Targeted epigenetic silencing of oncogenic Transcription Factors (PQ18)

致癌转录因子的靶向表观遗传沉默 (PQ18)

• 批准号: 8382851
• 负责人: PILAR BLANCAFORT
• 金额: $ 30.71万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2012-08-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8382851
• 关键词: Architecture; Automobile Driving; Binding; Biological Assay; Breast; Breast Cancer Cell; Cancer cell line; Catalytic Domain; Cell Culture Techniques; Cell Cycle; Cell Proliferation; Cell division; Cells; Chromatin; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA-Binding Proteins; Deposition; Development; Devices; Down-Regulation; Drug Design; Encapsulated; Engineering; Enzymes; Epigenetic Process; Epithelial Cells; Frequencies; G1 Arrest; Gene Expression; Gene Silencing; Generations; Genes; Growth; Heart; Human; Implant; In Vitro; Inherited; Injection of therapeutic agent; Lead; Life; Link; Longevity; Maintenance; Malignant Neoplasms; Malignant neoplasm of ovary; Mammary Neoplasms; Mammary gland; Measurement; Memory; Messenger RNA; Methods; Methylation; Modeling; Modification; Molecular; Monitor; Nature; Normal tissue morphology; Nude Mice; Oncogenes; Oncogenic; Pattern; Pharmaceutical Preparations; Phase; Phenotype; Physical condensation; Physiologic pulse; Play; Promoter Regions; Protein Engineering; RNA Interference; Recurrence; Refractory; Relapse; Relative (related person); Retroviral Vector; Reverse Transcriptase Polymerase Chain Reaction; Role; Shapes; Site; Small RNA; Sodium; Staging; Stem cells; Technology; Testing; Therapeutic Agents; Therapeutic Effect; Time; Tumor Cell Line; Work; Xenograft procedure; Zinc Fingers; activating transcription factor; base; cancer cell; cell growth; chemotherapy; chromatin immunoprecipitation; design; embryonic stem cell; in vitro Model; in vivo; knock-down; link protein; malignant breast neoplasm; mouse model; mutant; nanoparticle; neoplastic cell; novel therapeutics; pre-clinical; prevent; promoter; self-renewal; small molecule; stem cell differentiation; subcutaneous; therapeutic target; transcription factor; transmission process; tumor; tumor growth; tumor initiation; tumor progression; vector

DESCRIPTION (provided by applicant): Epigenetic modifications play a key role in tumor origin and progression. Oncogenic transcription factors (TFs) are frequently over-expressed in breast cancers, while being silenced in normal epithelial cells. TFs can switch entire transcriptional gene cascades, resulting in tumor initiation and progression. Since most TFs do not have intrinsic enzymatic activities and they lack small-molecule-binding pockets, these targets have been refractory to drug design. The oncogenic TFs Sox2 is over-expressed in breast cancers of advanced stage, while the gene is silenced and hyper-methylated in normal epithelial cells. As a stable repressive mark, DNAme catalyzed by DNA-methyltransferases, is regarded as a key player in epigenetic silencing. DNAme orchestrate other epigenetic modifications, shaping the architecture of the promoter and driving chromatin condensation and gene silencing. A hallmark of DNAme is that it is hereditary and thereby transmitted over cell generations. In many developmentally regulated TFs, such as Sox2, DNAme constitute an epigenetic switch, which changes cells from an active mitogenic state towards a G0/G1 arrest and differentiation. In this application, our objective is to target DNAme into the promoter of Sox2, which is highly expressed in breast cancer cell lines, with levels comparable or superior to embryonic stem cells. To direct specific DNAme, we will fuse engineered DNA-binding proteins made of sequence-specific Zinc Finger (ZF) domains with a catalytically active DNA-methyltransferase domain (Dnmt3a). Our objective is to restore the hereditable epigenetic silencing in the Sox2 promoter of the tumor cell in a pattern that is similar to breast epithelial cells. We hypothesize that ZFs-Dnmt3a fusions are able to target DNAme marks into the Sox2 oncogenic promoter, resulting in transmission of these marks over cell generations. This epigenetic memory will be accompanied by the maintenance of the transcriptional silencing and tumor cell growth inhibition. In Aim1 we propose the construction of 6ZF proteins linked to the Dnmt3a and inactive mutants, to assess whether these engineered proteins deposit specific silencing marks into the Sox2 promoter, resulting in oncogenic silencing. In Aim2 we monitor the longevity of the silencing implemented by the 6ZF- silencers. We will express the 6ZF constructs using inducible vectors to "pulse" and "chase" DNAme in cell culture and breast tumor models. Next, to move the technology towards a pre-clinical phase, we will deliver ATF mRNAs using nanoparticles that will be injected in mouse models of breast cancer (Aim 3). While RNAi technology can be used to knock-down oncogenes, its therapeutic effect is transient because of the short-lived time of the small RNA. The significance of this application is the potential of the ZF agent to induce an endogenous epigenetic reprogramming of the target TF, which is expected to maintain the longevity of the therapeutic effect. Thus, this work will be of vital importance to develop stable, inherited, oncogenic silencing methods, to suppress oncogenic expression in tumor cells. PUBLIC HEALTH RELEVANCE: Oncogenic Transcription Factors (TFs) play a critical role in the initiation and progression of human tumors. These genes are often epigenetically dysregulated, being aberrantly hypomethylated and over-expressed in cancer cells, relative to normal tissues. Due to their lack of small-molecule binding pockets, TFs are very refractory to small molecule-based approaches. Our work provides a new strategy to suppress the expression of these classically "undruggable" targets. We propose the design of zinc finger proteins linked to catalytically active DNA-methyltransferase domain to restore the epigenetic silencing these genes. By reprogramming the epigenetic silencing of tumor cells we hope to promote inherited changes in the epigenome of cancer cells and ultimately induce a more stable anti-tumor and therapeutic effect, helping minimize tumor relapse even if the ZF agent is discontinued. This work will provide sequence-specific molecular devices to stably restore the epigenetic silencing of oncogenic TFs. Our work will lead to the development of novel therapeutic agents for the treatment of aggressive breast and ovarian cancers, for which no cure is available.

