SEQUENCE-SPECIFIC CHROMATIN MODIFIERS; NOVEL PROTEIN THERAPEUTICS FOR B CELL LYMPHOMA

 描述（由申请人提供）：通过转录控制元件的遗传或表观遗传扰动来重编程基因表达已成为肿瘤发生的主要机制。使用比较“组学”方法，我们已经定义了原发性B细胞肿瘤（滤泡性淋巴瘤，FL）中的致病调节回路，将功能失调的增强子与其靶基因联系起来，包括许多参与细胞转化的基因。值得注意的是，大量FLARE与淋巴瘤相关的SNP重叠，或者具有影响增强子功能的获得性突变。我们的表观基因组为中心的方法还发现了两个以前不受重视的FL亚型，其特征基因与亚型特异性FLARE相关。这些发现为FL建立了不同的基于顺式转座的病因学，并为个性化表观遗传疗法提供了靶点。在这方面，我们已经开发了一种创新的策略，以靶向致病性RE逆转，使用序列特异性DNA结合蛋白拴染色质修饰剂（称为SSCM）。使用由锌指（ZF）蛋白融合到KRAB阻遏物结构域组成的原型SSCM，我们靶向了关键的NHL癌基因BCL 6，逆转了其致病性表达并引起广泛的细胞死亡。我们建议的目标是开发NHL的靶向表观遗传疗法，其可以作为蛋白质在体内递送。 这种翻译方法靶向肿瘤特异性RE，避免了广谱表观遗传抑制剂的副作用。在目标1中，将通过整合生物信息学分析与增强子功能的高通量测定来选择调节多个癌症相关基因的FLARE。这些多基因RE或控制中心是有吸引力的治疗靶点，因为其异常功能的逆转将同时影响几个基因的表达，降低肿瘤逃逸的可能性。在目标2中，我们将通过优化序列特异性结构域（锌指，TALE）和染色质修饰剂（激活或抑制）的组合来开发SSCM以靶向这些控制中心。同时，我们将开发靶向纳米载体方法，旨在通过结合肿瘤微环境中NHL或新生血管的表面标志物来改善SSCM的递送和疗效。目标3研究将利用NHL的小鼠异种移植模型来比较SSCM的直接蛋白递送与目标2中测试的靶向纳米载体平台，以最大化治疗功效，同时最小化正常细胞中的免疫原性和脱靶效应。为了实现我们的目标，申请人已经组建了一个基础，临床和生物信息学研究人员团队，他们已经在这个问题的基础方面进行了多年的富有成效的合作，定义了NHL的顺反子病因学。总的来说，这项工作预计将产生远远超出NHL的影响，引导使用靶向表观遗传疗法来逆转介导许多癌症的调节元件的致病性变化。

 DESCRIPTION (provided by applicant): Reprogramming gene expression via genetic or epigenetic perturbation of transcriptional control elements has emerged as a major mechanism of oncogenesis. Using a comparative "omics" approach, we have defined pathogenic regulatory circuits in primary B cell tumors (follicular lymphoma, FL), linking dysfunctional enhancers to their target genes, including many involved in cellular transformation. Remarkably, a significant number of FLAREs overlap lymphoma-associated SNPs or have acquired mutations that impact enhancer function. Our epigenome-centric approach also revealed two previously unappreciated FL subtypes whose signature genes correlate with subclass-specific FLAREs. These discoveries establish distinct cistrome-based etiologies for FL and provide targets for personalized epigenetic therapies. In this regard, we have developed an innovative strategy to target pathogenic REs for reversal, using sequence-specific DNA binding proteins tethered to chromatin modifiers (termed SSCMs). Using a prototype SSCM composed of a zinc-finger (ZF) protein fused to a KRAB repressor domain, we have targeted BCL6, a key NHL oncogene, reversing its pathogenic expression and causing widespread cell death. The goal of our proposal is the development of targeted epigenetic therapeutics for NHL that can be delivered as proteins in vivo. This translational approach targets tumor-specific REs, avoiding the side effects of broad-spectrum epigenetic inhibitors. In Aim 1, FLAREs that regulate multiple cancer-associated genes will be selected by integrating bioinformatic analyses with high-throughput assays of enhancer function. These multi-genic REs, or control hubs, are attractive therapeutic targets because reversal of their aberrant function will impact the expression of several genes simultaneously, reducing the potential for tumor escape. In Aim 2, we will develop SSCMs to target these control hubs by optimizing combinations of sequence-specific domains (zinc fingers, TALEs) and chromatin modifiers (activating or repressing). In parallel, we will develop targeted nanocarrier approaches designed to improve SSCM delivery and efficacy by binding to surface markers on NHL or neovasculature in the tumor microenvironment. Aim 3 studies will utilize a mouse xenograft model of NHL to compare direct protein delivery of SSCMs with targeted nanocarrier platforms tested in Aim 2, to maximize therapeutic efficacy while minimizing immunogenicity and off-target effects in normal cells. To accomplish our goals, the applicants have assembled a team of basic, clinical and bioinformatics researchers who have been collaborating productively for several years on the foundational aspects of this problem, defining the cistrome-based etiology of NHL. Collectively, this work is expected to have an impact well beyond NHL, shepherding the use of targeted epigenetic therapies to reverse pathogenic changes in regulatory elements that mediate many cancers.