Elucidating Oncogenic Mechanisms Underlying Wilms Tumor Using Kidney Organoids

项目摘要/摘要 肾母细胞瘤是最常见的儿童肾癌。对以Blastemal为主的肿瘤进行了分类 作为高风险并表现出对化疗治疗的抗药性，大幅增加了 与其他亚型相比，复发的可能性。目前的治疗包括切除整个受影响的 肾移植后化疗，反之亦然。儿科化疗增加发病风险 继发性癌症，而肾脏切除可以消除患者高达50%的功能肾单位。显然， 需要对以母细胞为主的Wilms瘤采取更安全和更有效的靶向治疗策略 减少或消除复发，同时保留宝贵的功能肾组织，以缓解 有可能出现慢性健康问题。我提出的研究的目的是阐明致癌机制。 以胚基细胞为主的肾母细胞瘤的形成以确定新的发展候选因素 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的前体细胞 胎儿肾脏的发育，包括转录因子SIX1，它在建立这种发育过程中起着关键作用 利基市场。对以胚基细胞为主的肿瘤进行测序，发现了一个复发的错义突变Q177R，位于 SIX1进化保守的DNA结合同源域中已知的DNA碱基接触残基。我 假设这种突变改变了直接的SIX1调控相互作用，扰乱了下游网络，推动了 基因表达异常，阻碍肾单位祖细胞分化，促进致癌状态。 利用包括CRISPR-Cas9基因组编辑和人类诱导多能干细胞在内的尖端技术 体外建立胚胎型肾母细胞瘤模型的目的：1)鉴定 SIX1-Q177R的致癌表型通过分化分析、细胞周期分析、 增殖和迁移分析以及2)定义SIX1-Q177R下游的调控网络中断 通过CHIP-SEQ、RNA-SEQ和免疫沉淀-质谱法。基因组与蛋白质组的整合 数据将精确定位由SIX1-Q177R直接调控的潜在致癌作用的靶基因 表型。这一开创性的工作将为理解分子 胚基为主的Wilms瘤的发生机制，指导研究 新的靶向疗法。此外，这项研究将为推动我们的 对人类肾脏发育的转录调控有基本的了解。 本研究将在支持和协作的实验室环境下进行 我的赞助人洛里·L·奥布莱恩博士是肾脏发育和肾单位调控方面的专家 祖细胞命运。我的联合赞助人阿尔伯特·S·鲍德温博士提供了额外的培训，他是 核因子-kB转录网络及其在癌症中的作用，本研究的完成和我的 培训计划将加强概念和实验研究技能，并促进专业发展。

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.