Epigenetic Preconditioning with Decitabine for Temozolomide Sensitization in Glioblastoma

地西他滨表观遗传预处理用于胶质母细胞瘤替莫唑胺敏化

• 批准号: 10260585
• 负责人: Raymund L Yong
• 金额: $ 8.48万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10260585
• 关键词: Acute Myelocytic Leukemia; Adjuvant; Adverse effects; Aftercare; Apoptosis; Apoptotic; Biological Markers; Brain Neoplasms; Categories; Cell Culture Techniques; Cell Line; Cells; Chemosensitization; Chronic Myelomonocytic Leukemia; Clinical; Clinical Trials; Combined Modality Therapy; Complement; DNA; DNA Adducts; DNA Double Strand Break; DNA Repair; DNA Repair Enzymes; Data; Decitabine; Detection; Development; Diagnosis; Differentiated Gene; Disease Progression; Dose; Double Strand Break Repair; Dysmyelopoietic Syndromes; Epigenetic Process; Evolution; Excision; Exhibits; Failure; Fractionated radiotherapy; Generations; Genes; Genetic Transcription; Glioblastoma; Glioma; Goals; Guanine; Immunotherapy; Implant; In Vitro; Induced Mutation; Lead; MGMT gene; MLH1 gene; MSH6 gene; Malignant Neoplasms; Mediating; Methylation; Mismatch Repair; Mismatch Repair Deficiency; Mus; Mutation; Newly Diagnosed; Operative Surgical Procedures; Oral; Patient Selection; Patients; Pattern; Pharmacodynamics; Phase I Clinical Trials; Precision therapeutics; Primary Brain Neoplasms; Prognosis; Promoter Regions; Proteins; Publishing; Purines; Recurrence; Regimen; Research; Resistance; Role; Signal Transduction; Solid; Solid Neoplasm; Specimen; Technology; Testing; Therapeutic; Thymine; Time; Tissues; Transcript; Tumor Debulking; Tumor Suppressor Genes; Tumor Tissue; Up-Regulation; Work; Xenograft Model; Xenograft procedure; alkyl group; base; bisulfite sequencing; chemoradiation; chemotherapeutic agent; clinical efficacy; cytotoxic; cytotoxicity; demethylation; desensitization; established cell line; gene repair; improved; in vivo; innovation; insight; interest; melanoma; mortality; mouse model; neoantigens; novel; patient derived xenograft model; patient subsets; preconditioning; predictive marker; promoter; prospective; protein expression; repaired; research clinical testing; response; single molecule real time sequencing; single-cell RNA sequencing; standard of care; stem cells; synergism; temozolomide; tissue biomarkers; tumor; tumor progression; tumorigenesis

