Investigating Oncogenic Cooperation between BMI-1 and EWS-FLI1

描述（由申请人提供）：尤文肉瘤家族肿瘤（ET）是高度侵袭性的骨和软组织肿瘤，迫切需要新的治疗方法。几乎所有的ET都表达融合癌基因EWS-FLI 1，这是一种转录因子，其确切的作用机制尚不清楚。不幸的是，ET的细胞起源仍然难以捉摸，这极大地阻碍了旨在确定新的治疗靶点的研究工作。本实验室和其他实验室的最新数据表明，ET是由神经嵴和/或间充质干细胞的恶性转化引起的。多梳基因BMI-1在许多人类癌症中高度表达，并在很大程度上通过CDKN 2A抑制来促进干细胞自我更新。我们最近发现BMI-1在ET中作为致癌基因发挥作用，但这是以CDKN 2A非依赖性方式介导的。重要的是，我们的数据表明BMI-1介导的细胞粘附变化可能促进肿瘤形成。在这个建议中，我们将测试的假设，ET来自神经嵴干细胞作为EWS-FLI 1和BMI-1之间的致癌合作的结果。我们的目标是确定BMI-1致癌活性在ET维持（目标1）和启动（目标2和3）中的机制。使用体外和体内试验，我们将确定ET细胞自我更新是否依赖于BMI-1。我们还将研究BMI-1过表达对局部和转移部位肿瘤植入的影响。通过评估EWS-FLI 1在表达不同水平BMI-1的细胞中表达的后果，我们将定义EWS-FLI 1/BMI-1合作的分子机制，并确定这些癌基因是否共同诱导原代干细胞恶性转化所必需和足够。我们的初步研究表明，与未转化的神经嵴和间充质干细胞相比，polycomb基因靶标在ET细胞中经常高度甲基化。我们将使用高度创新的人胚胎干细胞衍生的神经嵴干细胞分化模型与诱导型EWS-FLI 1表达载体相结合，以确定融合癌基因的激活是否导致发育途径的异常polycomb介导的沉默。BMI-1和polycomb活性在癌症生物学中的重要性现在已经得到了很好的证实。使用ET作为模型，这一建议将产生新的见解BMI-1在人类肿瘤的发生和发展的功能。BMI-1调节途径的鉴定将产生新的治疗干预靶点，这些靶点可用于有效劫持BMI-1活性的许多人类癌症。

DESCRIPTION (provided by applicant): Ewing sarcoma family tumors (ET) are highly aggressive bone and soft tissue tumors for which novel therapies are desperately needed. Almost all ET express the fusion oncogene EWS-FLI1, a transcription factor whose precise mechanism of action is unknown. Unfortunately, the cellular origin of ET remains elusive and this has greatly impeded research efforts aimed at identifying novel therapeutic targets. Recent data from our lab and others suggest the ET arise from malignant transformation of neural crest and/or mesenchymal stem cells. The polycomb gene BMI-1 is highly expressed by many human cancers and functions to promote stem cell self-renewal, in large part through CDKN2A repression. We have recently shown that BMI-1 acts as an oncogene in ET but that this is mediated in a CDKN2A-independent manner. Importantly, our data suggest that BMI-1-mediated changes in cell adhesion may promote tumor formation. In this proposal we will test the hypothesis that ET arise from neural crest stem cells as a result of oncogenic cooperation between EWS-FLI1 and BMI-1. It is our goal to define the mechanism of BMI-1 oncogenic activity in ET maintenance (Aim 1) and initiation (Aim 2 & 3). Using in vitro and in vivo assays we will determine if ET cell self-renewal is dependent on BMI-1. We will also investigate the impact of BMI-1 over-expression on tumor engraftment in local and metastatic sites. By assessing the consequences of EWS-FLI1 expression in cells that express variable levels of BMI-1, we will define the molecular mechanisms of EWS-FLI1/BMI-1 cooperation and determine if these oncogenes are together necessary and sufficient to induce malignant transformation of primary stem cells. Our preliminary studies have revealed that polycomb gene targets are frequently hyper-methylated in ET cells compared to untransformed neural crest and mesenchymal stem cells. We will use a highly innovative model of human embryonic stem cell-derived neural crest stem cell differentiation combined with an inducible EWS- FLI1 expression vector to determine if activation of the fusion oncogene leads to aberrant polycomb-mediated silencing of developmental pathways. The importance of BMI-1 and polycomb activity in cancer biology is now well established. Using ET as a model this proposal will generate novel insights into BMI-1 function in human tumor initiation and progression. Identification of BMI-1 -modulated pathways will generate novel targets for therapeutic intervention that can be exploited in the many human cancers that have effectively hijacked BMI-1 activity.