Chromatin regulators of stemness and therapy resistance in rhabdomyosarcoma

项目总结/摘要 横纹肌肉瘤（RMS）是最常见的儿童软组织肉瘤， 美国每年的患者。横纹肌肉瘤（RMS）患者的当前标准治疗 包括化疗、手术和/或放疗。然而，即使有这些组合疗法， 显著亚组的患者患有肿瘤复发、再发和转移，与极差的 预后差，5年生存率低。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肿瘤增殖细胞、关键分子生物学机制， 调节剂驱动RMS的治疗抗性和肿瘤复发，研究人员采用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，对RMS中肿瘤异质性和癌细胞演变动力学知之甚少。解剖 肿瘤间和肿瘤内的异质性，我已经使用单细胞转录组学来分析患者来源的 RMS样本。我在RMS肿瘤中发现了不同的细胞状态，包括增殖和间充质样增生。 具有较高TPC潜力的分化肌肉亚群，而几乎不具有TPC潜力的分化肌肉亚群， 转移到其他细胞状态。有了这些知识，以及条形码追踪技术的创新，我 建议剖析有助于细胞状态转换的分子机制，并同时评估 细胞表型随着其转录物、蛋白质和表观遗传学改变而沿着改变。一类重要的 和挑战性的分子在调节癌症的干性，进化后的治疗是染色质调节剂， 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细胞的增殖能力。 在单细胞水平上描绘癌细胞进化沿着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EZH 2及其催化产物 H3 K27 me 3将RMS细胞锁定在增殖细胞状态，并抑制其向其他分化状态的转变。 为了验证这一假设，我将首先评估EZH 2在条形码追踪调节细胞状态转换中的作用 和在EZH 2敲低的情况下的功能性茎测定（Aim 1）。另外，我还将分析 EZH 2和组蛋白H3赖氨酸27三甲基化的靶标，通过进行单细胞CUT&Tag，并询问 控制细胞状态转换的机制（目标2）。此外，我还将评估EZH 2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，沿着 细胞系和小鼠异种移植研究（Aim 3）。本研究的目的是调查 横纹肌肉瘤样本中的染色质调节因子，同时也承认肿瘤的异质性和细胞 细胞状态转换的可塑性。为了实现这些目标，我将说明一个全面的机制， RMS肿瘤的演变以及染色质调节剂如何在控制这一过程中发挥关键作用。

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.