High throughput assays for modulators of splicing switches during the EMT

描述(申请人提供)：上皮-间充质转化(EMT)的特征是上皮细胞失去细胞间黏附和细胞极性，并获得运动性和侵袭性。虽然EMT对肿瘤的发展至关重要，但它是肿瘤获得组织侵袭和转移能力的一种机制。因此，确定能够抑制EMT的新疗法是很重要的，但在高通量筛选(HTS)中开发的EMT抑制剂的分析方法很少。成纤维细胞生长因子受体2(FGFR2)剪接的变化发生在EMT过程中，我们使用一种创新的基于荧光素酶的剪接报告分析方法，先前进行了全基因组高通量cDNA表达筛选，以调节这种剪接开关。该筛查确定了上皮细胞类型特异性剪接调节因子ESRP1和ESRP2，证明了基于细胞的剪接分析在高通量、基于阵列的筛查中的可行性。在EMT过程中，一组广泛的ESRP调控的外显子切换剪接，表明在这一过程中发生了选择性剪接的全局变化。因此，这种剪接网络的变化是EMT的一个动态特征，ESRP调控靶的剪接变化可以作为EMT的生物标志物。在这一应用中，我们将使用ESRP调节的外显子开发更强大的下一代剪接报告分析，这些外显子在剪接中经历深刻的“开关”变化，并使用分子文库生产中心网络(MLPCN)配置它们用于HTS分析。在目标1中，我们将在我们建立的基于荧光素酶的报告基因的背景下，为HTS改编包含ESRP调节的外显子和侧翼内含子调控序列的现有微基因记者。记者将包括其包含被激活的外显子以及那些在EMT期间经历跳过的外显子。还将开发更多的记者，用于反屏幕，以确定HTS点击的优先顺序。在目标2中，这些筛选将被配置为以384井格式进行筛选，并将使用几个小化合物文库以及几个先前描述的化合物进行中试筛选，这些化合物已被证明起到剪接的一般调节剂的作用。这些化合物将在间充质细胞中筛选指示间充质向上皮转化(MET)反向过程的剪接变化，并在上皮细胞中筛选抑制或逆转可诱导的EMT。在第一年成功完成这一筹资机制的试点阶段(PAR-10-182)，将使我们能够使用MLPCN化合物资料库提交这些化验，以供更大规模的筛选阶段使用。这类筛选很有希望产生新的剪接小分子调节剂，包括一个广泛促进上皮特异性剪接途径的子集，以抑制或逆转EMT并阻止癌症转移。这类化合物可能包括那些影响信号通路或其他上游事件的化合物，这些上游事件可能会有效地激活抑制EMT的广泛转录和转录后基因表达程序。 公共卫生相关性：上皮向间充质转化(EMT)是癌细胞可以从原发部位逃逸并转移到远处的过程，因此是新型癌症治疗的靶点。我们已经确定了选择性剪接的调节因子，它控制着在EMT过程中丢失的上皮剪接网络，这表明间充质剪接计划可以促进EMT，这些剪接变化可以作为这一过程的生物标志物。目前的应用将使用创新的剪接分析来筛选抑制这种剪接过渡的新化合物，从而识别用于预防肿瘤转移的药物的先导化合物。

DESCRIPTION (provided by applicant): The epithelial-mesenchymal transition (EMT) is characterized by the loss of cell-cell adhesion and cell polarity in epithelial cells and the acquisition of motile and invasive properties. While essential for development, the EMT is one mechanism by which tumors can acquire the capability to undergo tissue invasion and metastasis. It is therefore important to identify novel therapies that can inhibit the EMT, but few assays for EMT inhibitors in high throughput screens (HTS) have developed. A change in fibroblast growth factor receptor 2 (FGFR2) splicing occurs during the EMT and using an innovative luciferase-based splicing reporter assay we previously carried out a genome-wide high throughput cDNA expression screen for regulators of this splicing switch. This screen identified the epithelial cell type specific splicing regulators ESRP1 and ESRP2 demonstrating the feasibility of cell-based splicing assays in high throughput, array-based screens. An extensive set of ESRP-regulated exons switch splicing during the EMT, indicating that global changes in alternative splicing occur during this process. A change in this splicing network is a thus a dynamic feature of the EMT and changes in splicing of ESRP-regulated targets can be used as a biomarker for the EMT. In this application we will develop more robust next generation splicing reporter assays using ESRP- regulated exons that undergo profound "switch-like" changes in splicing and configure them for HTS assays using the Molecular Libraries Production Centers Network (MLPCN). In Aim 1, we will adapt existing minigene reporters containing ESRP regulated exons and flanking intronic regulatory sequences for HTS in the context of our established luciferase-based reporter minigenes. The reporters will include exons whose inclusion is activated as well as those that undergo skipping during the EMT. Additional reporters will also be developed for use in counter-screens to prioritize HTS hits. In Aim 2, these screens will be configured for screening in 384 well format and pilot screens will be carried out using several small compound libraries as well as several previously described compounds that have been shown to function as general modulators of splicing. These compounds will be screened in mesenchymal cells for splicing changes indicative of the reverse process of mesenchymal to epithelial transition (MET) and in epithelial cells for inhibition or reversal of an inducible EMT. Successful completion of this pilot phase of this funding mechanism (PAR-10-182) in year one will enable us to submit these assays for the larger scale screening phase using the MLPCN library of compounds. Such screens hold great promise to yield novel small molecule regulators of splicing, including a subset that broadly promote epithelial-specific splicing pathways to inhibit or reverse the EMT and block cancer metastasis. Such compounds will potentially include those that affect signaling pathways or other upstream events that might potently activate broad transcriptional and post-transcriptional gene expression programs that inhibit the EMT. PUBLIC HEALTH RELEVANCE: The epithelial to mesenchymal transition (EMT) is the process by which cancer cells can escape from the primary site and metastasize to distant sites and is therefore a target for novel cancer therapies. We have identified regulators of alternative splicing that control an epithelial splicing network that is lost during the EMT, suggesting that a mesenchymal splicing program can promote the EMT and that these splicing changes serve as biomarkers for this process. The current application will use innovative splicing assays to carry out screens for novel compounds that inhibit this splicing transition and thereby identify lead compounds for drugs to prevent tumor metastasis.