Chemical Inhibitors of Rhabdomyosarcoma Self-Renawal

描述(申请人提供)：开发针对癌症自我更新途径的药物将为人类疾病提供新的有效治疗方法。重要的是，这些新药必须比传统药物鸡尾酒有更多的益处，并能更好地被患者耐受性更好，而非靶向效应更少。为此，斑马鱼已经成为癌症和化学发现的强大模型。利用斑马鱼横纹肌肉瘤的独特模型，我们以前已经证明斑马鱼肿瘤在分子上类似于人类胚胎横纹肌肉瘤(ERMS)，发现RAS通路在大多数人类ERMS中是活跃的，并在这种疾病中分离出一种自我更新的细胞类型。在这里，我们报告了新的转基因方法，它将荧光蛋白靶向特定的肿瘤细胞群，并可以在体内和实时显示myf5-GFP+肿瘤启动细胞。此外，斑马鱼ERMS对药物的反应方式与人类ERMS相似，我们证实mTOR和MAP-K途径对ERMS的持续生长至关重要。我们目前的斑马鱼ERMS模型的局限性之一是，每一只患病的动物都必须通过向单细胞期动物注射转基因DNA来产生。我们建议优化斑马鱼ERMS到幼体的细胞移植，这将创造大量受ERMS影响的动物储备。此外，将开发针对GAL4-VP16的条件转基因方法来发展肌肉种群，而第二个转基因将通过UAS启动子驱动激活的RAS的表达。通过与这两个转基因系杂交，RAS将在肌肉中特异表达，并导致ERMS。AIM 2将专注于使用化学遗传方法来识别FDA批准的调节ERM生长和改变肿瘤启动细胞总数的药物。受ERMS影响的鱼将用FDA批准的化合物进行治疗，并评估与对照动物相比肿瘤生长是否减少。对于抑制肿瘤生长的药物子集，ERMS肿瘤启动细胞将直接在活体动物中可视化，以评估药物对杀死ERMS肿瘤启动细胞的能力的影响。最后，将评估抑制肿瘤生长和减少斑马鱼肿瘤内自我更新细胞数量的化学物质在人类ERMS细胞系中的有效性。总之，我们的实验提供了新的方法来创造大量具有ERMS的斑马鱼，并直接在体内可视化肿瘤的生长和自我更新。我们的胚胎横纹肌肉瘤的荧光转基因斑马鱼模型与化学遗传方法相结合，将发现FDA批准的具有抗肿瘤活性的药物，这些药物可以特异性地去除自我更新的myf5+erms启动细胞。

DESCRIPTION (provided by applicant): Developing drugs that target cancer self-renewal pathways will provide new and efficacious treatments for human disease. Importantly, these new drugs must have increased benefit over conventional drug cocktails and be better tolerated by patients with fewer off-target effects. Toward this end, the zebrafish has emerged as both a powerful model of cancer and chemical discovery. Capitalizing on a unique zebrafish model of rhabdomyosarcoma, we have shown previously that zebrafish tumors are molecularly similar to human embryonal rhabdomyosaroma (ERMS), identified that the RAS pathway is active in a majority of human ERMS, and isolated a self-renewing cell type in this disease. Here, we report new transgenic approaches that target fluorescent proteins to specific tumor cell populations and can visualize myf5-GFP+ tumor-initiating cells in vivo and in real-time. Moreover, zebrafish ERMS respond to drugs in a similar manner as human ERMS, and we establish that both the mTOR and MAP-kinase pathway are critical for continued ERMS growth. One of the limitations of our current zebrafish ERMS model is that each diseased animal must be created by microinjection of transgenic DNA into one-cell stage animals. We propose to optimize cell transplantation of zebrafish ERMS into larvae which would create a large reserve of animals affected with ERMS. Additionally, conditional transgenic approaches will be developed that target GAL4-VP16 to developing muscle populations while a second transgene will drive expression of activated RAS by the UAS promoter. By crossing these two transgenic lines, RAS will be specifically expressed in muscle and lead to ERMS. Aim 2 will focus on using chemical genetic approaches to identify FDA approved drugs that modulate ERMS growth and alter the overall numbers of tumor-initiating cells. ERMS affected fish will be treated with FDA approved compounds and assessed for reduced tumor growth compared with control animals. For the subset of drugs that curb tumor growth, ERMS tumor-initiating cells will be directly visualized in live animals to assess drug effects on the ability to kill ERMS tumor-initiating cells. Lastly, chemicals that both curb tumor growth and reduce the number of self-renewing cells within the zebrafish tumor mass will be assessed for efficacy in human ERMS cell lines. In total, our experiments provide novel methods to create large numbers of zebrafish with ERMS and to directly visualize tumor growth and self-renewal in vivo. Our fluorescent transgenic zebrafish model of embryonal rhabdomyosarcoma when coupled with chemical genetic approaches will uncover FDA approved drugs with anti-tumor activity that specifically ablate self-renewing myf5+ ERMS-initiating cells.