Targeted epigenetic silencing of oncogenic Transcription Factors (PQ18)

描述（由申请人提供）：表观遗传修饰在肿瘤起源和进展中起关键作用。致癌转录因子（TF）在乳腺癌中经常过度表达，而在正常上皮细胞中沉默。TF可以切换整个转录基因级联，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口袋，因此这些靶标对于药物设计是难治的。Sox 2在晚期乳腺癌中过度表达，而在正常上皮细胞中该基因被沉默和过度甲基化。DNA甲基转移酶催化的DNAme作为一种稳定的抑制性标记，被认为是表观遗传沉默的关键因素。DNAme协调其他表观遗传修饰，塑造启动子的结构并驱动染色质凝聚和基因沉默。DNAme的一个标志是它是遗传性的，从而在细胞世代中传递。在许多发育调节的TF中，如Sox 2，DNAme构成表观遗传开关，其将细胞从活跃的促有丝分裂状态改变为G 0/G1停滞和分化。在本申请中，我们的目的是将DNAme靶向Sox 2的启动子，Sox 2在乳腺癌细胞系中高度表达，其水平与胚胎干细胞相当或上级。为了指导特异性DNAme，我们将融合由序列特异性锌指（ZF）结构域与催化活性DNA甲基转移酶结构域（Dnmt 3a）组成的工程化DNA结合蛋白。我们的目标是以类似于乳腺上皮细胞的模式恢复肿瘤细胞Sox 2启动子中可遗传的表观遗传沉默。我们假设ZFs-Dnmt 3a融合能够将DNAme标记靶向Sox 2致癌启动子，导致这些标记在细胞世代中的传递。这种表观遗传记忆将伴随着转录沉默和肿瘤细胞生长抑制的维持。在Aim 1中，我们提出了与Dnmt 3a和失活突变体连接的6 ZF蛋白的构建，以评估这些工程蛋白是否将特异性沉默标记存款到Sox 2启动子中，从而导致致癌沉默。在Aim 2中，我们监控6 ZF消音器的消音寿命。我们将使用诱导型载体在细胞培养物和乳腺肿瘤模型中表达6 ZF构建体以“脉冲”和“追踪”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递送ATF mRNA，这些纳米颗粒将被注射到乳腺癌小鼠模型中（Aim 3）。虽然RNAi技术可用于敲除癌基因，但由于小RNA的寿命短，其治疗效果是短暂的。该应用的重要性在于ZF试剂诱导靶TF的内源性表观遗传重编程的潜力，预期这将维持治疗效果的持久性。因此，这项工作将是至关重要的，以开发稳定的，遗传的，致癌沉默的方法，以抑制肿瘤细胞中的致癌基因的表达。 公共卫生相关性：致癌转录因子（TF）在人类肿瘤的发生和发展中起着关键作用。相对于正常组织，这些基因通常在癌细胞中表观遗传失调，异常低甲基化和过度表达。由于它们缺乏小分子结合口袋，TF对于基于小分子的方法是非常难处理的。我们的工作提供了一种新的策略来抑制这些经典的“不可药”目标的表达。我们建议设计锌指蛋白连接到催化活性的DNA甲基转移酶结构域，以恢复表观遗传沉默这些基因。通过重新编程肿瘤细胞的表观遗传沉默，我们希望促进癌细胞表观基因组的遗传变化，并最终诱导更稳定的抗肿瘤和治疗效果，即使停止ZF药物，也有助于最大限度地减少肿瘤复发。这项工作将提供序列特异性的分子装置，以稳定地恢复致癌TF的表观遗传沉默。我们的工作将导致开发用于治疗侵袭性乳腺癌和卵巢癌的新型治疗药物，这些癌症无法治愈。

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.