An inducible molecular memory system to unravel the mechanisms of drug resistance in head and neck cancer

诱导性分子记忆系统揭示头颈癌的耐药机制

• 批准号: 10523122
• 负责人: Robi D Mitra
• 金额: $ 15.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10523122
• 关键词: Adjuvant Therapy; Aftercare; Back; Benchmarking; Binding; Biological; Biological Markers; CRISPR interference; Cancer Etiology; Cancer Patient; Cause of Death; Cell Line; Cell Survival; Cells; Cetuximab; Chemicals; Chromium; Cytolysis; DNA; Development; Disease Outcome; Drug resistance; Enhancers; Epithelium; Event; Genes; Genetic Transcription; Genome; Genomics; Harvest; Head and Neck Cancer; Head and Neck Squamous Cell Carcinoma; Heterogeneity; Immunity; Learning; Malignant Epithelial Cell; Mediating; Memory; Mesenchymal; Messenger RNA; Methods; Modeling; Molecular; Morbidity - disease rate; Neoplasm Metastasis; Oncology; Organoids; Outcome; Pathway interactions; Patient Care; Pharmaceutical Preparations; Pharmacotherapy; Play; Prediction of Response to Therapy; Process; Reagent; Resistance; Role; Smoking History; Snails; Specimen; System; Tamoxifen; Technology; Testing; Time; Transforming Growth Factor beta; Transposase; Treatment Failure; clinically relevant; experience; experimental study; genome-wide; high risk; in vivo; interest; mortality; neoplastic cell; new therapeutic target; novel; patient derived xenograft model; predictive marker; prevent; programs; promoter; resistance mechanism; single-cell RNA sequencing; temporal measurement; therapy resistant; tool; transcription factor; transcriptome; treatment response; tumor

PROJECT SUMMARY HNSCC is the sixth leading cause of cancer-related mortality. Most deaths are caused by metastasis after treatment failure, but unfortunately, a firm understanding of the molecular pathways that drive treatment resistance remains elusive. For example, it is unclear why the vast majority of tumor cells are successfully eliminated by treatment, yet a few escape destruction. Are these cells in a privileged cell state that enables evasion of these drugs? Or does resistance emerge adaptively upon treatment? Given the morbidity and mortality associated with HNSCC, there is an urgent need to answer these questions, but this has been prevented by two major obstacles. First, HNSCC tumors are highly heterogeneous, so bulk genomic methods cannot discern subpopulations of cells that might give rise to resistant clones. Second, nearly all existing genomic methods are destructive and require specimen lysis at the time of measurement. This “destruction upon observation” has made it impossible to correlate molecular events that occurred in the past with the final fates of the cells in which these events took place. To overcome these barriers, we have recently utilized single-cell RNA- seq (scRNA-seq) to characterize heterogeneity among HNSCC tumors, defining a partial epithelial-to- mesenchymal (p-EMT) program which predicts HNSCC outcomes (Puram et al., Cell). We have also developed a novel single-cell ‘Calling Card’ (scCC) technology that can record the genome-wide interactions of any transcription factor (TF), creating a permanent molecular memory of all binding events that occur at a given moment or epoch (Moudgil et al., Cell). This allows transient molecular interactions to be captured non- destructively and read out later (e.g. after drug treatment), allowing us to “go back in time” and determine which cell states enabled a cell to resist treatment. We accomplish this by fusing any TF to the piggyBac transposase, which bestows the TF with the ability to direct transposon insertion into the genome near where it binds. We will use this technology to define the mechanisms by which HNSCC cells persist after cetuximab treatment and then evolve to produce resistant clones, identifying genes and pathways that can be targeted by adjuvant therapies. Specifically, we hypothesize there are subpopulations of tumor cells in a pre-existing p-EMT state that confers immunity to drug treatment. To test this hypothesis, we will first use our molecular memory tool to determine why some cells acquire p-EMT but others do not (Aim 1). This Aim is critical because EMT plays a key role in the development of cetuximab resistance, and our extensive experience with p-EMT will allow us to use this system to mature and benchmark our molecular memory tool. To probe pathways specific to cetuximab resistance, we will utilize scCC to evaluate HNSCC lines with cetuximab resistance and sensitivity and record both pre-existing and adaptive changes in cell state. We will validate these findings in vivo, establishing a set of genes and molecular pathways responsible for therapeutic resistance and thereby revealing new targets to overcome these mechanisms as well as biomarker predictors of treatment response.

项目摘要 HNSCC是癌症相关死亡的第六大原因。大多数死亡是由转移后， 治疗失败，但不幸的是，对驱动治疗的分子途径的坚定理解 抵抗仍然难以实现。例如，目前还不清楚为什么绝大多数肿瘤细胞成功地 通过治疗消除，但少数人逃脱了毁灭。这些单元是否处于特权单元状态， 这些药物的使用？还是治疗后会适应性地产生耐药性？鉴于发病率和 与HNSCC相关的死亡率，迫切需要回答这些问题，但这已经被 有两个主要障碍。首先，HNSCC肿瘤是高度异质性的，因此批量基因组方法 无法辨别可能产生抗性克隆的细胞亚群。其次，几乎所有现有的基因组 这些方法是破坏性的，并且在测量时需要样品溶解。这种“毁灭” “观察”使得不可能将过去发生的分子事件与生物的最终命运联系起来。 这些事件发生的细胞。为了克服这些障碍，我们最近利用单细胞RNA- seq（scRNA-seq）来表征HNSCC肿瘤之间的异质性，定义了部分上皮- 间充质（p-EMT）程序，其预测HNSCC结果（Puram等，Cell）。我们还开发了 一种新的单细胞“电话卡”（scCC）技术，可以记录任何 转录因子（TF），创建一个永久的分子记忆的所有结合事件发生在一个给定的 时刻或时期（Moudgil等人，细胞）。这使得瞬态分子相互作用被捕获非- 破坏性的，并读出后（例如药物治疗后），让我们“回到过去”，并确定 细胞状态使细胞能够抵抗治疗。我们通过将任何TF融合到piggyBac转座酶来实现这一点， 这赋予TF将转座子插入基因组中其结合位置附近的能力。我们将 使用该技术来确定HNSCC细胞在西妥昔单抗治疗后持续存在的机制， 进化以产生耐药克隆，识别可被辅助疗法靶向的基因和途径。 具体地说，我们假设存在预先存在的p-EMT状态的肿瘤细胞亚群， 对药物治疗有免疫力为了验证这一假设，我们将首先使用我们的分子记忆工具， 确定为什么一些细胞获得p-EMT，而另一些细胞不获得（Aim 1）。这一目标至关重要，因为EMT发挥着 在西妥昔单抗耐药的发展中起着关键作用，我们在p-EMT方面的丰富经验将使我们能够 用这个系统来成熟和测试我们的分子记忆工具。探索西妥昔单抗的特异性通路 我们将利用scCC评估具有西妥昔单抗抗性和敏感性的HNSCC系，并记录 细胞状态的预先存在的和自适应的变化。我们将在体内验证这些发现，建立一套 基因和分子途径负责治疗耐药性，从而揭示新的目标， 克服这些机制以及治疗反应的生物标志物预测因子。