(PQD1) lmpact of cytotoxic and targeted therapy on clonal evolution of CLL

项目摘要 克隆演变是癌症进展和复发的关键特征。我们最近的研究， 利用一种新开发的管道来估计携带每种病毒的癌细胞的比例， 通过整合全外显子组测序和局部拷贝在肿瘤内的体细胞突变 数量数据，将携带推定驱动突变的亚克隆的存在与不利的 慢性淋巴细胞白血病（CLL）的临床结果，并建议CLL治疗可能 加速克隆进化的过程（朗道等人，Cell 2013）。我们建议， 亚克隆突变是一个活跃的进化过程的指示。 我们现在寻求明确建立亚克隆突变对CLL生物学的影响， 疾病复发和临床结果的发展。这将通过纵向 分析从II期入组的患者中收集的系列样品的克隆结构， III期临床试验（因此统一治疗），解决治疗前景 CLL。特别是，我们将对接受以下治疗的患者的样本进行详细的遗传分析： 基于氟达拉滨的标准一线化疗（Aim 1）。与此同时，我们将研究 暴露于伊曲替尼（一种非常有前途的布鲁顿酪氨酸不可逆抑制剂）的患者样本 激酶，其预期是未来CLL疗法的基石（目的2）。分析 暴露于这两种类型治疗的样本将包括亚克隆 结构以及动态表型变化的评估（通过单细胞检测） RNA测序），以验证突变分析并确定转录网络。 耐药细胞，以揭示潜在的新的和有效的治疗组合。到 因果联系的影响，推定的驱动程序和治疗CLL克隆进化，我们将产生 研究治疗背景下克隆间动力学的体内模型（目的3）。我们将使用 变革性基因组编辑技术，以生成模拟白血病的细胞系 携带代表性CLL驱动突变的亚群，从而机械地解剖 个体遗传病变对进化景观的贡献。通过创建一个 克隆进化的动物模型，我们将有可能更有效地评估 新疗法对克隆选择的影响。总的来说，这些研究是 旨在建立一个框架，了解动态进化的作用， CLL的前景对这种目前无法治愈的疾病的诊断，预后和治疗。

Project Summary Clonal evolution is a key feature of cancer progression and relapse. Our recent study, which utilized a newly developed pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequencing and local copy number data, linked the presence of subclones harboring putative driver mutations with adverse clinical outcome in chronic lymphocytic leukemia (CLL) and suggested that CLL therapy may accelerate the process of clonal evolution (Landau et al., Cell 2013). We propose that presence of subclonal mutations that are putative drivers are indicative of an active evolutionary process. We now seek to definitively establish the impact of subclonal mutations on CLL biology, the development of disease relapse and clinical outcome. This will be achieved by longitudinal analysis of clonal structure of serial samples collected from patients enrolled on phase II and phase III clinical trials (and hence uniformly treated) that address the treatment landscape of CLL. In particular, we will perform detailed genetic analysis of samples from patients receiving standard-of-care first line fludarabine-based chemotherapy (Aim 1). In parallel, we will examine patient samples exposed to ibrutinib, a highly promising irreversible inhibitor of Bruton's tyrosine kinase which is anticipated to be a cornerstone of future CLL therapy (Aim 2). Analysis of samples exposed to both these types of therapies will include characterization of subclonal structure as well as assessment of the dynamic phenotypic changes (detected by single cell RNA-sequencing) to validate mutation analysis and determine the transcriptional networks of drug resistant cells in order to reveal potential novel and effective treatment combinations. To causally link the impact of putative drivers and therapy on CLL clonal evolution, we will generate an in vivo model to study interclonal dynamics in the setting of therapy (Aim 3). We will use transformative genome-editing techniques to generate cell lines that model leukemic subpopulations bearing representative CLL driver mutations and thereby mechanistically dissect the contribution of individual genetic lesions to the evolutionary landscape. By creating an animal model of clonal evolution, we will have the potential to more effectively evaluate preclinically the impact of novel therapeutics on clonal selection. In total, these studies are designed to establish a framework for understanding the role of the dynamic evolutionary landscape of CLL on the diagnosis, prognosis and treatment of this currently incurable disease.