Targeting AVIL, a novel oncogene in rhabdomyosarcoma

横纹肌肉瘤（RMS）是最常见的儿童软组织癌症。尽管加强了治疗， 晚期RMS患者的结果仍然令人沮丧。到目前为止，没有靶向治疗 available.显然需要更好的理解和治疗。在我们的初步研究中，我们发现了一种新的 癌基因AVIL在RMS中的表达。我们发现：1）AVIL在某些RMS中与管家基因MARS形成融合; 2)AVIL在我们检测的大多数剩余RMS中过表达，但在间质中几乎检测不到。 干细胞或正常肌肉组织; 3）RMS细胞在沉默MARS中对AVIL失调上瘾- AVIL（在携带融合蛋白的RMS细胞中）或沉默AVIL（在AVIL过表达的RMS中）几乎根除了肿瘤。 细胞，并显著抑制体内异种移植物，但对对照细胞没有影响; 4）相反， 过表达AVIL促进细胞增殖和迁移，使成纤维细胞形成灶，并转化成纤维细胞。 间充质干细胞; 5）AVIL表达增加的肉瘤患者预后较差;以及6） RMS细胞对我们抑制AVIL的小分子敏感。根据这些观察，我们的长期 假设AVIL是RMS的阿基里斯之踵，靶向它可能是治疗 疾病在这个应用中，我们提出了系统的调查AVIL作为一个新的目标对RMS。目的 1：确定体外靶向AVIL的功效。我们将研究它在细胞系中的作用 以及RMS PDX培养物的短期外植体。我们将测试靶向AVIL的tet诱导型shRNA的功效， 和我们通过小分子筛选鉴定的小分子化合物。自上次提交以来，我们还 产生了超过70种新化合物，它们是最初的命中化合物的衍生物。我们将在体外测试它们的功效。在 此外，我们将确定AVIL表达是否作为对AVIL抑制敏感性的生物标志物。目的 2：确定在动物RMS模型中靶向AVIL的功效。我们将使用shRNA和小分子 在异种移植物模型中使用AVIL抑制剂来测试体内靶向AVIL的功效和安全性。另外我们有 产生Avil转基因和敲除模型。我们将Avil转基因与各种Cre菌株杂交， 测试Avil过表达是否足以用于RMS肿瘤发生。我们还将使用Avil敲除小鼠 通过将它们与RMS小鼠模型杂交来测试Avil表达是否是RMS所必需的 小鼠肿瘤发生。目的3：研究AVIL抑制的下游靶点和机制， 鉴定潜在药效学生物标志物。我们将研究AVIL的分子机制， 调节FOXM1的稳定性，并确定负责其功能的F-肌动蛋白的结构域和确切活性。 致癌活性目的4：评估AVIL靶向先导化合物的最佳ADME（吸收，分布， 代谢、消除）、生物利用度和药代动力学性质。拟议的研究将有一个 对RMS的认识和治疗产生重大影响。这些发现将为将AVIL作为目标铺平道路 新的癌基因，并导致新的治疗方法的发展绝望的疾病。

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue cancer. Despite treatment intensification, the outcome for patients with advanced stage RMS has remained dismal. As of now, no targeted therapy is available. Better understanding and treatment are clearly needed. In our preliminary study, we identified a novel oncogene, AVIL in RMS. We found that, 1) AVIL forms a fusion with a house-keeping gene MARS in some RMS; 2) AVIL is overexpressed in the majority of remaining RMS we tested, yet hardly detectable in mesenchymal stem cells or normal muscle tissues; 3) RMS cells are addicted to AVIL dysregulation in that silencing MARS- AVIL (in RMS cells that harbor the fusion) or silencing AVIL (in AVIL overexpressed RMS) nearly eradicated the cells, and dramatically inhibited in vivo xenografts, but had no effect on control cells; 4) conversely, overexpressing AVIL promoted cell proliferation and migration, enabled fibroblasts to form foci, and transformed mesenchymal stem cells; 5) sarcoma patients with increased AVIL expression have worse prognosis; and 6) RMS cells are sensitive to our small molecules inhibiting AVIL. Based on these observations, our long-term hypothesis is that AVIL is an Achilles heel of RMS, and targeting it may be an effective approach for treating the disease. In this application, we propose the systematic investigation of AVIL as a novel target against RMS. Aim 1: Determine the efficacy of targeting AVIL in vitro. We will investigate its role in an expanded list of cell lines and short-term explants of RMS PDX cultures. We will test the efficacy of tet-inducible shRNAs targeting AVIL, and small molecule compounds we identified through small molecule screen. Since last submission, we also generated over 70 novel compounds derivative of the initial hit compounds. We will test their efficacy in vitro. In addition, we will determine whether AVIL expression serves as a biomarker for sensitivity to AVIL inhibition. Aim 2: Determine the efficacy of targeting AVIL in animal RMS models. We will use shRNA and small-molecule inhibitors in xenograft models to test both the efficacy and safety of targeting AVIL in vivo. In addition, we have generated both Avil transgenic and knockout models. We will cross Avil transgenic with various Cre strains to test whether Avil overexpression is sufficient for RMS tumorigenesis. We will also use Avil knockout mouse model by crossing them with RMS mouse models to test whether Avil expression is necessary for RMS tumorigenesis in mouse. Aim 3: Investigate the downstream targets and mechanisms of AVIL inhibition and identify potential pharmacodynamic biomarkers. We will investigate molecular mechanism under which AVIL regulates FOXM1 stability, and determine the domain and exact activity on F-actin that are responsible for its oncogenic activity. Aim 4: Assess AVIL-targeting lead compounds for optimal ADME (absorption, distribution, metabolism, elimination), bioavailability, and pharmacokinetic properties. The proposed study will have a significant impact on the understanding and treatment of RMS. The findings will pave ways to target AVIL as a novel oncogene, and lead to the development of novel therapeutic approaches for the desperate disease.