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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要胶质母细胞瘤 (GBM) 是最常见和最具侵袭性的原发性脑肿瘤，5 岁时死亡率为 95% 年。当前 GBM 的标准治疗范式建立于 2005 年，包括最大如果可能，进行安全的手术减瘤，然后同时进行分割放疗和每日口服替莫洛胺 (TMZ)，随后每月进行 TMZ 辅助治疗。疾病进展的中位时间 TMZ 上的作用时间很短 7 个月，并且通常是由 GBM 细胞对烷基 DNA 的获得性耐药介导的 TMZ 生成的加合物。具体来说，细胞的 DNA 修复机制无法检测碱基错配会导致逃避正常的细胞凋亡信号传导。该应用程序的目标是确定是否表观遗传剂地西他滨 (DAC) 是一种 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超甲基化 MLH1 和/或 MSH6 启动子的 GBM 将易于用 DAC 去甲基化，增加基因转录，从而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的预期衍生的 GBM 球状细胞系来识别表现出的细胞系 DAC 预处理后对 TMZ 的敏感性或抵抗力显着增加。在场和不在场在 DAC 预处理过程中，将询问这些细胞系以确定 MMR 基因的甲基化状态启动子以及相应的转录物和蛋白质水平。预期对 DNA 双链的下游影响将检查链断裂修复水平和肿瘤突变负荷。作为补充，我们将利用单细胞 RNA 测序来识别对 DAC 反应的其他新型组织生物标志物表观遗传预处理，这将使临床试验中合理的患者选择成为可能。在具体目标 2 中，我们将使用原位小鼠 GBM 异种移植模型来确定低剂量 DAC 组合的功效使用标准 TMZ 方案可提高小鼠存活率。小鼠将被植入 GBM 细胞系在我们的体外实验中，DAC 预处理后显示出 TMZ 敏感性或耐药性增加研究。治疗后，肿瘤将被移出、重新培养并检查 MMR 蛋白水平表达和肿瘤突变负荷以验证体外观察到的药效学。这其中的一个创新该项目是我们利用长读长第三代亚硫酸氢盐测序技术的独特方法 (SMRT-seq) 使用单个基因同时定量基因启动子区域中所有 CpG 的甲基化水平一套引物。与短读长测序技术或甲基化技术相比，这提供了改进的能力特异性 PCR，检测 DAC 等表观遗传因子产生的去甲基化模式。这部作品是重要的是，高达 30% 的 IDH 野生型 GBM，包括侵袭性 MGMT 未甲基化和复发形式，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。