Abstract 4733: Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma
Authors: Ignatius Myron , Lobbardi Riadh , Hayes Madeline , Chen Eleanor , McCarthy Karin , Nielsen G. Petur , Beleyea Brian , Linardic Corinne , Khan Javed , Keller Charles , Langenau David M. 
Published: August 2015

Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric muscle cancer with extremely poor prognosis at relapse. Work from our group has identified the tumor-propagating cell (TPC) in a transgenic zebrafish model of kRASG12D-induced ERMS that is responsible for driving continued tumor growth and relapse. The TPC is molecularly similar to an activated muscle satellite-cell and expresses myf5, c-met, and m-cadherin. Building on these observations, we have identified the Notch pathway as a potent enhancer of ERMS self-renewal and TPC number. Specifically, bulk tumor limiting dilution cell transplantation experiments revealed that TPCs are increased 10-fold in kRASG12D expressing ERMS that co-express activated intracellular Notch1 (ICN1). This increase in TPC number is partly the result of ICN1 expressing ERMS exhibiting a 3-fold expansion of relapse-driving myf5-GFP+/mylz2-mCherry-negative ERMS-cell population. Unexpectedly, cell transplantation experiments revealed that Notch pathway activation also conferred tumor-propagating ability to the myf5-GFP+/mylz2-mCherry+ mid-differentiated ERMS cells - a population of cells previously shown to lack self-renewal capacity. Single cell engraftment studies uncovered that NOTCH activation caused cells to oscillate between the TPC and mid-differentiated ERMS molecular states that was not observed in kRASG12D-expressing ERMS, suggesting that Notch has important roles in both self-renewal and cell state transitions.

Next, we validated our findings in human ERMS where NOTCH1 is highly expressed both in tumors and ERMS-cell lines. We show important roles for NOTCH in regulating self-renewal and differentiation in human ERMS. Specifically, human ERMS cells that expressed activated NOTCH1 had elevated sphere-colony formation, a surrogate for self-renewal in vitro. By contrast, shRNA knockdown of NOTCH1 resulted in decreased sphere-colony formation and robust terminal differentiation of ERMS cells into late-stage, myosin-expressing myoblasts. Moreover, we identified that NOTCH1 directly activated SNAI1 expression and was required for both efficient sphere formation and differentiation in ERMS cell lines. SNAI1 is commonly over-expressed in human ERMS and its expression is positively correlated with NOTCH1. Taken together, our data indicate that Notch signaling is an important modifier of human ERMS acting to regulate both TPC self-renewal and differentiation. Notch and/or SNAl1 pathway inhibition may have potential benefit for a subset of patients with relapsed ERMS.

Citation Format: Myron Ignatius, Riadh Lobbardi, Madeline Hayes, Eleanor Chen, Karin McCarthy, G. Petur Nielsen, Brian Beleyea, Corinne Linardic, Javed Khan, Charles Keller, David M. Langenau. Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2015-4733