Role of WT1 in mixed phenotype acute leukemia

项目概要/摘要 儿童混合表型急性白血病（MPAL）是一种罕见的高危白血病， 儿童白血病病例，预后特别差（生存率<50%）。这主要是因为缺乏 针对这种疾病独特的遗传和发育基础的定制疗法，突出了一个关键的 需要更好地了解MPAL生物学。我们最近发现，WT 1基因在近一半的 所有儿童T/髓系MPAL患者，大多数突变导致提前终止密码子， WT 1蛋白的C末端DNA结合域。这种特殊的变化被预测为具有功能性 结果，因为WT 1编码对早期造血重要的DNA结合转录因子， 发展此外，WT 1在基因表达、染色质结构、 和DNA甲基化通过与其他分子复合物的相互作用，突出了多种途径 在WT 1突变造血细胞中可能被破坏。重要的是，这些途径中的每一个都代表了 潜在的漏洞，可以利用或直接针对合理设计的治疗。研究 本奖学金中提出的将确定WT 1突变的发育和分子后果， 早期造血，为制定新的战略，以克服这一困难， 治疗儿童疾病。本研究提案的目的1将解决截断WT 1突变在 扰乱了早期造血基于CRISPR/Cas9的方法将用于引入患者相关的 人造血干细胞和祖细胞（HSPC）中内源性WT 1基因座的突变， 忠实地模拟T/髓样MPAL的遗传和发育起源。野生型和WT 1突变细胞将 在体外使用集落形成测定和在体内通过移植到人源化小鼠中进行分化。 流式细胞术和基因表达谱将鉴定不同的造血群体和基因表达谱。 被WT 1改变破坏的表达程序。本研究提案的目标2将使用蛋白质组学 和功能基因组学测定，以鉴定截短WT 1突变诱导的分子变化 人HSPC，补充目标1中鉴定的发育改变。具体而言，这些分析将 绘制野生型中动态蛋白质-蛋白质相互作用、染色质修饰和DNA甲基化模式 和WT 1突变型HSPC。这些数据的整合将使识别的分子机制 WT 1突变MPAL早期造血功能失调的潜在转录变化。总的来说，这些 这些研究将阐明反复发生的WT 1改变在发育和分子水平上的影响， T/髓系MPAL，并揭示了新的途径和依赖性，可用于治疗 效益

PROJECT SUMMARY/ABSTRACT Pediatric mixed phenotype acute leukemia (MPAL) is a rare, high-risk leukemia that accounts for 2-3% of pediatric leukemia cases and has a particularly poor prognosis (<50% survival). This is primarily due to lack of tailored therapies that target the unique genetic and developmental basis of this disease, highlighting a critical need to better understand MPAL biology. We recently discovered that the WT1 gene is mutated in nearly half of all pediatric T/myeloid MPAL patients, with most mutations causing premature termination codons that remove the C-terminal DNA binding domain of the WT1 protein. This particular alteration is predicted to have functional consequences, as WT1 encodes a DNA binding transcription factor important for early hematopoietic development. Furthermore, WT1 plays important roles in the regulation of gene expression, chromatin structure, and DNA methylation through interactions with other molecular complexes, highlighting the multiple pathways that may be disrupted in WT1-mutant hematopoietic cells. Importantly, each of these pathways represent potential vulnerabilities that can be exploited or directly targeted with rationally-designed therapy. The research proposed in this Fellowship will identify the developmental and molecular consequences of WT1 mutations in early hematopoiesis, providing a critical first step towards developing new strategies to combat this difficult to treat childhood disease. Aim 1 of this research proposal will address the role of truncating WT1 mutations in perturbing early hematopoiesis. A CRISPR/Cas9-based approach will be used to introduce patient-relevant mutations at the endogenous WT1 locus in human hematopoietic stem and progenitor cells (HSPCs) which will faithfully model the genetic and developmental origin of T/myeloid MPAL. Wild-type and WT1-mutant cells will be differentiated in vitro using colony forming assays and in vivo through transplantation into humanized mice. Flow cytometry and gene expression profiling will identify distinct hematopoietic populations and gene expression programs that are disrupted by WT1 alterations. Aim 2 of this research proposal will use proteomics and functional genomics assays to identify the molecular alterations induced by truncating WT1 mutations in human HSPCs, complementing the developmental alterations identified in Aim 1. Specifically, these assays will map dynamic protein-protein interactions, chromatin modifications, and DNA methylation patterns in wild-type and WT1-mutant HSPCs. Integration of these data will enable identification of the molecular mechanisms underlying transcriptional changes that deregulate early hematopoiesis in WT1-mutant MPAL. Overall, these studies will elucidate the developmental and molecular consequences of recurrent WT1 alterations in T/myeloid MPAL and reveal new pathways and dependencies that may be interrogated for therapeutic benefit.