A NOVEL EPIGENETIC IMMUNOTHERAPY FOR OVARIAN CANCER TREATMENT

项目摘要 卵巢癌是女性妇科恶性肿瘤死亡的主要原因， 仅今年美国就有超过14,000人因此死亡然而，目前的疗法不提供 对卵巢癌患者有意义的长期临床益处。因此，新的和更有效的疗法 卵巢癌的治疗是迫切需要的。我们发现卵巢癌细胞在表观遗传学上 下调自然杀伤细胞（NK）细胞所必需的自然杀伤组2D（NKG 2D）配体， 先天免疫系统来消灭癌细胞。基于这些发现，我们合理化了一种方法， 允许NKG 2D配体再表达的药物将增强NK细胞介导的卵巢癌根除 细胞，具有治疗价值。为了确定这种表观遗传药物的目标，当抑制时， 为了研究NKG 2D配体在卵巢癌细胞中的表达，我们进行了大规模的表观基因组范围的shRNA 筛选并鉴定端粒沉默1样破坏因子（DOT 1 L）作为NKG 2D配体ULBP 1的调节因子。 我们发现短发夹RNA（shRNA）或小分子抑制剂EPZ-5676抑制DOT 1 L， 显著增加NK细胞对卵巢癌细胞的根除。基于这些结果，我们假设 DOT 1 L通过调节NKG 2D配体的表达来调节NK细胞介导的卵巢癌根除。 癌细胞总体目标是严格确定DOT 1 L作为卵巢癌驱动因素的作用 肿瘤的生长和进展，以NK细胞依赖的方式发挥作用，并测试 用于治疗卵巢癌的抑制DOT 1 L的酶。目标1实验将集中于确定 DOT 1 L作为卵巢癌生长驱动因子的体内作用，并确定DOT 1 L的这种功能是否 依赖于NK细胞。为此，基于我们在免疫活性同基因小鼠模型中的结果，我们 将使用一种新的人源化小鼠模型，该模型具有含有NK细胞的功能性人类免疫系统， DOT 1 L抑制在更人类疾病相关的临床前小鼠模型设置中。我们还将确定 DOT 1 L的作用机制。为此，基于我们的初步结果，我们将测试NKG 2D的作用 配体和其他潜在的机制，如通过DOT 1 L调节ICAM 1，在调节 DOT 1 L对NK细胞介导的卵巢癌根除的作用目标2实验将确定是否 DOT 1 L的体内药理学抑制以NK细胞依赖性方式阻断卵巢癌生长。到 为此，我们将确定DOT 1 L抑制剂EPZ-5676是否通过NK细胞依赖性抑制卵巢肿瘤生长。 在卵巢癌的人源化和临床前患者来源的异种移植（PDX）模型中， 总的来说，我们的研究结果将确定DOT 1 L在调节NK细胞活性中的新功能 体内抗卵巢癌细胞，并验证DOT 1 L小分子抑制剂增强NK细胞功能 作为治疗卵巢癌的一种新的治疗方法。

PROJECT SUMMARY Ovarian cancer is the leading cause of deaths due to gynecological malignancies in women and will accounts for over 14,000 deaths in the United States this year alone. However, current therapies do not provide meaningful long-term clinical benefits to ovarian cancer patients. Therefore, new and more effective therapies for ovarian cancer treatment are urgently needed. We found that ovarian cancer cells epigenetically downregulate the Natural Killer Group 2 D (NKG2D) ligands that are necessary for Natural Killer (NK) cells of innate immune system to eradicate cancer cells. Based on these findings, we rationalized that an approach that will allow re-expression of NKG2D ligands will enhance NK cell-mediated eradication of ovarian cancer cells and will be of therapeutic value. To identify such epigenetic drug targets that when inhibited will increase the expression of NKG2D ligand in ovarian cancer cells, we performed a large-scale epigenome-wide shRNA screen and identified Disruptor of Telomeric Silencing 1-Like (DOT1L) as a regulator of NKG2D ligand ULBP1. We found that DOT1L inhibition by short-hairpin RNA (shRNA) or by a small-molecule inhibitor, EPZ-5676, significantly increased eradication of ovarian cancer cells by NK cells. Based on these results, we hypothesize that DOT1L by regulating the expression of NKG2D ligands regulates NK cell-mediated eradication of ovarian cancer cells. The overall objective is to rigorously determine the role of DOT1L as a driver of ovarian cancer tumor growth and progression that function in a NK cell-dependent manner and test the clinical value of pharmacologically inhibiting DOT1L for treating ovarian cancer. Aim 1 experiments will focus on determining the in vivo role of DOT1L as a driver of ovarian cancer growth and ascertain if this function of DOT1L is dependent on NK cells. To this end, based on our results in immunocompetent syngeneic mouse models, we will use a novel humanized mouse model with functional human immune system containing NK cells to test the DOT1L inhibition in a more human disease relevant pre-clinical mouse model setting. We will also determine the mechanism of DOT1L action. To this end, based on our preliminary results, we will test the role of NKG2D ligands and other potential mechanisms, such as regulation of ICAM1 by DOT1L, in regulating the ability of DOT1L on NK cell-mediated eradication of ovarian cancer. Aim 2 experiments will determine if pharmacological inhibition of DOT1L in vivo blocks ovarian cancer growth in a NK cell-dependent manner. To this end, we will determine if DOT1L inhibitor, EPZ-5676, inhibits ovarian tumor growth in a NK cell-dependent manner in humanized and in the pre-clinical patient-derived xenograft (PDX) models of ovarian cancer. Collectively, the results of our studies will identify a novel function of DOT1L in regulating NK cell activity against ovarian cancer cells in vivo and validate DOT1L small-molecule inhibitors to enhance NK cell function as a new therapeutic approach for treating ovarian cancer.!