Elucidating Oncogenic Mechanisms Underlying Wilms Tumor Using Kidney Organoids

使用肾脏类器官阐明肾母细胞瘤的致癌机制

• 批准号: 10543184
• 负责人: Matthew Jared Stevenson
• 金额: $ 3.92万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543184
• 关键词: 4 year old; Address; Adult; Affect; Affinity Chromatography; Alleles; Automobile Driving; Binding; Bioinformatics; Biological Models; CRISPR/Cas technology; Cancer Biology; Cell Cycle; Cell Line; Cells; ChIP-seq; Child; Childhood; Chronic; Chronic Kidney Failure; Classification; Colorectal Cancer; Complex; DNA; DNA Binding; DNA Sequence Alteration; Data; Data Set; Development; Diagnosis; Engineering; Environment; Excision; Exhibits; Fetal Kidney; Foundations; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Transcription; Genome engineering; Genomics; Goals; Health; Heterozygote; Homeodomain Proteins; Human; Human Engineering; Immunoprecipitation; In Vitro; Investigation; Kidney; Knock-in; Knowledge; Laboratories; Malignant - descriptor; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant childhood renal neoplasm; Malignant neoplasm of ovary; Mass Spectrum Analysis; Migration Assay; Missense Mutation; Modeling; Molecular; Mus; Mutation; NF-kappa B; Nephroblastoma; Nephrons; Oncogenic; Organoids; Patients; Pattern; Phenotype; Play; Proliferating; Proteins; Proteomics; Recurrence; Relapse; Research; Resistance; Risk; Role; Second Primary Cancers; Specificity; System; Technology; Therapeutic; Tissue-Specific Gene Expression; Tissues; Training; Transcriptional Regulation; Work; aerobic glycolysis; base; cancer initiation; cdc Genes; chemotherapy; cofactor; data integration; experimental study; genome editing; high risk; homeodomain; human model; induced pluripotent stem cell; innovation; insight; malignant breast neoplasm; man; migration; mutant; nephrogenesis; nephron progenitor; new therapeutic target; novel; overexpression; programs; protein protein interaction; relapse risk; skills; stem cells; targeted treatment; transcription factor; transcriptome sequencing; treatment strategy; tumor; tumorigenesis; tumorigenic

PROJECT SUMMARY/ABSTRACT Wilms tumor is the most common pediatric kidney cancer. Blastemal-predominant tumors are classified as high-risk and have demonstrated resistance to chemotherapeutic treatments, drastically increasing the likelihood for relapse compared to other subtypes. Current treatment consists of removal of the entire affected kidney followed by chemotherapy, or vice versa. Pediatric chemotherapy increases the risk for developing secondary cancers, while kidney removal eliminates up to 50% of a patient’s functioning nephrons. Clearly, safer and more effective targeted therapeutic strategies for blastemal-predominant Wilms tumor are needed to reduce or eliminate relapse while simultaneously retaining precious functional kidney tissue to mitigate the potential for chronic health issues. The goal of my proposed research is to elucidate the oncogenic mechanisms underlying blastemal-predominant Wilms tumor formation to identify novel candidate factors for the development of targeted therapies. These tumors express markers normally restricted to the nephron progenitor niche during fetal kidney development, including the transcription factor SIX1 which plays a critical role in establishing this niche. Sequencing of blastemal-predominant tumors identified a recurring missense mutation, Q177R, at a known DNA base-contacting residue within the evolutionary-conserved DNA binding homeodomain of SIX1. I hypothesize this mutation alters direct SIX1 regulatory interactions disrupting downstream networks, driving aberrant gene expression, obstructing nephron progenitor differentiation, and promoting a pro-oncogenic state. Utilizing cutting-edge technologies including CRISPR-Cas9 genome editing and human induced pluripotent stem cell-derived kidney organoids to model blastemal-predominant Wilms tumor in vitro, I aim to: 1) identify oncogenic phenotypes resulting from SIX1-Q177R through differentiation analysis, cell cycle profiling, proliferation, and migration assays and 2) define regulatory network disruptions downstream of SIX1-Q177R through ChIP-seq, RNA-seq and immunoprecipitation-mass spectrometry. Integration of genomic and proteomic data will pinpoint target genes directly regulated by SIX1-Q177R that are potentially contributing to oncogenic phenotypes. This pioneering work will provide a substantial foundation for understanding the molecular mechanisms contributing to the development of blastemal-predominant Wilms tumor, guiding the investigation of novel targeted therapies. Furthermore, this research will contribute vital knowledge to advance our fundamental understanding of the transcriptional control of human kidney development. This research will take place in a supportive and collaborative laboratory environment under the direction of my sponsor Dr. Lori L. O’Brien, an expert in kidney development and the regulatory control of nephron progenitor cell fate. With additional training provided by my co-sponsor Dr. Albert S. Baldwin, a leader in the field of NF-kB transcriptional networks and their roles in cancer, completion of this research and execution of my training plan will strengthen conceptual and experimental research skills and enhance professional development.

项目总结/摘要 肾母细胞瘤是最常见的小儿肾癌。胚细胞为主的肿瘤被分类为 作为高风险，并已证明耐化疗治疗，大大增加了 与其他亚型相比复发的可能性。目前的治疗方法包括切除整个受影响的 先肾后化疗，反之亦然。儿童化疗会增加患上 继发性癌症，而肾脏切除消除了高达50%的患者的功能肾单位。很显然， 需要更安全和更有效的胚细胞为主的肾母细胞瘤的靶向治疗策略， 减少或消除复发，同时保留宝贵的功能性肾组织， 潜在的慢性健康问题。我所提出的研究目标是阐明致癌机制 潜在的胚细胞为主的肾母细胞瘤形成，以确定新的候选因素的发展 有针对性的治疗。这些肿瘤表达的标记物通常局限于肾单位祖细胞龛， 胎儿肾脏发育，包括转录因子SIX1，它在建立这一过程中起着关键作用。 利基胚细胞占优势的肿瘤的测序确定了一个复发的错义突变，Q177R，在一个突变位点。 SIX1的进化保守的DNA结合同源结构域内的已知DNA碱基接触残基。我 假设这种突变改变了直接SIX1调节相互作用，破坏了下游网络， 异常基因表达、阻碍肾单位祖细胞分化和促进原癌状态。 利用CRISPR-Cas9基因组编辑和人类诱导多能干细胞等尖端技术 细胞来源的肾类器官体外模型胚细胞占主导地位的Wilms肿瘤，我的目的是：1）确定 通过分化分析，细胞周期分析， 增殖和迁移测定和2）确定SIX1-Q177 R下游的调节网络破坏 通过ChIP-seq、RNA-seq和免疫沉淀-质谱。基因组和蛋白质组的整合 数据将精确定位由SIX1-Q177R直接调控的靶基因，这些基因可能导致致癌性。 表型这一开创性的工作将为理解分子生物学提供坚实的基础。 机制有助于胚细胞为主的肾母细胞瘤的发展，指导调查 新的靶向治疗。此外，这项研究将为推动我们的发展贡献重要的知识 对人类肾脏发育的转录控制有基本的了解。 这项研究将在一个支持和合作的实验室环境下进行， 我的担保人洛里·L医生奥布莱恩是肾脏发育和肾单位调控方面的专家， 祖细胞命运通过我的共同赞助人Albert S.鲍德温，一个领导人在 NF-kB转录网络及其在癌症中的作用领域，完成这项研究并执行我的 培训计划将加强概念和实验研究技能，促进专业发展。