Investigating Oncogenic Cooperation between BMI-1 and EWS-FLI1

描述（申请人提供）：Ewing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-FLI1，这是一种其确切作用机制尚不清楚的转录因子。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。我们实验室和其他实验室最近的数据表明，ET起源于神经嵴和/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抑制CDKN2A来促进干细胞自我更新。我们最近表明，BMI-1在ET中作为致癌基因，但这是通过cdkn2a独立的方式介导的。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的形成。在本提案中，我们将验证ET是由神经嵴干细胞作为EWS-FLI1和BMI-1之间的致癌合作的结果而产生的假设。我们的目标是确定BMI-1在ET维持（Aim 1）和启动（Aim 2和3）中的致癌活性机制。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过表达对肿瘤在局部和转移部位植入的影响。通过评估EWS-FLI1在表达不同水平BMI-1的细胞中表达的后果，我们将确定EWS-FLI1/BMI-1合作的分子机制，并确定这些致癌基因是否共同诱导原代干细胞恶性转化的必要和充分条件。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，多梳基因靶点在ET细胞中经常被超甲基化。我们将使用一个高度创新的人胚胎干细胞衍生的神经嵴干细胞分化模型，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异常的多梳介导的发育途径沉默。BMI-1和多梳蛋白活性在癌症生物学中的重要性现已得到充分证实。以ET为模型，这一提议将对BMI-1在人类肿瘤发生和发展中的功能产生新的见解。鉴定BMI-1调节通路将产生新的治疗干预靶点，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.