Identification of first-in-class epigenetic inhibitors that target Thymine DNA Glycosylase (TDG) for future precision therapy of metastatic melanoma

摘要 使用抗PD 1、抗PD-L1和抗CTLA 4抗体的免疫检查点阻断（ICB）是一种令人兴奋的， 转移性黑色素瘤的有效新疗法。不幸的是，ICB仅对1/3的患者有效， 因此，迫切需要先进的治疗方法。这就需要对以下问题有一个基本的认识： 为什么三分之二的病例对治疗没有反应？ 通过检查TCGA皮肤黑色素瘤（SKCM）数据集，我们发现约40%的转移性黑色素瘤患者 黑色素瘤病例表现出显著的全基因组DNA低甲基化， 致癌信号通路和抗PD 1免疫疗法的先天抗性。低甲基化 这种黑色素瘤亚类的表观基因组也与胸腺嘧啶DNA的上调呈正相关， 糖基化酶（TDG）和10 - 11易位酶，依次使甲基-CpG脱甲基 网站.这些观察结果提出了通过升高的泰特/TDG活性导致低甲基化的可能性， 致癌激活和抗PD 1抗性。 我们发表了在黑色素瘤细胞中TDG敲低导致DNA超甲基化并停止增殖 在培养和异种移植中。重要的是，在TDG敲低后， 黑色素瘤细胞与SKCM中低甲基化CpG位点显著相关。这提供了临床相关性 我们的体外研究表明，抑制TDG可以逆转转移性肝癌的低甲基化表观基因组， 黑色素瘤，潜在地抑制致癌途径并使它们对ICB敏感。这一点，加上 TDG在成年小鼠中不是必需的，这一事实表明TDG有一个潜在的宽治疗窗口 正常（成人）和癌细胞之间的抑制剂。我们筛选了一个虚拟的化学品库 化合物MC 1具有中等效力的TDG抑制活性。在验证了我们的HTS测定后，我们建议 筛选活动，以确定具有更高效力、特异性和化学特性的新先导化合物， 最大化我们未来药物开发的机会在这里，我们将： 1)筛选TDG小分子抑制剂的化学文库，然后进行严格的命中验证测定， 鉴定新型和更有效TDG抑制剂。 2)使用计算方法对MC 1进行SAR研究，并单独筛选> 100亿的化合物 虚拟库，以鉴定具有TDG抑制活性的有效新支架。 这项研究的结果将导致范式转移表观遗传疗法的发展， TDG抑制剂作为一类DNA高甲基化剂，可纠正低甲基化黑素瘤亚类 使其对ICB敏感

ABSTRACT Immune checkpoint blockade (ICB) with anti-PD1, anti-PD-L1 and anti-CTLA4 antibodies is an exciting and effective new treatment for metastatic melanoma. Unfortunately, ICB is effective in only 1/3 of the patients, and therefore advanced therapeutic approaches are urgently needed. This requires a fundamental understanding of why 2/3 of cases are not responsive to treatment. By examining the TCGA skin cutaneous melanoma (SKCM) dataset, we found that ~40% of metastatic melanoma cases exhibit prominent genome-wide DNA hypomethylation that correlates with upregulation of oncogenic signaling pathways and innate resistance to anti-PD1 immunotherapy. The hypomethylated epigenome of this melanoma subclass is also positively correlated with upregulation of Thymine DNA Glycosylase (TDG) and Ten Eleven Translocation enzymes that work sequentially to demethylate methyl-CpG sites. These observations raise the possibility that hypomethylation via elevated TET/TDG activity contributes to oncogenic activation and anti-PD1 resistance. We published that TDG knockdown in melanoma cells leads to DNA hypermethylation and stopped proliferation in culture and in xenografts. Importantly, CpG sites that became hypermethylated after TDG knockdown in melanoma cells significantly correlated with hypomethylated CpG sites in SKCM. This provides clinical relevance to our in vitro studies and suggests that inhibiting TDG can reverse the hypomethylated epigenome of metastatic melanomas, potentially suppressing oncogenic pathways and sensitizing them to ICB. This, combined with the fact that TDG is not essential in adult mice, suggests there is a potentially wide therapeutic window for TDG inhibitors between normal (adult) and cancer cells. We screened a virtual chemical library and identified a lead compound, MC1, with TDG inhibitory activity at moderate potency. Having validated our HTS assay, we propose screening campaigns to identify new leads with increased potency, specificity and chemical properties that will maximize our chances for future drug development. Here we will: 1) Screen chemical libraries for small molecule inhibitors of TDG, followed by stringent hit validation assays, to identify novel and more potent inhibitors of TDG. 2) Use computational methods to perform SAR studies on MC1, and to separately screen a >10 billion compound virtual library to identify potent new scaffolds with TDG inhibitory activity. Outcomes from this research will lead to the development of paradigm-shifting epigenetic therapy, based on TDG inhibitors as first-in-class DNA hypermethylating agents, that correct hypomethylated melanoma subclass and sensitize it to ICB.