描述（由申请人提供）：表观遗传修饰在肿瘤的发生和发展中起关键作用。致癌转录因子（TFs）在乳腺癌中经常过度表达，而在正常上皮细胞中是沉默的。tf可以改变整个转录基因级联反应，导致肿瘤的发生和发展。由于大多数tf不具有内在的酶活性，并且它们缺乏小分子结合袋，因此这些靶点难以进行药物设计。致癌TFs Sox2在晚期乳腺癌中过度表达，而该基因在正常上皮细胞中沉默和超甲基化。作为一个稳定的抑制标记，dna甲基转移酶催化的dna被认为是表观遗传沉默的关键参与者。DNAme协调其他表观遗传修饰，塑造启动子的结构，驱动染色质凝聚和基因沉默。DNAme的一个特点是它是遗传性的，因此在细胞世代之间传播。在许多发育调节的tf中，如Sox2， DNAme构成一个表观遗传开关，将细胞从活跃的有丝分裂状态改变为G0/G1停滞和分化。在这项应用中，我们的目标是将DNAme靶向到Sox2的启动子中，Sox2在乳腺癌细胞系中高度表达，其水平与胚胎干细胞相当或更高。为了指导特异性DNAme，我们将融合由序列特异性锌指结构域（ZF）组成的工程dna结合蛋白与催化活性dna甲基转移酶结构域（Dnmt3a）。我们的目标是恢复肿瘤细胞中Sox2启动子的可遗传表观遗传沉默，其模式与乳腺上皮细胞相似。我们假设ZFs-Dnmt3a融合能够将DNAme标记靶向到Sox2致癌启动子中，从而导致这些标记在细胞世代中传递。这种表观遗传记忆将伴随着转录沉默的维持和肿瘤细胞生长的抑制。在Aim1中，我们提出构建与Dnmt3a和失活突变体连接的6ZF蛋白，以评估这些工程蛋白是否将特定的沉默标记沉积到Sox2启动子中，从而导致致癌沉默。在目标2中，我们监测了6ZF-消声器实现的消声的寿命。我们将使用诱导载体表达6ZF构建体，在细胞培养和乳腺肿瘤模型中“脉冲”和“追逐”DNAme。接下来，为了将该技术推向临床前阶段，我们将使用纳米颗粒将ATF mrna注射到乳腺癌小鼠模型中（目标3）。虽然RNAi技术可以用于敲除癌基因，但由于小RNA的时间很短，其治疗效果是短暂的。这一应用的意义在于ZF药物有可能诱导靶TF的内源性表观遗传重编程，这有望维持治疗效果的持久性。因此，这项工作对于开发稳定的、遗传的、致癌的沉默方法来抑制肿瘤细胞中的致癌表达至关重要。