Genetic evolution of glioblastomas during radiation and temozolomide therapy

 描述（由申请人提供）：胶质母细胞瘤（GBM）在基因组学上得到了很好的表征，但具有异质性，对所有现有治疗策略均表现出显著的耐药性。最有效的治疗方法是放射治疗（RT）和烷化剂替莫唑胺（TMZ），但进展通常发生在开始这些治疗后的几个月内。这种深刻的耐药性背后的机制仍然未知，但遗传异质性可能是一个主要因素，正如在其他癌症中所显示的那样。不幸的是，人们对GBM基因组如何随着治疗而进化知之甚少。这些信息将有助于指导制定策略，以避免耐药性的发展，并确定复发性疾病的最佳治疗方法。我们假设，与RT和TMZ治疗后复发的GBM的体细胞遗传特征与治疗前GBM有很大不同，并且这些差异指向GBM抵抗这些治疗的机制。为了测试这一点，我们建议使用创新的基因组分析工具和患者衍生的模型系统来识别和功能验证与耐药性相关的复发性遗传变化。我们的合作联盟已经收集了前所未有数量的配对治疗前和治疗后人类肿瘤（>200）。我们还创建了100多个患者来源的GBM模型，这些模型将被用于测试复发性耐药驱动因素的出现。来自患者样品和模型的初步数据表明，在治疗期间发生了实质性的肿瘤演变，并将TP53、CHEK 2和其他合理的靶点鉴定为耐药的候选介体。对数据的集体分析将用于解决两个目标。在目标1中，我们将使用配对的治疗前和治疗后肿瘤样本的全外显子组测序来测试辐射和替莫唑胺治疗导致人类肿瘤中一致的遗传变化的假设，以确定群体结构的大规模变化，并使用单细胞测序来评估这些治疗对微观异质性的影响。在目标2中，我们将使用患者来源的细胞系（PDCL）和患者来源的异种移植物（PDX）检验治疗后GBM中鉴定的遗传变化在功能上有助于GBM中的RT和TMZ抗性的假设。我们将使用深度测序确定辐射和替莫唑胺对这些模型及其基因组层次的影响，并测试体外和体内耐药候选驱动因素的影响。我们将决定 抗性克隆在处理之前存在，或者使用创新的单细胞条形码方法随机诱导，以确定克隆亚结构的进化在重复实验中是否一致。这些研究将全面了解GBM标准治疗期间的遗传进化。它们将为复发性疾病中靶向治疗的分配提供诊断方法，并确定驱动耐药性的遗传变化。这些新的耐药驱动因素的治疗靶向可能代表了一种合理的方法，可以大大改善我们现有的GBM患者的护理标准。

 DESCRIPTION (provided by applicant): Glioblastomas (GBMs) are genomically well characterized, yet heterogeneous, and exhibit profound resistance to all existing treatment strategies. The most effective therapeutics are radiation therapy (RT) and the alkylating agent temozolomide (TMZ), but progression typically occurs within months after initiating these treatments. The mechanisms underlying this profound resistance remain unknown, but genetic heterogeneity is likely a major contributor, as has been shown in other cancers. Unfortunately, little is known about how GBM genomes evolve with treatment. This information would be useful to guide development of strategies to avoid the development of resistance and to identify optimal therapeutic approaches in the recurrent setting. We hypothesize that somatic genetic profiles of GBMs that recur after treatment with RT and TMZ differ substantially from pre-treatment GBMs, and that the differences point to mechanisms by which GBMs resist these treatments. To test this, we propose to identify and functionally validate recurrent genetic changes associated with resistance using innovative genomic analysis tools and patient derived model systems. Our collaborative consortium has collected an unprecedented number of paired pre- and post-treatment human tumors (>200). We have also created more than 100 patient derived GBM models that will be treated to test for the emergence of recurrent resistance drivers. Preliminary data from both patient samples and models indicate substantial tumor evolution occurs during treatment and identify TP53, CHEK2 and other rational targets as candidate mediators of resistance. Collective analysis of the data will be used to address two Aims. In Aim 1, we will test the hypothesis that treatment with radiation and temozolomide leads to consistent genetic changes in human tumors using whole exome sequencing of paired pre- and post-treatment tumor samples to determine large-scale changes in population structures and single cell sequencing to evaluate the effects of these treatments on microheterogeneity. In Aim 2, we will test the hypothesis that genetic changes identified in post-treatment GBMs functionally contribute to RT and TMZ resistance in GBM using patient derived cell lines (PDCL) and patient derived xenografts (PDX). We will determine the effects of radiation and temozolomide on these models and their genomic hierarchies using deep sequencing and test the effects of candidate drivers of resistance both in vitro and in vivo. We will determine whether resistant clones exist prior to treatment or are stochastically induced using an innovative single cell barcoding approach to determine whether the evolution of clonal substructures is consistent across replicate experiments. These studies will create a comprehensive understanding of genetic evolution during standard-of-care therapy for GBM. They will inform diagnostic approaches for assignment of targeted therapeutics in the recurrent setting and identify genetic changes driving resistance. Therapeutic targeting of these novel resistance drivers could represent a rational approach to substantially improve our existing standard of care for GBM patients.