Cancer Epigenetics: A novel PRC2 Dysregulation Mechanism in Multiple Myeloma

项目摘要/摘要 多发性骨髓瘤(MM)是一种常见的、破坏性的血液系统癌症，总体存活率低，预后不良。 需要开发新的治疗方法。最近对多发性骨髓瘤患者样本的全基因组图谱显示 多发性骨髓瘤发病机制下的组蛋白甲基化的整体扰动模式；尤其是，增强的 H3K27me3(组蛋白H3，赖氨酸27的三甲基化)水平与异常基因沉默和 随着MM进展到攻击性阶段。H3K27me3是由多梳抑制物2产生的 (PRC2)，一种使用EZH2或EZH1作为酶亚基的组蛋白甲基转移酶复合体。 然而，MM患者样本的直接测序未发现EZH2、EZH1或其他PRC2突变 基因。H3K27me3在多发性骨髓瘤发病机制中的作用机制尚不清楚。 我们最近发现PhD Finger Protein 19(PHF19)基因的异常扩增和过度表达 我们以前报道过编码一种新的PRC2调节因子，在多发性骨髓瘤患者中频繁发生，并预测 临床预后差。我们还发现PHF19的异常表达，以及它与PRC2的相互作用， 是多发性骨髓瘤体外和体内致瘤所必需的。从机制上讲，PHF19显著增强了 染色质占据PRC2，导致MM细胞H3K27高甲基化。有趣的是，MM不同于 许多癌症是由于EZH1的高表达所致，EZH1是一种研究较少的H3K27甲基转移酶，它 显著促进MM细胞的生长和H3K27me3的失调。我们假设异常的PHF19 扩增和过度表达代表了一种重要的多发性骨髓瘤促进机制，使人具有攻击性 EZH1和EZH2 H3K27甲基过激活的肿瘤特征和表观遗传学扰动 转移酶。PHF19介导多发性骨髓瘤的分子事件及机制剖析 致瘤性应该为新的抗多发性骨髓瘤策略提供关键的见解。为了实现这个目标，我们将决定 PHF19过表达是否是促进严重度癌发生的必要条件和充分条件 多发性骨髓瘤模型包括患者来源的异种移植模型(AIM 1)；我们将表征PHF19的作用 多发性骨髓瘤细胞中PRC2染色质占位的过度表达和表观基因的失控 无偏见的芯片测序和转录组分析方法(目标2)；第三，我们将使用组合的 遗传学(CRISPR/Cas9)和药理学(抑制剂)方法确定EZH1和EZH2 是PHF19介导的多发性骨髓瘤发生的关键效应因子(目标3)。对拟议的研究具有重要意义的是 我们将评估一种独特的EZH1和EZH2小分子抑制剂对MM的治疗效果 最近被我们的实验室发现。我们希望全面描述一种重要的，但尚未探索的致癌因素 通过用严格的癌症模型从功能上表征PHF19在多发性骨髓瘤中的通路，获得了一个机制 了解并确定PHF19的致癌效应因子，以进行潜在的治疗干预。我们会 还利用尖端技术，包括CRISPR-Cas9介导的基因编辑技术和 独特的小分子抑制剂。因此，提案的完成应导致对 多发性骨髓瘤的发生机制及治疗方法的创新和靶向 致命的癌症。

PROJECT SUMMARY/ABSTRACT Multiple myeloma (MM) is a common, devastating hematological cancer with a low rate of overall survival and a need for development of new treatments. Recent genome-wide profiling of MM patient samples has revealed a globally perturbed pattern of histone methylations underlying MM pathogenesis; in particular, an enhanced level of H3K27me3 (tri-methylation of histone H3, lysine 27) correlates well with aberrant gene silencing and with MM progression to the aggressive stage. H3K27me3 is produced by Polycomb Repressive Complex 2 (PRC2), a histone methyltransferase complex that employs either EZH2 or EZH1 as an enzymatic subunit. However, direct sequencing of MM patient samples identified no mutation of EZH2, EZH1 or other PRC2 genes. The mechanisms responsible for H3K27me3 dysregulation during pathogenesis of MM remain elusive. We recently found that aberrant amplification and over-expression of PHD Finger Protein 19 (PHF19), a gene previously reported by us to encode a new regulator of PRC2, occurs frequently in MM patients and predicts a poor clinical prognosis. We also found that aberrantly expressed PHF19, as well as its interaction with PRC2, is required for tumorigenicity of MM in vitro and in vivo. Mechanistically, PHF19 dramatically enhances chromatin occupancy of PRC2, causing H3K27 hypermethylation in MM cells. Intriguingly, MM differs from many cancers by the high expression of EZH1, a less studied H3K27 methyltransferase enzyme, which significantly contributes to MM cell growth and H3K27me3 dysregulation. We hypothesize that aberrant PHF19 amplification and overexpression represents an important MM-promoting mechanism that confers aggressive tumor features and epigenetic perturbations through hyper-activation of EZH1 and EZH2 H3K27 methyl- transferase enzymes. Dissecting the molecular events and mechanisms underlying PHF19-mediated MM tumorigenicity should provide critical insights into new anti-MM strategies. Towards this goal, we will determine whether overexpression of PHF19 is necessary and sufficient to promote tumorigenesis in rigorous cancer models of MM including a patient-derived xenograft model (aim 1); we will characterize effects of PHF19 overexpression on chromatin occupancy of PRC2 and epigenetic gene deregulations in MM cells through unbiased ChIP sequencing and transcriptome profiling approaches (aim 2); and third, we will use a combined genetic (CRISPR/cas9) and pharmacological (inhibitor) approach to determine whether both EZH1 and EZH2 are crucial effectors of PHF19-mediated tumorigenesis in MM (aim 3). Significant to the proposed research is that we will evaluate anti-MM therapeutic effects of a unique small-molecule inhibitor of EZH1 and EZH2 recently discovered by our laboratories. We expect to fully delineate an important, yet unexplored oncogenic pathway in MM by functionally characterizing PHF19 with rigorous cancer models, gain a mechanistic understanding, and determine oncogenic effectors of PHF19 for potential therapeutic interventions. We will also utilize cutting-edge techniques including the CRISPR-cas9 mediated gene editing technology and a unique small-molecule inhibitor. Therefore, completion of the proposal should result in a new understanding for mechanism of MM tumorigenesis and should yield innovative, targeted therapeutics for the treatment of this deadly cancer.