Role of EZH2 as a Driver and Therapeutic Target of Hepatocellular Carcinoma

项目摘要 在美国，肝细胞癌（HCC）每年造成超过30，000例死亡，并且目前， 治疗提供可忽略的临床益处。表观遗传学沉默HCC肿瘤抑制基因的因素是 预期促进肿瘤发展，因此可能提供新的治疗靶点。组蛋白 甲基转移酶EZH 2是多梳抑制复合物2（PRC 2）的催化亚基，PRC 2是一种主调节因子， 表观遗传沉默EZH 2在多种恶性肿瘤中突变或过表达，并且已被证明 在某些癌症中起致癌基因的作用。已经开发了EZH 2的有效小分子抑制剂， 并在某些EZH 2驱动的癌症的临床试验中显示出有希望的结果，最近获得了FDA 批准用于治疗上皮样肉瘤和滤泡性淋巴瘤。基于对HCC患者的分析 通过对EZH 2和EZH 2样本以及涉及培养的HCC细胞和HCC小鼠模型的实验，我们发现EZH 2 是HCC癌基因，是肿瘤发展的内在驱动因素。EZH 2的这种内在致癌活性是 至少部分是由于ERK特异性磷酸酶DUSP 6的表观遗传沉默，导致ERK特异性磷酸酶DUSP 6的激活。 丝裂原活化蛋白（MAP）激酶途径，刺激细胞增殖。此外，我们有 在我们小组发表的两项研究中，EZH 2抑制了自然杀伤细胞（NK）的能力， 细胞通过表观遗传学沉默编码ULBP 1的基因来根除培养的HCC细胞，ULBP 1是一种肿瘤细胞表面 NK细胞的活化配体，并通过表观遗传学沉默该基因来抑制NK细胞向HCC细胞的迁移 编码趋化因子CXCL 10。因此，我们发表的和初步的结果表明，EZH 2驱动HCC 肿瘤发展通过细胞内在机制（MAP激酶途径的激活， 细胞增殖）和细胞外在机制（抑制NK细胞介导的肿瘤根除）。在这 我们的目标是建立EZH 2在HCC小鼠模型中的这两种作用，确定EZH 2在HCC小鼠模型中的作用。 EZH 2促进肿瘤生长的分子途径，并评估EZH 2作为HCC药物靶点。 在具体的目标1中，我们将确定EZH 2通过抑制NK细胞介导的抗肿瘤活性来促进HCC肿瘤生长。 在人源化小鼠中的肿瘤免疫，并将CXCL 10和ULBP 1定义为关键的EZH 2靶点， 表观遗传沉默抑制NK细胞介导的HCC根除。在具体目标2中，我们将建立DUSP 6 作为一个关键的EZH 2靶基因，其表观遗传沉默通过增加 ERK 1/2磷酸化。在具体的目标3中，我们将评估EZH 2单独和联合给药的药理学抑制。 与其他抗癌剂联合作为潜在的HCC治疗。总的来说，实验的结果 本申请中提出的方法将阐明EZH 2通过细胞内在和细胞外在途径 促进HCC肿瘤生长，并评估HCC的新治疗方法。

PROJECT SUMMARY Hepatocellular carcinoma (HCC) accounts for over 30,000 deaths annually in the United States and current therapies provide negligible clinical benefit. Factors that epigenetically silence HCC tumor suppressor genes are expected to promote tumor development and thus may provide novel therapeutic targets. The histone methyltransferase EZH2 is the catalytic subunit of polycomb repressive complex 2 (PRC2), a master regulator of epigenetic silencing. EZH2 is mutated or overexpressed in a variety of malignancies and has been shown to function as an oncogene in some cancers. Efficacious small molecule inhibitors of EZH2 have been developed and have shown promising results in clinical trials for certain EZH2-driven cancers and recently received FDA approval for the treatment of epithelioid sarcoma and follicular lymphoma. Based upon analysis of HCC patient samples, and experiments involving cultured HCC cells and mouse models of HCC, we have found that EZH2 is an HCC oncogene that is an intrinsic driver of tumor development. This intrinsic oncogenic activity of EZH2 is due, at least in part, to epigenetic silencing of an ERK-specific phosphatase, DUSP6, resulting in activation of a mitogen-activated protein (MAP) kinase pathway that stimulates cellular proliferation. Furthermore, we have demonstrated, in two published studies from our group, that EZH2 suppresses the ability of natural killer (NK) cells to eradicate cultured HCC cells by epigenetically silencing the gene encoding ULBP1, a tumor cell-surface activating ligand for NK cells, and suppresses NK cell migration to HCC cells by epigenetically silencing the gene encoding the chemokine CXCL10. Thus, our published and preliminary results suggest that EZH2 drives HCC tumor development through both a cell intrinsic mechanism (activation of a MAP kinase pathway that stimulates cellular proliferation) and a cell extrinsic mechanism (inhibition of NK cell-mediated tumor eradication). In this application our objectives are to establish these two roles of EZH2 in HCC mouse models, determine the molecular pathways through which EZH2 promotes tumor growth, and evaluate EZH2 as an HCC drug target. In specific aim 1, we will establish that EZH2 promotes HCC tumor growth by suppressing NK cell-mediated anti- tumor immunity in humanized mice, and define the roles of CXCL10 and ULBP1 as critical EZH2 targets whose epigenetic silencing suppresses NK cell-mediated eradication of HCC. In specific aim 2, we will establish DUSP6 as a critical EZH2 target gene whose epigenetic silencing promotes HCC tumor growth through increased ERK1/2 phosphorylation. In specific aim 3, we will evaluate pharmacological inhibition of EZH2 alone and in combination with other anti-cancer agents as a potential HCC therapy. Collectively, the results of the experiments proposed in this application will elucidate the cell intrinsic and cell extrinsic pathways through which EZH2 promotes HCC tumor growth, and evaluate new therapeutic approaches for HCC.