A role for notch in self renewal in embryonal rhabdomyosarcoma

自我更新的肿瘤增殖细胞驱动肿瘤持续生长并导致复发。如果过程 肿瘤细胞的自我更新被关闭，那么肿瘤就会消退，病人就会复发。 自由了这一更新提案的目标是确定细胞和分子机制， Notch调节肿瘤增殖潜能和肿瘤增殖细胞状态的可塑性 胚胎性横纹肌肉瘤（ERMS），一种破坏性的儿科肌肉恶性肿瘤。复发是主要的 ERMS患者面临的临床问题，只有不到40%的复发患者能够存活。进展 在这个项目上，我们结合了斑马鱼ERMS模型的体内实验和体外实验， 使用ERMS细胞系和原代组织的研究证实了Notch途径激活增加细胞增殖的假设。 肿瘤增殖细胞（TPC）池，但令人惊讶的是，体内细胞移植实验发现， Notch使得非TPC能够去分化为TPC。使用人类患者样品、ERMS细胞系和 斑马鱼中的相关数据已经鉴定了人ERMS中的关键Notch调节靶，包括SNAI 1， MEF2C、PAX7和MYF5。我的提案中的初步数据显示，RAS驱动的ERMS包含 ERMS增殖细胞的分子上不同的群体，其表达高水平的myf 5，但缺乏分化 肌肉标志物表达。这些细胞可以在活体荧光转基因斑马鱼中直接可视化， 前所未有的机会来可视化活动物的自我更新。根据这些意见，我的建议将 确定Notch改变斑马鱼肿瘤增殖潜力的细胞和分子机制 人类ERMS具体而言，目标1将评估Notch通路激活是否改变对称性与不对称性 通过对活的荧光转基因鱼进行动态实时成像，观察ERMS繁殖细胞亚组分的分裂。 一个子目标将使用谱系追踪方法来确定去分化的频率和动力学，以制备TPC。 在ERMS。目的2：通过有限稀释法，证明NOTCH 1在人ERMS中扩增TPC 将低传代人原代ERMS细胞移植到免疫受损小鼠中。目标3将评估 下游NOTCH1效应基因SNAI1、PAX7、MYF5和MEF2C扩增的分子机制 自我更新，驱动去分化和阻断终末分化。总的来说，我的建议提供了一个 全面的策略来询问Notch途径如何调节ERMS自我更新， 作为临床相关Notch途径抑制剂， 增殖细胞频率、去分化和最终复发。

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 updated proposal is to define the cellular and molecular mechanisms by which Notch regulates tumor-propagating potential and plasticity of the tumor propagating cell state 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. Progress on this project using a combination of in vivo experiments in the zebrafish ERMS model and in in vitro experiments using ERMS cell lines and primary tissues has validated the hypothesis that Notch pathway activation increases the pool of tumor- propagating cells (TPCs), but rather surprisingly in vivo cell transplantation experiments finds that Notch enables the dedifferentiation of non-TPCs into TPCs. Using human patient samples, ERMS cell lines and correlative 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 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. 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 1 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. A sub aim will use lineage tracing methods to define the frequency and dynamics of dedifferentiation to make TPCs in ERMS. Aim 2 Will show that NOTCH1 expands TPCs in vivo in human ERMS by utilizing limiting dilution cell transplantation of low passage human primary ERMS cells into immune compromised mice. Aim 3 will assess the molecular mechanisms by which downstream NOTCH1 effector genes SNAI1, PAX7, MYF5 and MEF2C expands self-renewal, drives dedifferentiation and blocks terminal differentiation. 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, dedifferentiation and ultimately relapse.