Chemical Inhibitors of Rhabdomyosarcoma Self-Renawal

描述（由申请人提供）：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的鸡尾酒药物有更大的益处，并且更容易被患者耐受，脱靶效应更少。为此，斑马鱼已经成为癌症和化学发现的有力模型。利用一种独特的斑马鱼横纹肌肉瘤模型，我们先前已经证明斑马鱼肿瘤在分子上与人类胚胎横纹肌肉瘤（ERMS）相似，发现RAS通路在大多数人类胚胎横纹肌肉瘤中是活跃的，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，将荧光蛋白靶向特定的肿瘤细胞群，并可以在体内实时观察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，我们确定mTOR和map -激酶途径对ERMS的持续生长都至关重要。我们目前的斑马鱼ERMS模型的局限性之一是，每只患病动物必须通过向单细胞期动物中微量注射转基因DNA来创建。我们建议将斑马鱼ERMS细胞移植到幼体中，以建立大量的ERMS动物储备。此外，将开发有条件的转基因方法，将GAL4-VP16靶向于发育中的肌肉群体，而第二种转基因将通过UAS启动子驱动活化RAS的表达。通过杂交这两个转基因系，RAS将在肌肉中特异性表达并导致ERMS。目标2将侧重于使用化学遗传学方法来鉴定FDA批准的调节ERMS生长和改变肿瘤起始细胞总数的药物。受ERMS影响的鱼将使用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减少的情况。对于抑制肿瘤生长的药物子集，将在活体动物中直接观察ERMS肿瘤启动细胞，以评估药物杀死ERMS肿瘤启动细胞的能力。最后，在斑马鱼肿瘤中抑制肿瘤生长和减少自我更新细胞数量的化学物质将被评估对人类ERMS细胞系的有效性。总的来说，我们的实验提供了一种新的方法，可以用ERMS制造大量的斑马鱼，并直接观察肿瘤在体内的生长和自我更新。我们的胚胎性横纹肌肉瘤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，可以特异性地切除自我更新的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. PUBLIC HEALTH RELEVANCE: Identification of FDA approved drugs that suppress cancer self-renewal will provide new drug combinations for the treatment of human malignancy. Using a zebrafish model of embryonal rhabdomyosarcoma and chemical genetic approaches, we will identify drugs that specifically target self-renewing ERMS cells for destruction.