Generation of TWIST1 reporters through characterization of TWIST1 dependent netwo

描述(申请人提供)：胶质母细胞瘤(GBM)是最恶性和最常见的固有脑肿瘤。尽管进行了积极的治疗，但这种疾病总是致命的，患者平均存活不到一年。针对这种致命疾病的进展需要识别特定的分子靶点和工具，以快速筛选抑制侵袭性胶质瘤干细胞(GSC)功能的治疗剂。我们首先确定了Twist1在GBM侵袭中的作用，并在体外实验中证明了Twist1基因敲除抑制了人脑胶质瘤干细胞的活性。稳定地抑制Twist1在人GSCs或小鼠转化的神经干细胞和祖细胞中的表达显示出显著的体内肿瘤生长抑制。我们的结果表明Twist1在GSC的致瘤性中起着关键作用，提示抑制Twist1可能具有治疗意义。然而，到目前为止，Twist1抑制剂的产生几乎是不可能的，因为1)转录因子很难靶向；2)对Twist1途径的了解非常有限；以及3)Twist1活性的可靠报告不存在。我们方法的创新之处在于为这一障碍开发了一种“变通办法”。这项可行性研究非常适合R21机制，因为它将研究GSCs中的Twist1途径，并将产生重要的新研究工具，以促进在GSCs中开发Twist1抑制剂。结合基因表达微阵列的GSCs和Twist1基因敲除参考芯片测序来确定直接的Twist1靶点，通路分析将确定通过与Twist1直接和间接相互作用而调节的基因/网络。对所得数据的生物信息学分析将导致识别与TWST1活性相关的候选转录调控基序。然后，这些基序将被用来产生报告结构，为GSCs中Twist1途径的活性调节提供信息。通过激活或抑制基因来反映Twist1调节的双重报告有望在随后的TW途径抑制剂的高通量筛选中提高这一方法的敏感性和特异性。由于Twist1参与了胶质瘤的发展和多种恶性肿瘤的侵袭和转移，因此Twist1抑制剂的产生有望对临床肿瘤学领域产生广泛的影响。

DESCRIPTION (provided by applicant): Glioblastomas (GBM) are the most malignant and common intrinsic brain tumor. Despite aggressive treatment the disease is uniformly fatal and patients survive on average less than a year. Progress against this lethal disease requires the identification of specific molecular targets and tools for rapid screening of therapeutic agents that inhibit invasive glioma stem cell (GSC) function. We first identified the role of TWIST1 in GBM invasion and demonstrated that TWIST1 knockdown inhibits human glioma stem cell activity in in vitro assays. Stable knockdown of TWIST1 expression in human GSCs or mouse-transformed neural stem and progenitor cells demonstrated significant inhibition of tumor growth in vivo. Our results show the critical role of TWIST1 in GSC tumorigenicity, suggesting that inhibition of TWIST1 may have therapeutic significance. However, the generation of TWIST1 inhibitors has to date proven nearly impossible because 1) transcription factors are difficult to target; 2) there is very limited knowledge of the TWIST1 pathway; and, 3) reliable reporters of TWIST1 activity do not exist. The innovation of our approach is to develop a "workaround" to this roadblock. This feasibility study is ideally suited for the R21 mechanism, as it will investigate the TWIST1 pathway in GSCs and will generate important new research tools to facilitate developing TWIST1 inhibitors in GSCs. Using a combined analysis of gene expression microarrays of GSCs with TWIST1 knockdown referenced to CHIP-sequencing to define direct TWIST1 targets, pathway analysis will identify genes/networks regulated through direct and indirect interactions with TWIST1. Bioinformatic analysis of the resulting data will lead to identification of candidate transcriptional regulatory motifs associated with TWST1 activity. These motifs will then be used to generate reporter constructs that inform on regulation of TWIST1 pathway activity in GSCs. Dual reporters that reflect TWIST1 regulation through activation or repression of genes are expected to increase the sensitivity and specificity of this approach in subsequent high-throughput screening for TW pathway inhibitors. Because TWIST1 is involved in the progression of gliomas and in the invasion and metastasis of a large variety of malignant tumors, the generation of TWIST1 reporters to screen for TWIST1 inhibitors is expected to have broad impact on the field of clinical oncology.