Role of WT1 in mixed phenotype acute leukemia

WT1在混合表型急性白血病中的作用

基本信息

批准号：
10314019
负责人：
Lindsey Montefiori
金额：
$ 6.64万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2023-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10314019
关键词：
Acute Lymphocytic Leukemia Acute leukemia Address Apoptosis Binding Biological Assay Biology Biotin Blood Cells C-terminal CD34 gene CRISPR/Cas technology Cancer Model Cell Separation Cell model Cells Child Childhood Childhood Leukemia Chromatin Chromatin Structure Complement Complex DNA Binding DNA Binding Domain DNA Methylation Dependence Development Disease Epigenetic Process Event Exhibits Exons Fellowship Flow Cytometry Frequencies Future Gene Expression Gene Expression Profiling Gene Expression Regulation Genes Genetic Genetic Enhancement Genetic Models Genetic Transcription Genomics Goals Guide RNA Hematopoiesis Hematopoietic Hematopoietic stem cells Human Impairment In Vitro Knock-out Knowledge Label Lentivirus Leukemic Cell Lymphoid Lymphoid Cell Malignant - descriptor Malignant Childhood Neoplasm Maps Mediating Methodology Modeling Molecular Molecular Target Mutate Mutation Myelogenous Myeloid Cells Pathway interactions Patients Phenotype Play Population Prevalence Process Prognosis Proteins Proteomics Recurrence Regimen Regulation Regulator Genes Research Research Proposals Research Training Resources Role Sampling Surface Antigens Terminator Codon Testing Therapeutic Therapeutic Intervention Transcriptional Regulation Transplantation Treatment outcome Umbilical Cord Blood Uncertainty WT1 gene Zinc Fingers base bisulfite sequencing cell type chromatin modification combat data integration embryo tissue epigenetic regulation functional genomics genomic data hematopoietic differentiation high risk histone modification humanized mouse improved outcome in vivo individualized medicine insertion/deletion mutation leukemia leukemogenesis loss of function methylation pattern mouse model mutant new therapeutic target novel novel therapeutics nuclease pre-clinical premature progenitor programs protein protein interaction stem targeted treatment therapeutic target therapy design transcription factor transcriptome transcriptome sequencing tumor vector whole genome

项目摘要

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.

项目摘要/摘要小儿混合表型急性白血病（MPAL）是一种罕见的高风险白血病，占2-3％小儿白血病病例，预后较差（存活率<50％）。这主要是由于缺乏针对该疾病的独特遗传和发育基础的量身定制疗法，突出了关键需要更好地了解MPAL生物学。我们最近发现，WT1基因在几乎一半所有儿科T/髓样MPAL患者，大多数突变导致过早终止密码子去除 WT1蛋白的C末端DNA结合结构域。预计这种特殊变化具有功能后果，因为WT1编码了早期造血的DNA结合转录因子很重要发展。此外，WT1在调节基因表达，染色质结构，通过与其他分子复合物相互作用的DNA甲基化，突出了多种途径在WT1突变的造血细胞中可能会破坏。重要的是，这些途径中的每一个代表可以通过理性设计的治疗来利用或直接针对的潜在漏洞。研究在此奖学金中提出的将确定WT1突变的发展和分子后果早期的造血，为制定新策略以应对这一难以打击这种难以打击这种困难的第一步治疗儿童疾病。本研究建议的目标1将解决截断WT1突变在扰动早期造血。基于CRISPR/CAS9的方法将用于引入与患者相关的方法人造血茎和祖细胞（HSPC）中内源性WT1基因座的突变，该突变将忠实地对T/髓样MPAL的遗传和发育起源进行建模。野生型和WT1突变细胞将使用菌落形成测定法和体内通过移植到人源化小鼠中进行分化。流式细胞仪和基因表达谱分析将确定不同的造血种群和基因被WT1改变破坏的表达程序。本研究建议的目标2将使用蛋白质组学和功能基因组学测定法，以识别截断WT1突变引起的分子改变人类HSPC，补充AIM 1中确定的发展变化。具体来说，这些测定法 MAP动态蛋白 - 蛋白质相互作用，染色质修饰和DNA甲基化模式在野生型中和WT1突变的HSPC。这些数据的整合将使能够鉴定分子机制在WT1突变MPAL中解除早期造血的基本转录变化。总体而言，这些研究将阐明复发性WT1改变的发展和分子后果 T/髓样MPAL并揭示可能被询问治疗的新途径和依赖项益处。