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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要 /摘要横纹肌肉瘤（RMS）是影响数百个的最常见的儿童软组织肉瘤每年在美国的患者。横纹肌肉瘤（RMS）患者的当前标准治疗包括化学疗法，手术和 /或辐射。但是，即使有这些治疗性的组合，大量患者子集患有肿瘤复发，复发和转移，与非常恶化有关预后和惨淡的5年生存率。这仍然是改善患者结局的主要障碍横纹肌肉瘤。为了更好地理解诸如肿瘤传播细胞之类的机制，关键分子研究人员采用RMS细胞系的调节剂，促进RMS的治疗性耐药性和肿瘤解体的调节剂，转基因动物模型和异种移植研究研究了RMS的潜在肿瘤传播细胞（TPC）。然而，对于RMS中的肿瘤异质性和癌细胞演化动力学知之甚少。剖析肿瘤间和肿瘤内异质性，我已经使用单细胞转录组学来介绍患者衍生的 RMS的样本。我在RMS肿瘤中发现了不同的细胞态，包括增殖和间充质样 TPC潜力较高的亚群，而分化的肌肉亚群几乎没有向其他细胞状态过渡。有了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过渡的分子机制，并同时评估细胞表型随着其转录本，蛋白质和表观遗传学的改变而变化。一类重要在调节癌症干性方面的具有挑战性的分子，疗法后的进化是染色质调节剂，它是染色质调节剂需要在有限的单元线模型中进行深层测序。单细胞多组学的技术创新，包括单细胞RNA，单细胞ATAC，单细胞切割和标签以及细胞谱系条形码跟踪很大程度上会降低在单细胞水平上介绍癌细胞演化以及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及其催化产品 H3K27ME3锁定RMS细胞处于增殖性细胞状态，并抑制其过渡到其他分化状态。为了检验这一假设，我将首先评估EZH2在通过条形码跟踪调节细胞状态过渡中的作用在EZH2敲低的背景下进行功能STEM测定（AIM 1）。独立地，我还将介绍直接 EZH2和组蛋白H3赖氨酸27三甲基化的靶标通过进行单细胞切割和TAG和询问控制细胞状态过渡的机制（AIM 2）。此外，我还将评估EZH2抑制剂与化学疗法和放射线的合作，利用独特的免疫功能障碍斑马鱼模型沿通过细胞系和小鼠异种移植研究（AIM 3）。拟议研究的目标是调查横纹肌肉瘤样品中的染色质调节剂，同时也确认肿瘤杂种性和细胞细胞状态过渡中的可塑性。通过实现这些目标，我将说明一种关于如何 RMS肿瘤的发展以及染色质调节剂如何在控制这一过程中起关键作用。