DESCRIPTION (provided by applicant): Self-renewing tumor-propagating cells drive continued tumor growth and are responsible for relapse. If the process by which tumor cells self-renew could be turned off, then tumors would regress and patients would remain relapse free. The goal of this proposal is to determine a role for Notch in regulating tumor-propagating potential in embryonal rhadomyosarcoma (ERMS), a devastating pediatric malignancy of the muscle. Relapse is the major clinical problem facing patients with ERMS, with less than 40% of relapse patients surviving their disease, highlighting the need to identify molecular pathways that drive self-renewal and tumor re-growth at relapse. My hypothesis is that Notch pathway activation increases the pool of tumor-propagating cells by altering cell fate decisions following cell division, leading to increased symmetric cell divisions and subsequently larger fractions of relapse associated clones. Notch has been implicated as an important modulator of self-renewal in normal muscle stem cells by controlling symmetric versus asymmetric divisions and the pathway is commonly activated in ERMS through overexpression of NOTCH1 and 3. Preliminary data within my proposal shows that RAS-driven ERMS contain a molecularly distinct population of ERMS-propagating cells that express high levels of myf5 but lack differentiated muscle marker expression. These cells can be directly visualized in live, fluorescent-transgenic zebrafish, allowing unprecedented access to visualize self-renewal in live animals. Moreover, I have shown that Notch and RAS synergize to increase both tumor size and the overall pool of ERMS-propagating cells 25-fold when compared with ERMS that lack Notch pathway activation. Increased ERMS-propagating potential is accompanied by enhanced expression of pax7, myf5 and c-Met and in the context of muscle stem cells, both pax7 and myf5 are important transcriptional regulators. Building on these observations, my proposal will determine the cellular and molecular mechanisms by which Notch alters tumor-propagating potential in both zebrafish and human ERMS. Specifically, Aim 1a will assess if Notch confers tumor-propagating potential to a molecularly definable subpopulation of ERMS cells. Aim1b will assess if Notch pathway activation alters symmetric vs. asymmetric divisions in the ERMS-propagating cell subfraction by dynamic real-time imaging of live, fluorescent transgenic fish. Aim 2 will extend these findings to human disease. Aim 2a will assess if Notch pathway enhances tumor-propagating cell frequencies in vitro through use of sphere colony forming assays in primary human and established cell lines. Aim 2b will utilize limiting dilution cell transplantation of low passage human primary ERMS cells into immune compromised mice, comparing tumors with high and low Notch activity and correlating the frequency of ERMS-propagating cells within the tumor mass. Aim 3 will assess in ERMS tumor cell lines and low passage human primary ERMS, if Notch regulates self-renewal by directing the expression of important muscle transcriptional regulators PAX7 and MYF5 both of which are upregulated in human and zebrafish ERMS. In total, my proposal provides a comprehensive strategy to interrogate how the Notch pathway regulates ERMS self-renewal and will likely have immense therapeutic significance as clinically-relevant Notch pathway inhibitors would likely reduce tumor- propagating cell frequency, self-renewal and ultimately relapse.

PUBLIC HEALTH RELEVANCE: Relapse is the major clinical problem facing children with Embryonal Rhabdomyosarcoma (ERMS) and is driven by self-renewing tumor propagating cells. This proposal will assess the role of Notch in regulating self-renewal within ERMS- propagating cells.