Chromatin regulators of stemness and therapy resistance in rhabdomyosarcoma

横纹肌肉瘤干性和治疗耐药性的染色质调节因子

• 批准号: 10622041
• 负责人: Yun Wei
• 金额: $ 17.82万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622041
• 关键词: Affect; Aftercare; Animal Model; Bar Codes; Binding; Biological Assay; Catalytic Domain; Cell Cycle; Cell Differentiation process; Cell Line; Cell Lineage; Cell Proliferation; Cells; Chemotherapy and/or radiation; Childhood Soft Tissue Sarcoma; Chromatin; Clinic; Collaborations; Colony-Forming Units Assay; Complex; Cyclophosphamide; Dactinomycin; EZH2 gene; Epigenetic Process; Evolution; Frequencies; Gene Expression; Genetic Transcription; Goals; Heterogeneity; Histone H3; Histones; Image; Immune; Impairment; Inter-tumoral heterogeneity; Knowledge; Lysine; Malignant Neoplasms; Mesenchymal; Messenger RNA; Methylation; Modeling; Modification; Molecular; Mus; Muscle; Muscle Cells; Muscle Fibers; Mutate; Myoblasts; NOTCH3 gene; Nature; Neoplasm Metastasis; Operative Surgical Procedures; Pathway interactions; Patient-Focused Outcomes; Patients; Pediatric Neoplasm; Pharmaceutical Preparations; Phenotype; Play; Polycomb; Process; Prognosis; Proliferating; Proteins; RNA; Radiation; Recurrence; Recurrent tumor; Relapse; Repressor Proteins; Research; Research Personnel; Resistance; Resolution; Rhabdomyosarcoma; Role; Sampling; Stress; Survival Rate; Tail; Techniques; Testing; Time; Transcript; Transcription Coactivator; Transgenic Animals; Undifferentiated; United States; Vincristine; Work; XCL1 gene; Xenograft procedure; Zebrafish; cancer cell; cell killing; chemotherapy; childhood sarcoma; cost; deep sequencing; drug testing; dynamical evolution; gene repression; histone methylation; improved; in vivo; inhibitor; innovation; irradiation; knock-down; mouse model; multiple omics; myocyte-specific enhancer-binding-factor 2C; neoplastic cell; new therapeutic target; novel; novel therapeutics; patient derived xenograft model; patient subsets; pharmacologic; pre-clinical; preclinical efficacy; profiles in patients; progenitor; programs; single-cell RNA sequencing; standard care; stem; stemness; synergism; therapy resistant; transcription factor; transcriptomics; tumor; tumor growth; tumor heterogeneity

PROJECT SUMMARY / ABSTRACT Rhabdomyosarcomas (RMS) are the most common childhood soft-tissue sarcomas affecting hundreds of patients in the United States annually. Current standard treatments for rhabdomyosarcoma (RMS) patients include chemotherapy, surgery, and /or radiation. However, even with these combinations of therapeis, significant subsets of patients suffer tumor recurrence, relapse, and metastasis, associated with extremely worse prognosis and dismal 5-year survival rate. This remains the major hurdle to improve the patient outcomes with rhabdomyosarcomas. To better understand the mechanisms such as tumor-propagating cells, critical molecular regulators that drives therapy resistance and tumor-relapse in RMS, researchers has employed RMS cell lines, transgenic animal models, and xenograft studies to study the potential tumor-propagating cells (TPCs) for RMS. Yet, little is known about the tumor heterogeneity and cancer cell evolution dynamics in RMS. To dissect the inter-tumoral and intra-tumoral heterogeneity, I have used the single-cell transcriptomics to profile patient-derived samples of RMS. I uncovered distinct cell states in RMS tumors, including proliferation and a mesenchymal-like subpopulations that have higher TPC potential, whereas the differentiated muscle subpopulation that barely transits towards other cell states. With this knowledge, and the innovation of barcode tracing techniques, I propose to dissect molecular mechanisms that contribute to cell state transitions, and concurrently assess the cell phenotypes changes along with its transcripts, proteins, and epigenetics alterations. One class of important and challenging molecules in regulating cancer stemness, evolution post therapies is chromatin regulators, which requires deep sequencing in limited cell line models. The technical innovation of single-cell multiomics, including single-cell RNA, single-cell ATAC, single-cell CUT&Tag, and cell lineage barcode tracing largely decrease the cost and time needed to profile cancer cell evolution along with epigenetic modifications at single-cell levels. With effective collaboration with computational biologists, I hypothesize that EZH2 and its catalytic product H3K27me3 lock RMS cells in the proliferative cell state and inhibit their transition into other differentiated states. To test this hypothesis, I will first assess the role of EZH2 in regulating cell state transitions with barcode tracing and functional stem assays in the context of EZH2 knockdown (Aim 1). Independently, I will also profile the direct targets of EZH2 and histone H3 lysine 27 trimethylation by performing single-cell CUT&Tag, and interrogate mechanism that controls cell state transition (Aim 2). In addition, I will also assess the EZH2 inhibitors in collaboration with chemotherapy and radiation utilizing the unique immune-compromised zebrafish models along with cell line and mouse xenograft studies (Aim 3). The goals of the proposed research are to investigate chromatin regulators in rhabdomyosarcoma samples while also acknowledging the tumor-heterogeneity and cell plasticity in cell state transitions. By achieving these aims, I will illustrate a comprehensive mechanism as to how RMS tumors evolve and how chromatin regulators play critical roles in controlling this process.

