Deciphering synergistic combinatorial targets in glioma

描述(申请人提供)：胶质母细胞瘤是最恶性的原发胶质瘤。尽管医学上取得了进步，但对传统疗法来说，它仍然难以奏效。最近对导致胶质瘤发生的分子事件的了解的进展为胶质瘤的治疗提供了替代的治疗靶点。针对受体酪氨酸激酶(RTK)和PTEN-PI3K-AKT通路上的各种信号转导节点的几个新的小分子已经被开发出来，目前正处于不同的测试阶段，包括EGFR抑制剂Tarceva、Iressa和AEE788以及mTOR抑制剂如雷帕霉素和CCI-779。然而，当单独使用时，它们在早期阶段的L/11临床试验中只取得了有限的成功，很可能是因为其他代偿或侧支通路否定了抑制单个信号结节的治疗效果。合乎逻辑的下一步是找出可以阻断这些通路的药物，这样当它们联合使用时，就能产生更强的抗肿瘤效果。我们的长期目标是确定特定的分子制剂，当联合使用时，将改善胶质母细胞瘤的治疗结果。这项提议寻求使用现代siRNA基因沉默技术来识别其失活与小分子抑制剂结合时将产生协同抗肿瘤活性的靶点。以下是实现这一目标的具体目标。具体目标1：确定分子靶点或途径，其干扰与先导药物治疗相结合将导致致命性表型。我们的工作假设是，沉默重叠的通路或补偿激活的小分子抑制剂(先导药物)治疗将导致细胞死亡。利用siRNA文库和信号通路药物，全基因组微阵列筛选将被用来识别当沉默时将使细胞对药物诱导的细胞死亡敏感的靶点或通路。我们希望为测试的四种药物中的每一种确定一个或多个目标。具体目标2：在体外验证其失活使肿瘤细胞对药物诱导的细胞死亡敏感的靶点或途径。我们的工作假设是，抑制目标1中确定的靶点将在体外协同增强先导药物的抗肿瘤效果。目标1中确定的靶点将通过以下方式得到验证：1)将单个siRNA的靶标失活与先导药物治疗相结合；2)将靶标灭活与市售药物和先导药物治疗相结合；3)将显性-阴性结构的靶标灭活与先导药物治疗相结合；4)评估铅药物对胶质瘤细胞系的作用；5)用反相蛋白裂解物阵列(RPPA)进行体外靶点评估。具体目的3：评价先导药物联合靶基因灭活的体内治疗效果。我们的工作假设是，当在动物模型中进行测试时，在体外验证的一个或多个靶点将发挥药物协同作用。靶基因失活药物治疗的体内疗效将在颅内和皮下动物模型中进行测试。稳定表达针对靶基因的siRNA的肿瘤细胞将被植入动物体内，随后进行铅药物治疗。对于那些可以通过使用小分子灭活的靶点，接受未经修饰的肿瘤细胞的动物将被药物和小分子治疗。将评估肿瘤大小和动物存活率，以评估联合治疗的疗效。下游效应基因的活性将通过反相蛋白裂解物阵列(RPPA)进行检测。我们预计，这些结果不仅将促进我们对COMP信号通路与特定靶向治疗反应之间的关联的了解，还将增加联合治疗胶质母细胞瘤的选择范围。

DESCRIPTION (provided by applicant): Glioblastomas is the most malignant form of primary gliomas. Despite the advance in medicine, it remains refractory to conventional therapies. Recent progress in understanding of molecular events leading to glioma genesis provides alternative therapeutic targets for management of gliomas. Several novel small molecules targeting various signaling nodes along receptor tyrosine kinases (RTKs) and PTEN-PI3K-akt pathway have been developed and are currently in different stages of testing, including EGFR inhibitors like Tarceva, Iressa, and AEE788 as well as mTOR inhibitors such as Rapamycin and CCI-779. However, they have met with only limited success in early phase l/ll clinical trials when used alone, most likely because other compensatory or collateral pathways negate the therapeutic efficacy of inhibiting a single signal node. The logical next step, is to identify drugs that would block these pathways so that when given in combination, they elicit enhanced anti-tumor effect. Our long-term goal is to identify specific molecular agents that, when used in combination, would improve the therapeutic outcome for glioblastoma. This proposal seeks to use modern siRNA gene-silencing techniques to identify targets whose inactivation when combined with small molecule inhibitors would confer synergistic anti-tumor activity. Following are specific aims to achieve this goal. Specific Aim 1: To identify the molecular targets or pathways whose disruption in combination with lead drug treatment will result in a lethal phenotype. Our working hypothesis is that silencing overlapping pathways or compensatory activated in response to treatment with a small molecule inhibitor (lead drug) will result in cell death. Using both a siRNA library and a signaling pathway drug, a genome-wide microarray screen will be employed to identify targets or pathways that when silenced will sensitize cells to drug-induced cell death. We expect to identify one or more targets for each of the four drugs tested. Specific Aim 2: To validate the targets or pathways whose inactivation sensitizes tumor cells to drug-induced cell death in vitro. Our working hypothesis is that inhibiting the targets identified in aim 1 will synergistically enhance the anti-tumor effect of the lead drug in vitro. Targets identified in aim 1 will be validated by 1) combining inactivation of targets by individual siRNA with lead drug treatment; 2) combining inactivation of targets with commercially available drug and lead drug treatment; 3) combining inactivation of targets by a dominant-negative constructs with lead drug treatment;. 4) assessment of lead drug effect on a battery of glioma cell lines; and 5) In vitro target evaluation with reverse phase protein lysate array (RPPA). Specific Aim 3: To evaluate the combined therapeutic efficacy of lead drug and target gene inactivation in vivo. Our working hypothesis is that one or more of the targets validated in vitro will exert drug-synergy when tested in animal models. The in vivo therapeutic efficacy of drug treatment with target gene inactivation will be tested in both intracranial and subcutaneous animal models. Tumor cells stably expressing siRNA against the target gene will be implanted into animals followed by lead drug treatment. For those targets whose inactivation could be achieved by using small molecules, animals receiving unmodified tumor cells will be treated with drug and small molecules. Tumor size and animal survival will be assessed to evaluate the therapeutic efficacy of combination treatments. The activity of downstream effector genes will be examined by reverse phase protein lysate array (RPPA). We expect that the results will not only advance our knowledge of the association between comp signal pathways and response to a particular targeted therapy but also increase the range of options for treating glioblastoma with combination therapy.