Cancer Epigenetics: A novel PRC2 Dysregulation Mechanism in Multiple Myeloma

项目总结/摘要 多发性骨髓瘤（MM）是一种常见的、毁灭性的血液癌症，总体生存率低， 需要开发新的治疗方法。最近MM患者样本的全基因组分析显示， MM发病机制下组蛋白甲基化的整体扰动模式;特别是， H3 K27 me 3（组蛋白H3，赖氨酸27的三甲基化）水平与异常基因沉默密切相关， MM进展到侵袭性阶段。H3 K27 me 3由Polycomb Repressive Complex 2产生 （PRC 2），一种采用EZH 2或EZH 1作为酶亚基的组蛋白甲基转移酶复合物。 然而，MM患者样品的直接测序未鉴定出EZH 2、EZH 1或其他PRC 2突变。 基因.在MM发病过程中负责H3 K27 me 3失调的机制仍然难以捉摸。 我们最近发现PHD指蛋白19（PHF 19）的异常扩增和过度表达， 我们以前报道过编码PRC 2的一种新的调节因子，经常发生在MM患者中，并预测了一种新的调节因子。 临床预后差。我们还发现异常表达的PHF 19，以及它与PRC 2的相互作用， 是MM体外和体内致瘤性所必需的。从机制上讲，PHF 19大大增强了 PRC 2的染色质占据，导致MM细胞中的H3 K27超甲基化。有趣的是，MM不同于 EZH 1是一种研究较少的H3 K27甲基转移酶， H3 K27 me 3的表达显著促进MM细胞生长和H3 K27 me 3失调。我们假设异常的PHF 19 扩增和过表达代表了一个重要的MM促进机制，赋予侵袭性 通过EZH 1和EZH 2 H3 K27甲基- 转移酶剖析PHF 19介导的MM的分子事件和机制 致瘤性应该为新的抗MM策略提供重要的见解。为了实现这一目标，我们将确定 PHF 19的过表达是否是促进恶性肿瘤发生的必要和充分条件 MM模型，包括患者来源的异种移植模型（目的1）;我们将表征PHF 19的作用 PRC 2在MM细胞中的染色质占有率和表观遗传基因失调的过度表达， 无偏的ChIP测序和转录组分析方法（目的2）;第三，我们将使用组合的 基因（CRISPR/cas9）和药理学（抑制剂）方法来确定EZH 1和EZH 2 是MM中PHF 19介导的肿瘤发生的关键效应子（目的3）。对拟议研究的重要意义是 我们将评估一种独特的EZH 1和EZH 2小分子抑制剂的抗MM治疗效果 最近被我们的实验室发现。我们希望能完全描绘出一个重要的，但尚未探索的致癌基因， 通过用严格的癌症模型功能性地表征PHF 19在MM中的通路，获得了一种机制， 了解并确定PHF 19的致癌效应物，用于潜在的治疗干预。我们将 还利用包括CRISPR-cas9介导的基因编辑技术和 独特的小分子抑制剂。因此，完成该提案后， MM肿瘤发生的机制，并应产生创新的，有针对性的治疗方法，用于治疗这种疾病 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.