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的发生和发展中起着关键作用。癌基因转录因子(Tf)在乳腺癌中经常过度表达，而在正常上皮细胞中沉默。转铁蛋白可以切换整个转录基因的级联，导致肿瘤的发生和发展。由于大多数转录因子不具有固有的酶活性，而且它们缺乏小分子结合口袋，这些靶点一直难以进行药物设计。致癌基因TFS Sox2在晚期乳腺癌中高表达，而在正常上皮细胞中沉默和高甲基化。作为一种稳定的抑制标记，DNA-甲基转移酶催化的DNAME被认为是表观遗传沉默的关键分子。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的一个特点是它是遗传的，因此可以通过细胞世代传播。在许多发育调节的转录因子中，如Sox2，DNAME构成了一个表观遗传开关，使细胞从活跃的有丝分裂状态转变为G0/G1停滞和分化。在这项应用中，我们的目标是将DNAME靶向Sox2的启动子，Sox2在乳腺癌细胞系中高表达，其水平与胚胎干细胞相当或更高。为了指导特定的DNAME，我们将把序列特异的锌指(ZF)结构域与具有催化活性的DNA-甲基转移酶结构域(DNMT3A)组成的DNA结合蛋白融合在一起。我们的目标是以一种类似于乳腺上皮细胞的模式恢复肿瘤细胞Sox2启动子中可遗传的表观遗传沉默。我们假设ZFS-DNMT3A融合能够将DNAME标记靶向Sox2致癌启动子，导致这些标记在细胞代间传递。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Aim1中，我们建议构建与DNMT3A连锁的6ZF蛋白和非活性突变体，以评估这些工程蛋白是否在Sox2启动子中沉积了特定的沉默标记，从而导致致癌沉默。在AIM2中，我们监测由6ZF-消音器实现的消音的寿命。我们将使用诱导性载体在细胞培养和乳腺肿瘤模型中表达6ZF结构，以“脉冲”和“追逐”DNAME。下一步，为了将这项技术推向临床前阶段，我们将使用纳米颗粒传递ATF mRNAs，并将其注射到乳腺癌小鼠模型中(目标3)。虽然RNAi技术可以用来敲除癌基因，但由于小RNA的寿命很短，其治疗效果是暂时的。这一应用的意义在于ZF制剂有可能诱导靶TF的内源性表观遗传重编程，这有望保持治疗效果的长寿。因此，这项工作对于发展稳定的、遗传的、致癌的沉默方法，抑制肿瘤细胞中的致癌表达将是至关重要的。 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。与正常组织相比，这些基因通常是表观遗传失调的，在癌细胞中异常低甲基化和过度表达。由于它们缺乏小分子结合口袋，因此对基于小分子的方法非常难以实现。我们的工作提供了一种新的策略来抑制这些经典的“不能下药”的靶点的表达。我们建议设计连接到催化活性DNA-甲基转移酶结构域的锌指蛋白，以恢复这些基因的表观遗传沉默。通过对肿瘤细胞的表观遗传沉默重新编程，我们希望促进癌细胞表观基因组的遗传变化，最终产生更稳定的抗肿瘤和治疗效果，即使ZF制剂停止使用，也有助于将肿瘤复发降至最低。这项工作将提供序列特异的分子装置，以稳定地恢复致癌因子的表观遗传沉默。我们的工作将导致开发新的治疗药物来治疗侵袭性乳腺癌和卵巢癌，这些癌症目前还没有治愈的方法。

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.