Therapeutically relevant targets of Twist1 dimers in glioma

 描述(申请人提供)：胶质母细胞瘤(GBM)是最致命的人类癌症之一，但尽管经过数十年的广泛努力，中位生存期仍为一年或更短。因为侵袭性和干性是导致胶质瘤恶性的相互关联的属性，所以共同调节这些表型的基因是强有力的治疗靶点。其中一个候选基因是Twist1(TW)，它是一种bHLH转录因子(TF)，对上皮间充质转化(EMT)、癌和正常发育中的侵袭、干性和转移起核心作用。在胶质瘤中，TW在较高级别的肿瘤中表达上调，并在体外促进GBM细胞的侵袭和干性，以及在体内促进胶质瘤干细胞(GSCs)的存活和生长。这些发现有力地支持了它作为GBM治疗靶点的潜力，但通过传统方法证明很难找到TFS的药物抑制剂。因此，这项建议的目的是开发间接方法，通过对TW的恶性活动至关重要的下游基因来靶向GSCs中的TW。TW必须与其他bHLH蛋白形成同源或异源二聚体来调节基因转录，而特定的二聚体基序定义了独特的表型和基因表达亚网络。激活独特的基因表达模式或亚网络，也映射到特定的恶性表型的特定TW二聚体配对，将被用来识别对TW激活GSC侵袭和生存至关重要的靶点。此外，TW的磷酸化调节二聚体伙伴亲和力和GBM细胞的侵袭行为。基于这些观察，这项提议将检验TW二聚体特定通路(子网络)可以识别治疗靶点以抑制TW调节的恶性肿瘤的假设。目的1利用蛋白质组学分析与侵袭性和干性增加或减少相关的TW bHLH家族结合伙伴。为了促进这一筛选，将通过表达亲侵袭野生型和抗侵袭TW磷酸化突变体来引入差异侵袭和二聚体亲和力。在AIM2候选二聚体中，将测试被验证为调节TW结合和侵袭性或干细胞样表型的二聚体，以确定它们对体内肿瘤致瘤性和恶性表型(侵袭性、增殖性、存活率和血管生成)的影响。在AIM3中，基因表达阵列和芯片序列将定义TW二聚体特定的亚网络，并识别直接和间接调节的TW靶点。通过对GO功能类别、途径和公共GBM数据库中表达的综合生物信息学分析对候选靶点进行排序，然后将在体外和体内进行测试，以验证在选定的GSC系中与TW抑制的等价性。这是一项创新 该方法有望识别TW二聚体特异的基因表达亚网络和靶点，从功能上复制TW抑制对侵袭性、GSC致瘤性和胶质瘤恶性程度的影响。这一策略有望对目前被认为“无法用药”的其他TF具有治疗作用。有意义的是，这些研究将提供新的机制洞察力，对理解TW在发育、EMT和TW促进恶性肿瘤的许多其他癌症中的作用具有广泛的意义。

 DESCRIPTION (provided by applicant): Glioblastoma (GBM) is among the most lethal human cancers, but despite decades of extensive effort, median survivals remain one year, or less. Because invasion and stemness are inter-connected properties that drive much of glioma malignancy, genes that co-regulate these phenotypes are robust therapeutic targets. One such candidate is TWIST1 (TW), a bHLH transcription factor (TF) central to epithelial mesenchymal transition (EMT), invasion and stemness and metastasis in carcinomas and normal development. In gliomas TW is upregulated in higher grade tumors and promotes GBM cell invasion and stemness in vitro and viability and growth of glioma stem cells (GSCs) in vivo. These findings strongly support its potential as a therapeutic target for GBM but identifying drug inhibitors of TFs has proven difficult by traditional means. The purpose of this proposal then is t develop indirect methods for targeting TW in GSCs through downstream genes essential to its malignant activity. TW must form a homo or heterodimers with other bHLH proteins to regulate gene transcription and the specific dimer motif defines unique phenotypes and gene expression sub-networks. The activation of unique gene expression patterns, or sub-networks, specific TW dimer partners which also map to specific malignant phenotypes will be leveraged to identify targets critical for TW activation of GSC invasion and survival. Further, phosphorylation of TW regulates dimer partner affinities and invasive behavior of GBM cells. Based on these observations this proposal will test the hypothesis that TW dimer specific pathways (sub-networks) can identify therapeutic targets to inhibit TW-regulated malignancy. AIM 1 employs proteomics to profile TW bHLH family binding partners associated with increased or decreased invasion and stemness. To facilitate this screen differential invasion and dimer affinity will be introduced through expression of pro- invasive wild-type and an anti-invasive TW phospho-mutant. In AIM2 candidate dimers validated to regulate TW binding and invasive or stem-like phenotypes will be tested to establish their impact on tumorigenicity and malignant phenotypes (invasion, proliferation, survival and angiogenesis) in vivo. In AIM3 gene expression arrays and ChIP-seq will define TW dimer specific sub-networks and identify direct and indirectly regulated TW targets. Candidate targets rank-ordered by comprehensive bioinformatic analysis of GO functional categories, pathways and expression in public GBM databases will then be tested in vitro and in vivo to validate equivalency with TW inhibition in selected GSC lines. This innovative approach is expected to identify TW dimer specific gene expression sub-networks and targets that functionally phenocopy the effects of TW inhibition on invasion, GSC tumorigenicity and glioma malignancy. This strategy is expected to have therapeutic relevance that translates to other TFs currently considered "undruggable". Of significance, these studies will provide new mechanistic insight with broad implications for understanding the role of TW in development, EMT and the many other cancers in which TW promotes malignancy.