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脑肿瘤，其死亡率为95%。 好几年了。目前的GBM标准治疗模式是在2005年建立的，包括最大 在可能的情况下，安全的手术清除，随后配合分割放射治疗和每日口服 替唑胺(TMZ)，随后每月一次的佐剂TMZ周期。疾病进展的中位时间 在TMZ上是短短7个月，通常是由GBM细胞对烷基DNA的获得性抵抗力介导的 TMZ生成的加合物。具体地说，细胞的DNA修复机制无法检测碱基 错配导致逃避正常的细胞凋亡信号。此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去甲基化试剂，可以改善或恢复碱基错配 检测和修复，以增加基底膜对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将服从DAC的去甲基化， 增加基因转录，从而提高错配修复蛋白的活性。在具体目标1中，我们将使用 我们可以利用大量的潜在来源的GBM球状细胞系来鉴定表现出 DAC预适应后显著增加对TMZ的敏感性或抵抗力。在在场和不在时 在DAC预适应中，将询问这些株系以确定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的影响加倍- 将检查链断裂修复水平和肿瘤突变负担。作为补充，我们将 利用单细胞RNA测序确定对DAC有反应的其他新的组织生物标志物 表观预适应，这将使临床试验的合理患者选择。在具体目标2中，我们 将使用小鼠原位GBM异种移植模型来确定小剂量DAC联合应用的疗效 与标准的TMZ方案相比，可提高小鼠的存活率。小鼠将被植入GBM细胞系 在我们的体外实验中显示出在DAC预适应后TMZ的敏感性或耐药性增加 学习。在治疗之后，肿瘤将被移植、再培养并检查MMR蛋白水平。 表达和肿瘤突变负荷，以验证体外观察的药效学。这是一种创新 项目是我们利用长时间阅读的第三代亚硫酸盐测序技术的唯一方法 (SMRT-SEQ)同时定量一个基因启动子区域中所有CPGS的甲基化水平 一套底漆。与短读测序技术或甲基化相比，这提供了更好的能力- 特异性聚合酶链式反应，以检测由表观遗传剂如DAC产生的去甲基化模式。这项工作是 意义重大，因为高达30%的IDH-野生型GBM，包括侵略性的MGMT未甲基化和 复发形式，可能对以错配修复为目标的表观遗传预适应方法有反应 基因上调。