Generation of TWIST1 reporters through characterization of TWIST1 dependent netwo

描述（由申请人提供）：胶质母细胞瘤（GBM）是最恶性和最常见的内源性脑肿瘤。尽管进行了积极的治疗，这种疾病仍然是致命的，患者平均存活不到一年。针对这种致命疾病的进展需要鉴定特异性分子靶标和工具，用于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TWIST 1在GBM侵袭中的作用，并在体外试验中证明了TWIST 1敲低抑制人胶质瘤干细胞活性。在人GSC或小鼠转化的神经干细胞和祖细胞中稳定敲除TWIST 1表达证明了体内肿瘤生长的显著抑制。我们的研究结果表明TWIST 1在GSC致瘤性中的关键作用，表明抑制TWIST 1可能具有治疗意义。然而，TWIST 1抑制剂的产生迄今已被证明几乎是不可能的，因为1）转录因子难以靶向; 2）对TWIST 1途径的了解非常有限;以及3）不存在可靠的TWIST 1活性报告基因。我们方法的创新之处在于为这一障碍制定一个“变通办法”。 这项可行性研究非常适合R21机制，因为它将研究GSC中的TWIST 1通路，并将产生重要的新研究工具，以促进在GSC中开发TWIST 1抑制剂。使用参考CHIP测序的具有TWIST 1敲低的GSC的基因表达微阵列的组合分析来定义直接TWIST 1靶标，途径分析将鉴定通过与TWIST 1的直接和间接相互作用调节的基因/网络。对所得数据的生物信息学分析将导致识别与TWST 1活性相关的候选转录调控基序。然后，这些基序将用于产生报告构建体，其告知GSC中TWIST 1途径活性的调节。通过激活或抑制基因来反映TWIST 1调节的双报告基因有望在随后的TW途径抑制剂的高通量筛选中增加这种方法的灵敏度和特异性。由于TWIST 1参与神经胶质瘤的进展以及多种恶性肿瘤的侵袭和转移，因此产生TWIST 1报告基因以筛选TWIST 1抑制剂预计将对临床肿瘤学领域产生广泛影响。

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.