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

鉴定针对胸腺嘧啶 DNA 糖基化酶 (TDG) 的一流表观遗传抑制剂，用于未来转移性黑色素瘤的精准治疗

• 批准号: 10310527
• 负责人: ALFONSO BELLACOSA
• 金额: $ 9.35万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10310527
• 关键词: Adult; Analysis of Variance; Behavior; Biological Assay; Biology; Cell Cycle Arrest; Cell Line; Chemicals; Clinical; Collaborations; Computing Methodologies; Cutaneous Melanoma; DNA; DNA Methylation; DNA Modification Methylases; DNA Repair; DNA Repair Enzymes; Data; Data Set; Development; Dioxygenases; Drug Kinetics; Enhancers; Enzymes; Epigenetic Process; Equilibrium; Exhibits; Family; Future; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Hematologic Neoplasms; Human; Hypermethylation; Immunization; Immunotherapy; In Vitro; Lead; Libraries; Malignant Neoplasms; Mass Spectrum Analysis; Melanoma Cell; Metastatic Melanoma; Methods; Methylation; Molecular; Mus; Neoplasm Metastasis; Neoplasms; Oncogene Activation; Oncogenes; Oncogenic; Outcomes Research; Pathway interactions; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Precision therapeutics; Primary Neoplasm; Publishing; Repression; Research; Resistance; Sampling; Signal Pathway; Site; Skin; Small Molecule Chemical Library; Specificity; Structure; Testing; The Cancer Genome Atlas; Therapeutic; Thymine DNA Glycosylase; Toxicology; Tumor Promoters; Tumor Suppressor Proteins; Up-Regulation; Validation; Work; Xenograft procedure; analog; anti-CTLA4 antibodies; anti-PD-1; anti-PD-L1; anti-cancer; base; cancer cell; chemical property; clinically relevant; counterscreen; demethylation; design; drug development; drug discovery; epigenetic drug; epigenetic therapy; epigenome; epigenomics; genome-wide; immune checkpoint blockade; improved; inhibitor; knock-down; lead optimization; melanoma; novel; novel strategies; overexpression; personalized cancer therapy; pre-clinical; precision medicine; repair enzyme; response; scaffold; screening; small hairpin RNA; small molecule; small molecule inhibitor; small molecule libraries; targeted treatment; tool; tumor; virtual; virtual library; virtual screening

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.

摘要 用抗PD1、抗PD-L1和抗CTLA4抗体进行免疫检查点阻断(ICB)是一种令人兴奋的和 转移性黑色素瘤的有效新疗法。不幸的是，ICB只对三分之一的患者有效，而且 因此，迫切需要先进的治疗方法。这需要从根本上理解 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~40%的转移性 黑色素瘤病例表现出明显的全基因组DNA低甲基化，与 致癌信号通路与抗PD1免疫治疗的先天抵抗。低甲基化的 这个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。 糖基酶(TDG)和11种依次作用于甲基-CpG去甲基化的转位酶 网站。这些观察结果增加了通过升高TET/TDG活性而导致低甲基化的可能性 致癌激活和抗PD1耐药。 我们发表了黑色素瘤细胞中TDG基因敲除导致DNA高甲基化并停止增殖 在培养和异种移植中。重要的是，在TDG基因敲除后，CpG位点变得超甲基化 黑色素瘤细胞与SKCM中CpG低甲基化位点显著相关。这提供了临床上的相关性 我们的体外研究表明，抑制TDG可以逆转转移瘤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其对ICB敏感。这一点，再加上 TDG在成年小鼠中不是必需的，这一事实表明TDG有一个潜在的广阔的治疗窗口 正常(成人)和癌细胞之间的抑制剂。我们筛选了一个虚拟化学图书馆，并确定了一条线索 化合物MC1，具有中等效力的TDG抑制活性。在验证了我们的HTS测试后，我们建议 筛选活动，以确定具有更高效力、特异性和化学性质的新线索 最大限度地增加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HIT验证分析，以 发现新的和更有效的TDG抑制剂。 2)使用计算方法对MC1进行SAR研究，并分别筛选a&gt；100亿化合物 虚拟文库用于鉴定具有TDG抑制活性的有效新支架。 这项研究的结果将导致基于以下方面的范式转换表观遗传疗法的发展 TDG抑制剂作为一流的DNA超甲基化药物，纠正低甲基化黑色素瘤亚类 并使其对ICB敏感。