Role of WT1 in mixed phenotype acute leukemia

项目摘要/摘要 儿童混合型急性白血病(MPAL)是一种罕见的高危白血病，占 儿童白血病病例，预后特别差(50%存活率)。这主要是由于缺乏 针对这种疾病的独特遗传和发育基础的量身定制的治疗方法，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，WT1基因在近一半的 所有儿童T/髓系MPAL患者，大多数突变导致过早终止密码子移除 WT1蛋白的C-末端DNA结合域。这一特殊的改变被预测具有功能性 后果，因为WT1编码一种对早期造血重要的DNA结合转录因子 发展。此外，WT1在基因表达、染色质结构、 和DNA甲基化通过与其他分子复合体的相互作用，突出了多条途径 这可能会在WT1突变的造血细胞中被破坏。重要的是，这些途径中的每一条都代表 可以通过合理设计的治疗方法来利用或直接针对的潜在漏洞。这项研究 将确定WT1突变的发育和分子后果 早期造血，为制定抗击这一困难的新战略提供了关键的第一步 治疗儿童疾病。这项研究提案的目标1将解决截断WT1突变在 扰乱了早期的造血。将使用基于CRISPR/CAS9的方法来引入与患者相关的 人类造血干细胞和祖细胞(HSPC)内源性WT1基因突变将 忠实地模拟T/髓系MPAL的遗传和发育起源。野生型和WT1突变细胞将 通过克隆形成实验在体外分化，并通过移植到人源化的小鼠体内进行分化。 流式细胞术和基因表达谱将识别不同的造血群体和基因 被WT1改变打乱的表达程序。这项研究提案的目标2将使用蛋白质组学 和功能基因组学分析确定截断WT1突变引起的分子变化 人类HSPC，补充目标1中确定的发育变化。具体地说，这些检测将 绘制野生型的动态蛋白质-蛋白质相互作用、染色质修饰和DNA甲基化模式图 和WT1突变型HSPC。这些数据的整合将使分子机制的识别成为可能 WT1突变的MPAL早期造血功能的潜在转录变化。总的来说，这些 研究将阐明反复发生的WT1改变在发育和分子水平上的影响。 T/髓系MPAL，并揭示可能被询问用于治疗的新途径和依赖 利益。

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.