Genotypic Interactions in Brain Cancer Heterogeneity

项目摘要 多形性胶质母细胞瘤(GBM)患者12-15个月平均生存期的微小改善 尽管几十年来神经外科和放射治疗取得了进展，但许多临床试验 新的治疗方法，以及对驱动分子机制的更多了解。一个核心问题是 这种侵袭性肿瘤的异质性混淆了成功的治疗。这种异质性 表现为混合细胞学亚型，基因典型为突变和基因扩增， 在转录水平上则是基因表达的区域差异。因此，多个和空间上不同的 在单个GBM中存在异型群体，使得任何病变或路径特异性治疗变得更少 有效。虽然在理解细胞内在机制方面已经付出了相当大的努力 治疗耐药，对体内异质肿瘤细胞之间的相互作用知之甚少 这些肿瘤导致了这种癌症的顽固性。在GBM中，扩增的 表皮生长因子受体是一种标志性突变，存在于60%的病例中，通常发生在 不同的方式，并经常与结构变化联系在一起。其中最常见的是 突变的EGFRvIII(也被称为∆EGFR)导致与肿瘤有关的结构性活性突变受体 增强能力。这种能力在扩增的wtEGFR中是缺乏的，尽管它的肿瘤更普遍 表情。通过在体内模拟这种类型的遗传异质性，我们已经确定了IL-6旁分泌 EGFRvIII活性驱动的信号机制不仅可以将表达wtEGFR的细胞招募到加速状态 增殖，但也促进EGFR靶向的治疗耐药通过激活一个亲生存 炎症性NF-κB/BRD4信号轴。鉴于NF-κB/BRD4在血管重构中的核心作用 染色质超级增强子我们假设EGFRvIII/wtEGFR亚群相互作用不仅增强 侵袭性肿瘤生长，但也促使这些方面的基因表达同步 通过共享增强子重塑的异型细胞。 这一更新项目的总体目标是剖析和瞄准GBM EGFR/EGFRvIII 异质性通过协调NF-κB/BRD4介导的细胞重塑来驱动治疗耐药 表观遗传景观。将进行以下实验：1)基因组编辑，以创建 用于机械化学生物学研究的药物选择性BRD4等位基因；2)将该修饰的BRD4等位基因用作 细胞因子刺激的NF-κB/BRD4增强子染色质结构和功能分析工具 异型和亚群消融的胶质瘤中的景观；3)基因和药物抑制 确定了EGFR和EGFRvIII共有的NF-κB/BRD4介导的遗传或表观遗传脆弱性 异型细胞，以提高治疗效果。

Project Summary Minimal improvement in the 12-15 month average survival of patients with glioblastoma multiforme (GBM) has been achieved despite decades of advances in neurosurgery and radiation therapy, many clinical trials for novel therapeutics, and increased understanding of the driving molecular mechanisms. A central issue that confounds successful treatment is the heterogeneous nature of this aggressive tumor. This heterogeneity presents phenotypically as mixed cytological subtypes, genotypically as mutations and gene amplifications, and transcriptionally as regional differences in gene expression. As a result, multiple and spatially distinct heterotypic populations exist within a single GBM, making any lesion- or pathway-specific therapy less effective. While considerable effort has been placed on understanding cell intrinsic mechanisms conferring therapeutic resistance, much less is known about the interactions between heterogeneous tumor cells within these neoplasms that contribute to the recalcitrant nature of this cancer. In GBM, amplification of the epidermal growth factor receptor, a hallmark mutation present in 60% of cases, often occurs in a heterogeneous manner and is frequently associated with structural alterations. The most common of these alterations, EGFRvIII, (also known as ∆EGFR) results in a constitutively active mutant receptor with tumor enhancing capability. This ability is lacking from amplified wtEGFR despite its more pervasive tumor expression. By modeling this type of genetic heterogeneity in vivo, we have determined that an IL-6 paracrine signaling mechanism driven by EGFRvIII activity can not only recruit wtEGFR-expressing cells into accelerated proliferation, but also promote EGFR-targeted therapeutic resistance through activation of a pro-survival inflammatory NF-κB/BRD4 signaling axis. Given the central role of NF-κB/BRD4 in the remodeling of chromatin super enhancers we postulate that EGFRvIII/wtEGFR sub-population interactions not only enhance aggressive tumor growth, but also prompt the synchronization of aspects of gene expression in these heterotypic cells through shared enhancer remodeling. The overall goal of this renewal project is to dissect and target the mechanisms whereby GBM EGFR/EGFRvIII heterogeneity drives therapeutic resistance through orchestration of NF-κB/BRD4-mediated remodeling of the epigenetic landscape. The following lines of experimentation will be carried out: 1) genome editing to create a drug-selective BRD4 allele for mechanistic chemical biology studies; 2) use of this modified BRD4 allele as a tool for chromatin structure and functional analysis of the cytokine-stimulated NF-κB/BRD4 enhancer landscape in heterotypic and subpopulation-ablated gliomas; 3) genetic and pharmacological inhibition of identified NF-κB/BRD4-mediated genetic or epigenetic vulnerabilities shared among EGFR and EGFRvIII heterotypic cells to enhance therapeutic efficacy.