Investigating Oncogenic Cooperation between BMI-1 and EWS-FLI1

描述(申请人提供)：尤文肉瘤家族肿瘤(ET)是一种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-FLI1，这是一种转录因子，其确切作用机制尚不清楚。不幸的是，ET的细胞来源仍然难以捉摸，这极大地阻碍了旨在确定新的治疗靶点的研究努力。来自我们实验室和其他实验室的最新数据表明，ET源于神经脊和/或间充质干细胞的恶性转化。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CDKN2A抑制发挥促进干细胞自我更新的功能。我们最近发现BMI-1在ET中起癌基因的作用，但这是以CDKN2A不依赖的方式介导的。重要的是，我们的数据表明BMI-1介导的细胞黏附的变化可能促进肿瘤的形成。在这个提案中，我们将检验这一假设，即ET来自神经脊干细胞，这是EWS-FLI1和BMI-1之间癌基因合作的结果。我们的目标是明确BMI-1致癌活性在ET维持(目标1)和启动(目标2和3)中的作用机制。通过体外和体内实验，我们将确定ET细胞的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-FLI1在表达不同水平BMI-1的细胞中的表达后果，我们将确定EWS-FLI1/BMI-1协同作用的分子机制，并确定这些癌基因一起是否共同诱导原代干细胞恶性转化。我们的初步研究表明，与未转化的神经脊和间充质干细胞相比，多梳基因靶标在ET细胞中经常超甲基化。我们将使用一种高度创新的人类胚胎干细胞来源的神经脊干细胞分化模型，结合可诱导的EWS-FLI1表达载体来确定融合癌基因的激活是否会导致多梳介导的发育途径异常沉默。BMI-1和多梳联活性在癌症生物学中的重要性现在已经得到了充分的证实。使用ET作为模型，这一建议将对BMI-1在人类肿瘤发生和发展中的作用产生新的见解。识别BMI-1调节的通路将产生新的治疗干预靶点，可用于许多有效劫持BMI-1活性的人类癌症。

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.