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.

项目摘要胶质母细胞瘤（GBM）是最常见，最具侵略性的原发性脑肿瘤，死亡率为95％。年。当前的GBM标准治疗范例是在2005年建立的，由最大在可能的情况下，安全的手术延伸 Temzolomide（TMZ），随后每月辅助TMZ的周期。疾病进展的中位时间在TMZ上是短短的7个月，经常通过GBM细胞对烷基-DNA获得的耐药性介导 TMZ产生的加合物。具体而言，细胞的DNA修复机械无法检测基础的失败不匹配导致逃避正常的凋亡信号传导。此应用程序的目的是确定是否 DNA脱甲基剂表观遗传剂Decitabine（DAC）可以改善或恢复基础不匹配检测和修复以提高GBM对TMZ的敏感性。我们的中心假设是具有高甲基化的MLH1和/或MSH6启动子的GBM可以与DAC脱甲基化，增加基因转录，因此不匹配修复蛋白活性。在特定目标1中，我们将使用我们可以使用的大量前瞻性GBM球形细胞系，以识别显示的线条 DAC预处理后，显着提高了对TMZ的敏感性或抗性。在存在和缺席的情况下在DAC预处理中，这些线将被审问以确定MMR基因的甲基化状态启动子以及相应的转录本和蛋白质水平。预期下游对DNA双重影响将检查链断裂修复水平和肿瘤突变负担。对此的补充，我们将采用单细胞RNA测序来识别对DAC反应性的其他新型组织生物标志物表观遗传学的预处理，这将使患者为临床试验进行合理的选择。在特定的目标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的功效具有标准TMZ方案来改善小鼠的存活率。小鼠将被GBM细胞系植入证明在我们的体外DAC预处理后表现出提高的TMZ敏感性或抗性研究。治疗后，肿瘤将被培养，再培养和检查MMR蛋白水平表达和肿瘤突变负担以验证体外观察到的药效学。对此的创新项目是我们利用长阅读第三代Bisulfite测序技术的独特方法（SMRT-SEQ）与单个基因启动子区域中所有CPG的甲基化水平同时与一个单一的甲基化水平一组底漆。与短读测序技术或甲基化相比，这提供了提高的能力 - 特定的PCR，以检测由DAC等表观遗传剂产生的脱甲基化模式。这项工作是意义重大，因为多达30％的IDH WildType GBM，包括侵略性的Mgmt未甲基化和经常性形式，可能对针对不匹配修复的表观遗传预处理方法有反应基因上调。