subfraction by dynamic real-time imaging of live, fluorescent transgenic fish.A sub aim will use lineage tracing methods to define the frequency and dynamics of dedifferentiation to make TPCsin ERMS. Aim 2 Will show that NOTCH1 expands TPCs in vivo in human ERMS by utilizing limiting dilution celltransplantation of low passage human primary ERMS cells into immune compromised mice. Aim 3 will assess themolecular mechanisms by which downstream NOTCH1 effector genes SNAI1, PAX7, MYF5 and MEF2C expandsself-renewal, drives dedifferentiation and blocks terminal differentiation. In total, my proposal provides acomprehensive strategy to interrogate how the Notch pathway regulates ERMS self- renewal and will likely haveimmense therapeutic significance as clinically-relevant Notch pathway inhibitors would likely reduce tumorpropagating cell frequency, dedifferentiation and ultimately relapse.