High Throughput Screening for Modulators of STAT5 (RMI)

描述(由申请人提供)：在确定癌症靶向治疗的途径时，三个要求是最重要的。该途径必须在肿瘤细胞中被激活，必须服从于药物调节，并且在正常细胞中抑制该途径不能导致过度的毒性。STAT家族转录因子是完全满足这些要求的靶标。特别是，STAT3和STAT5在广泛的人类肿瘤中被不适当地激活，它们的抑制在正常组织中可以耐受，几乎没有有害影响。我们已经开发了一个系统来筛选STAT5的抑制剂，我们已经确定了一些有希望的化合物。然而，STAT5在许多血液病癌症中被激活，通常不被我们已经确定的STAT3调节器抑制。鉴于我们在识别STAT3抑制剂方面取得的成功，以及它们与STAT5缺乏交叉反应，本提案的目标是开发一种高通量筛选系统来识别STAT5抑制剂。我们将建立稳定的细胞系，在其中引入由β-酪蛋白启动子驱动的荧光素酶报告基因。荧光素酶活性是由该结构诱导的，以响应激活的STAT5，但不响应其他STATS或其他转录因子。我们将使用三个细胞系统：T47D人乳腺癌细胞，其中STAT5可以通过内源性催乳素受体激活；NKL人白血病细胞，其中STAT5在IL-2治疗后被激活；以及BA/F3细胞，其中STAT5在IL-3作用下被激活。筛查将在哈佛医学院的化学和细胞生物学研究所进行，我们在那里有一个长期的合作，以确定其他统计数据的调节器。在最初的筛选中，我们将确定抑制或诱导STAT5依赖的荧光素酶表达的化合物。虽然这项工作的主要目标是确定STAT5抑制剂，但激活STATS的化合物也可能具有治疗益处，特别是在乳腺癌中。然后，我们将在稳定表达NF-kappaB依赖的荧光素酶结构的细胞中进行二次筛选，以测试这些调节器对STAT途径的特异性，这是我们之前为早期筛选构建的。对STATS依赖的基因激活表现出特异性的化合物将被评估其机械性能，并将在适当的体外和动物模型中进行研究。通过这种经过验证和结构化的方法，我们计划在一年的资助期结束前确定STAT5的10种特定抑制剂和5种特定激活剂。最终目标是开发出可作为人类癌症靶向疗法的化合物。

DESCRIPTION (provided by applicant): In identifying pathways for the targeted therapy of cancer, three requirements are paramount. The pathway must be activated in tumor cells, it must be amenable to pharmacological modulation, and inhibition of the pathway in normal cells must not lead to undue toxicity. STAT family transcription factors are targets that fulfill these requirements fully. In particular, STAT3 and STAT5 are activated inappropriately in a wide array of human tumors, and their inhibition can be tolerated in normal tissue with little detrimental effect. We have developed a system to screen for inhibitors of STAT5, and we have identified a number of promising compounds. However, STAT5, which is activated in many hematologic cancers, is generally not inhibited by the STAT3 modulators we have identified. Given the success we have had in identifying STAT3 inhibitors, and their lack of cross-reactivity with STAT5, the goal of this proposal is to develop a high throughput screening system to identify inhibitors of STAT5. We will create cell lines in which stable transfection is used to introduce a luciferase reporter gene driven by the beta-casein promoter. Luciferase activity is induced from this construct in response to activated STAT5, but not in response to other STATs or other transcription factors. We will use three cellular systems:T47D human breast cancer cells, in which STAT5 can be activated through the endogenous prolactin receptor; NKL human leukemia cells in which STAT5 is activated following IL-2 treatment; and, Ba/F3 cells, a hematopoietic cell line in which STAT5 is activated in response to IL-3. The screen will be conducted at the Institute of Chemistry and Cell Biology at Harvard Medical School, where we have a longstanding collaboration to identify modulators of other STATs. In the initial screen, we will identify compounds that inhibit or induce STAT5-dependent luciferase expression. Although the primary goal of this work is to identify STAT5 inhibitors, compounds that activate STATS may also have therapeutic benefit, particularly in breast cancer. These modulators will then be tested for specificity for the STAT pathway by performing a secondary screen in cells stably expressing NF-kappaB dependent luciferase constructs, which we had constructed previously for our earlier screens. Compounds that show specificity for STATS-dependent gene activation will then be evaluated for mechanistic properties, and will be studied in appropriate in vitro and animal models. Through this validated and structured approach, we plan to identify ten specific inhibitors and five specific activators of STAT5 by the end of the one year funding period. The ultimate goal is the development of compounds that will act as targeted therapies for human cancers.