Generation of TWIST1 reporters through characterization of TWIST1 dependent netwo

描述（申请人提供）：胶质母细胞瘤（GBM）是恶性程度最高、最常见的脑肿瘤。尽管进行了积极的治疗，这种疾病仍然是致命的，患者平均存活时间不到一年。对抗这种致命疾病的进展需要确定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敲低可以抑制人胶质瘤干细胞的活性。稳定敲低TWIST1在人GSCs或小鼠转化的神经干和祖细胞中的表达，在体内可显著抑制肿瘤生长。我们的研究结果表明TWIST1在GSC致瘤性中起关键作用，提示抑制TWIST1可能具有治疗意义。然而，迄今为止，产生TWIST1抑制剂几乎是不可能的，因为1)转录因子难以靶向；2)对TWIST1通路的了解非常有限；3)不存在可靠的TWIST1活动报告。我们方法的创新之处在于为这个障碍开发了一个“变通方法”。这项可行性研究非常适合R21机制，因为它将研究GSCs中的TWIST1通路，并将为开发GSCs中的TWIST1抑制剂提供重要的新研究工具。利用TWIST1敲低的GSCs基因表达微阵列结合chip测序来确定TWIST1的直接靶点，途径分析将确定通过与TWIST1直接和间接相互作用调控的基因/网络。对所得数据的生物信息学分析将导致与TWST1活性相关的候选转录调控基序的鉴定。然后，这些基序将用于生成报告结构，以告知GSCs中TWIST1通路活性的调节。通过激活或抑制基因来反映TWIST1调控的双报告基因有望在随后的TW通路抑制剂高通量筛选中提高该方法的敏感性和特异性。由于TWIST1参与胶质瘤的进展以及多种恶性肿瘤的侵袭和转移，因此产生TWIST1报告基因来筛选TWIST1抑制剂有望在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.