Project Summary Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a 95% rate of mortality at 5 years. The current standard therapeutic paradigm for GBM was established in 2005 and consists of maximum safe surgical debulking when possible, followed by concomitant fractionated radiotherapy and daily oral temzolomide (TMZ), and subsequently monthly cycles of adjuvant TMZ. Median time to disease progression on TMZ is a short 7 months, and is frequently mediated by acquired resistance by GBM cells to the alkyl-DNA adducts generated by TMZ. Specifically, the failure of the cell's DNA repair machinery to detect base mismatches leads to evasion of normal apoptotic signaling. The goal of this application is to determine whether the epigenetic agent decitabine (DAC), a DNA demethylating agent, can improve or restore base mismatch detection and repair in order to increase GBM sensitivity to TMZ. Our central hypothesis is that a subset of GBM with hypermethylated MLH1 and/or MSH6 promoters will be amenable to demethylation with DAC, increasing gene transcription and consequently mismatch repair protein activity. In Specific Aim 1, we will use the large bank of prospectively derived GBM spheroid cell lines at our disposal to identify lines that exhibit significantly increased sensitivity or resistance to TMZ after DAC preconditioning. In the presence and absence of DAC preconditioning, these lines will be interrogated to determine the methylation status of MMR gene promoters and the corresponding transcript and protein levels. Expected downstream effects on DNA double- strand break repair levels and tumor mutational burden will be examined. Complementary to this, we will employ single cell RNA sequencing to identify other, novel tissue biomarkers of responsiveness to DAC epigenentic preconditioning, which will enable rational patient selection for clinical trials. In Specific Aim 2, we will use an orthotopic mouse GBM xenograft model to determine the efficacy of low-dose DAC in combination with the standard TMZ regimen in improving mouse survival. Mice will be implanted with GBM cell lines demonstrated to exhibit increased TMZ sensitivity or resistance after DAC preconditioning in our in vitro studies. Following treatment, tumors will be explanted, recultured, and examined for levels of MMR protein expression and tumor mutational burden to validate pharmacodynamics observed in vitro. An innovation of this project is our unique approach of leveraging long-read third-generation bisulfite sequencing technology (SMRT-seq) to quantitate methylation levels of all CpGs in a gene promoter region simultaneously with a single set of primers. This provides improved ability, compared to short-read sequencing technology or methylation- specific PCR, to detect patterns of demethylation produced by epigenetic agents such as DAC. This work is significant because up to 30% of IDH-wildtype GBM, including the aggressive MGMT unmethylated and recurrent forms, may be responsive to an epigenetic preconditioning approach targeted at mismatch repair gene upregulation.

项目摘要 胶质母细胞瘤（GBM）是最常见和最具侵袭性的原发性脑肿瘤，在5岁时死亡率为95 年目前GBM的标准治疗模式建立于2005年， 可能时进行安全的手术减瘤，随后伴随分次放疗和每日口服 替莫唑胺（TMZ），随后每月一次的辅助TMZ。至疾病进展的中位时间 TMZ的治疗时间只有短短的7个月，并且通常由GBM细胞对烷基DNA的获得性抗性介导。 TMZ生成的加合物。具体来说，细胞的DNA修复机制无法检测到碱基， 错配导致逃避正常的凋亡信号传导。此应用程序的目标是确定是否 表观遗传学试剂地西他滨（DAC），一种DNA去甲基化剂，可以改善或恢复碱基错配 检测和修复，以增加GBM对TMZ的敏感性。我们的中心假设是， 具有高甲基化MLH 1和/或MSH 6启动子的GBM将适合于用DAC去甲基化， 增加基因转录并因此增加错配修复蛋白活性。在具体目标1中，我们将使用 我们可以使用大量前瞻性衍生的GBM球状体细胞系库来鉴定表现出 在DAC预处理后显著增加对TMZ的敏感性或抗性。在存在和不 DAC预处理后，这些细胞系将被询问以确定MMR基因的甲基化状态 启动子和相应的转录本和蛋白质水平。对DNA双链的预期下游效应 检测链断裂修复水平和肿瘤突变负荷。作为补充，我们将 使用单细胞RNA测序来鉴定对DAC反应性的其他新的组织生物标志物 表观遗传预处理，这将使临床试验的合理患者选择。在Aim Specific 2中，我们 将使用原位小鼠GBM异种移植物模型来确定低剂量DAC与 在提高小鼠存活率方面的作用。小鼠将被植入GBM细胞系 证明在我们的体外DAC预处理后， 问题研究治疗后，肿瘤将被切除，再培养，并检查MMR蛋白的水平 表达和肿瘤突变负荷，以验证体外观察到的药效学。这种创新 项目是我们利用长读第三代亚硫酸氢盐测序技术的独特方法 在一个实施方案中，使用SMRT-seq（SMRT-seq）来同时定量基因启动子区中所有CpG的甲基化水平，其中使用单个的甲基化探针。 引物组。与短读段测序技术或甲基化相比，这提供了改进的能力。 特异性PCR，以检测表观遗传因子如DAC产生的去甲基化模式。这项工作是 这是显著的，因为高达30%的IDH-野生型GBM，包括侵袭性MGMT未甲基化和 复发性形式，可能对针对错配修复的表观遗传预处理方法有反应 基因上调