项目概要/摘要 横纹肌肉瘤 (RMS) 是最常见的儿童软组织肉瘤，影响数百名儿童 美国每年都有患者。横纹肌肉瘤 (RMS) 患者的现行标准治疗 包括化疗、手术和/或放疗。然而，即使采用这些治疗组合， 相当一部分患者患有肿瘤复发、复发和转移，与病情极其恶化有关 预后和惨淡的 5 年生存率。这仍然是改善患者治疗效果的主要障碍 横纹肌肉瘤。为了更好地理解肿瘤增殖细胞、关键分子等机制 导致 RMS 治疗耐药和肿瘤复发的调节因子，研究人员采用了 RMS 细胞系， 转基因动物模型和异种移植研究，以研究 RMS 的潜在肿瘤增殖细胞 (TPC)。 然而，人们对 RMS 中的肿瘤异质性和癌细胞进化动态知之甚少。剖析 肿瘤间和肿瘤内异质性，我使用单细胞转录组学来分析患者来源的异质性 RMS 样本。我发现了 RMS 肿瘤中不同的细胞状态，包括增殖和间质样细胞状态 具有较高 TPC 潜力的亚群，而几乎没有分化的肌肉亚群 过渡到其他细胞状态。凭借这些知识以及条形码追踪技术的创新，我 提议剖析有助于细胞状态转变的分子机制，并同时评估 细胞表型随着其转录物、蛋白质和表观遗传学的改变而变化。一类重要的 在调节癌症干细胞性方面具有挑战性的分子是染色质调节剂，它是治疗后的进化 需要在有限的细胞系模型中进行深度测序。单细胞多组学的技术创新，包括 单细胞RNA、单细胞ATAC、单细胞CUT&Tag和细胞谱系条形码追踪大大降低了 分析癌细胞进化以及单细胞水平的表观遗传修饰所需的成本和时间。 通过与计算生物学家的有效合作，我假设 EZH2 及其催化产物 H3K27me3 将 RMS 细胞锁定在增殖细胞状态并抑制它们向其他分化状态的转变。 为了验证这个假设，我将首先评估 EZH2 在通过条形码追踪调节细胞状态转换中的作用 以及 EZH2 敲低背景下的功能性干分析（目标 1）。独立地，我还将介绍直接 通过执行单细胞 CUT&Tag 来确定 EZH2 和组蛋白 H3 赖氨酸 27 三甲基化的靶标，并询问 控制细胞状态转换的机制（目标 2）。此外，我还将评估 EZH2 抑制剂 利用独特的免疫受损斑马鱼模型与化疗和放疗合作 细胞系和小鼠异种移植研究（目标 3）。拟议研究的目标是调查 横纹肌肉瘤样本中的染色质调节因子，同时也承认肿瘤异质性和细胞 细胞状态转变的可塑性。通过实现这些目标，我将说明一个全面的机制 RMS 肿瘤的进化以及染色质调节因子如何在控制这一过程中发挥关键作用。