Tracing mechanisms of cellular heterogeneity and drug resistance in cancer

 描述(由申请人提供)癌症治疗面临的一个主要挑战是肿瘤的异质性和抗药性克隆的选择。人们普遍认为，由于分支亚克隆进化，肿瘤具有大量的遗传异质性。最近，人们发现在同基因肿瘤群体中也存在异质性。细胞的异质性被归因于基因表达固有的随机过程和表观遗传变异性。在乳腺癌细胞中观察到了表型转换，这可以在扰动后重建干细胞、腔和基础表型亚群的平衡。此外，许多非小细胞肺癌患者对酪氨酸激酶抑制剂表现出可逆性耐药。我假设细胞处于不同的、半可遗传的表观遗传状态。 肿瘤的异质性。我还预测，这种表观遗传可塑性的存在是由于组蛋白修饰的不完全重置。由于缺乏追踪单个肿瘤细胞命运的技术，细胞异质性一直是一个相对未被探索的现象。在这项建议中，我使用一种新的谱系跟踪系统Tracer来研究肿瘤细胞异质性的机制，在这种系统中，每个细胞都写下自己独特的条形码，同时记录祖先细胞的进化历史。我建议将这项变革性的技术与共用的慢病毒CRISPR/gRNA文库相结合，以提供以前在共用基因扰动研究中无法获得的单细胞分辨率。使用这些工具，我将量化半稳定表观遗传状态之间的相互转化，并定义这些状态在选择压力下的遗传力。这些实验将阐明癌细胞的细胞异质性和可逆耐药的机制，并识别表观遗传耐药基因。示踪剂研究的长期目标是确定控制肿瘤进化轨迹的机制，并为旨在减缓或指导可预测的肿瘤进化路径的药物设计提供信息，最终目标是将癌症从致命疾病转变为慢性病。

 DESCRIPTION (provided by applicant) A major challenge facing cancer treatment is tumor heterogeneity and selection of drug resistant clones. It is widely accepted that tumors possess large amounts of genetic heterogeneity due to branching subclonal evolution. Recently, it was discovered that heterogeneity also exists within isogenic tumor populations. Cellular heterogeneity has been attributed to stochastic processes intrinsic to gene expression and to epigenetic variability. Phenotypic switching has been observed in breast cancer cells that can reestablish equilibrium of subpopulations of stem, luminal, and basal phenotypes following perturbation. Moreover, many NSCLC patients show a reversible drug tolerance to Tyrosine Kinase Inhibitors. I hypothesize that distinct, semi- heritable epigenetic states underlie cellular heterogeneity in tumors. I also predict this epigenetic plasticity exists due to incomplete resetting of histone modifications. Cellular heterogeneity has been a relatively unexplored phenomena due to a lack of technologies that can trace the fates of individual tumor cells. In this proposal I examine the mechanisms of cellular heterogeneity in tumors using a novel lineage tracking system, TRACER, where each cell writes its own unique barcode while recording the evolutionary history of ancestral cells. I propose to combine this transformative technology with pooled lentiviral CRISPR/gRNA libraries to provide a single cell resolution previously unavailable within pooled gene perturbation studies. Using these tools, I will quantitate the interconversion between semi-stable epigenetic states and define the heritability of these states while under selective pressures. The proposed experiments will illuminate the mechanisms of cellular heterogeneity and reversible drug tolerance in cancer cells and identify epigenetic drug resistance genes. The long term goal of TRACER studies is to identify the mechanisms that control the evolutionary trajectories of tumors and inform the design of drugs aimed at slowing or directing predictable paths of tumor evolution, with the ultimate goal of converting cancer from a lethal to a chronic disease.