Deciphering synergistic combinatorial targets in glioma

描述（由申请人提供）：胶质母细胞瘤是原发性胶质瘤中最恶性的一种。尽管医学进步，它仍然难以用传统的治疗方法。最近的进展，在理解的分子事件，导致胶质瘤的发生提供了替代的治疗目标的管理胶质瘤。已经开发了几种靶向沿着受体酪氨酸激酶（RTK）和PTEN-PI 3 K-akt通路的各种信号传导节点的新型小分子，并且目前处于不同的测试阶段，包括EGFR抑制剂如特罗凯（Tarceva）、易瑞沙（Iressa）和AEE 788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I/II期临床试验中仅获得有限的成功，这很可能是因为其他代偿或侧支途径否定了抑制单个信号节点的治疗功效。合乎逻辑的下一步是确定可以阻断这些途径的药物，以便在联合给药时，它们会引起增强的抗肿瘤作用。我们的长期目标是确定特定的分子药物，当联合使用时，将改善胶质母细胞瘤的治疗结果。该提案寻求使用现代siRNA基因沉默技术来鉴定当与小分子抑制剂组合时其失活将赋予协同抗肿瘤活性的靶标。以下是实现这一目标的具体目标。具体目标1：确定分子靶点或通路，其与先导药物治疗联合破坏将导致致死表型。我们的工作假设是，沉默重叠的途径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。使用siRNA文库和信号通路药物，将采用全基因组微阵列筛选来鉴定靶点或通路，当沉默时，这些靶点或通路将使细胞对药物诱导的细胞死亡敏感。我们希望为四种测试药物中的每一种确定一个或多个靶标。具体目的2：验证其失活可使肿瘤细胞对体外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中确定的靶点将协同增强先导药物的体外抗肿瘤作用。目标1中鉴定的靶标将通过以下方式进行验证：1）将单个siRNA的靶标失活与先导药物治疗相结合; 2）将市售药物和先导药物治疗与靶标失活相结合; 3）将显性阴性构建体的靶标失活与先导药物治疗相结合。4)评价先导药物对一组胶质瘤细胞系的作用;和5）用反相蛋白裂解物阵列（RPPA）进行体外靶向评价。具体目的3：评价先导药物与靶基因失活的体内联合治疗效果。我们的工作假设是，一个或多个在体外验证的目标将发挥药物协同作用时，在动物模型中进行测试。将在颅内和皮下动物模型中测试靶基因失活的药物治疗的体内治疗功效。将稳定表达针对靶基因的siRNA的肿瘤细胞植入动物体内，然后进行先导药物治疗。对于那些可以通过使用小分子实现失活的靶点，接受未修饰肿瘤细胞的动物将用药物和小分子进行治疗。将评估肿瘤大小和动物存活率，以评价联合治疗的疗效。下游效应基因的活性将通过反相蛋白裂解物阵列（RPPA）来检查。我们希望这些结果不仅能提高我们对comp信号通路与特定靶向治疗反应之间关联的认识，还能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.