Elucidation of the molecular mechanisms of the RBM15-MKL1 fusion protein in acute megakaryoblastic leukemia

阐明RBM15-MKL1融合蛋白在急性巨核细胞白血病中的分子机制

• 批准号: 10597365
• 负责人: Madeline Young Mayday
• 金额: $ 5.02万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597365
• 关键词: 4 year old; Acute Megakaryocytic Leukemias; Adenosine; Automobile Driving; Binding; Cancer Biology; Cell Line; Cells; Child; Chimeric Proteins; Complex; Computer Analysis; Cytotoxic Chemotherapy; DNA Sequence Alteration; Data; Deposition; Development; Diagnosis; Disease; FUS-1 Protein; Gene Expression; Genes; Goals; Health; Hematopoiesis; Immunoprecipitation; In Vitro; Investigation; Knowledge; Leukemic Cell; Maintenance; Malignant - descriptor; Malignant Childhood Neoplasm; Malignant Neoplasms; Mediating; Megakaryocytes; Messenger RNA; Mission; Modeling; Modification; Molecular; Mus; Mutation; Neonatal; Neonatal Leukemia; Oncogenic; Pathway interactions; Patient-Focused Outcomes; Pattern; Phenotype; Physiological; Play; Property; Protein Binding Domain; Proteins; Public Health; RNA; RNA Binding; RNA-Binding Proteins; Reader; Recurrence; Reporting; Research; Role; Scientific Advances and Accomplishments; Survival Rate; Techniques; Testing; Transcript; Transcription Coactivator; United States National Institutes of Health; WNT Signaling Pathway; Western Blotting; Work; acute megakaryoblastic leukemia cell; beta catenin; candidate selection; cell growth; chromatin remodeling; crosslink; crosslinking and immunoprecipitation sequencing; epitranscriptome; epitranscriptomics; experimental study; improved; improved outcome; in vivo; infancy; insight; interest; knock-down; leukemia; leukemogenesis; neonate; novel therapeutics; posttranscriptional; recruit; side effect; targeted treatment; therapeutic target; transcriptome; transcriptome sequencing; transcriptomics; tumorigenesis

PROJECT SUMMARY/ABSTRACT Acute megakaryoblastic leukemia (AMKL) is a form of cancer most prevalent in children under four years old. Targeted AMKL-specific treatment options are limited, and survival rates remain variable. A major obstacle to improving therapy options for AMKL is the dearth of data regarding the mechanisms that contribute to AMKL leukemogenesis. Of several known causative genomic alterations in AMKL, the t(1;22) translocation, which encodes the RBM15-MKL1 (RM) fusion protein, is considered a neonatal mutation as it is always diagnosed in children younger than 6 months old. RNA-binding motif protein 15 (RBM15) is required for recruitment to RNA of the N6-methyladenosine (m6A) writer complex and subsequent epitranscriptomic modification of the transcripts. Megakaryoblastic leukemia 1 (MKL1) is a transcriptional coactivator and is involved in gene expression and megakaryocyte maturation. RM retains all functional domains of both proteins and, despite our understanding of these proteins, the properties of RM itself remain poorly understood. The goal of this proposal is to investigate the molecular mechanisms by which RBM15’s association with RNA in the context of the RM fusion protein contributes to leukemogenesis. Based on our preliminary data, we hypothesize that RM alters gene expression via binding to RNA and promoting modification of the epitranscriptome which promotes oncogenesis via aberrant Wnt signalling. To test this hypothesis, two Aims are proposed. The first Aim is to identify RNAs bound and m6A modified by RM using enhanced crosslinking and immunoprecipitation sequencing techniques. This will determine which transcripts are targeted by RM and which adenosine residues are modified following RM binding, and functional analysis will determine pathways important to leukemogenesis. Computational integration with RNA-seq will provide insight into the fate of RNAs targeted by the RM fusion protein in contrast to control RBM15. The second Aim is to investigate the role of candidate proteins in RM- mediated leukemogenesis by knocking down select candidates in a physiologically relevant murine megakaryoblastic cell line model. In vitro and in vivo assessment of leukemogenesis will determine the requirement of these candidates for the survival of leukemia cells in vitro and for maintenance of disease in vivo. These experiments will provide insight into the transcriptomic and epitranscriptomic effects of RM that are critical to the mechanistic function of the fusion protein. A better understanding of the mechanisms driving RM-mediated AMKL will significantly broaden knowledge of AMKL leukemogenesis and provide potential novel therapeutic avenues to improve outcomes and survival rates of neonates with this disease.

项目总结/摘要 急性巨核细胞白血病（AMKL）是一种最常见于四岁以下儿童的癌症。 靶向AMKL特异性治疗选择有限，生存率仍然可变。的主要障碍 改善AMKL的治疗选择是缺乏有关AMKL机制的数据 白血病发生在AMKL的几种已知致病基因组改变中，t（1;22）易位， 编码RBM 15-MKL 1（RM）融合蛋白，被认为是一种新生儿突变，因为它总是被诊断为 6个月以下的儿童。RNA结合基序蛋白15（RBM 15）是募集RNA所必需的 的N6-甲基腺苷（m6 A）写入器复合物和随后的 成绩单巨核细胞白血病1（MKL 1）是一种转录辅激活因子，参与基因 表达和巨核细胞成熟。RM保留了两种蛋白质的所有功能结构域，尽管我们 尽管对这些蛋白质的了解不多，但RM本身的性质仍然知之甚少。这项提案的目的是 研究RBM 15与RNA结合的分子机制， 融合蛋白有助于白血病的发生。根据我们的初步数据，我们假设RM改变了 通过与RNA结合并促进表转录组的修饰的基因表达， 通过异常Wnt信号传导的肿瘤发生。为了验证这一假设，提出了两个目标。第一个目标是 使用增强的交联和免疫沉淀鉴定RM结合的RNA和修饰的m6 A 测序技术。这将决定RM靶向哪些转录本以及哪些腺苷残基 在RM结合后被修饰，功能分析将确定对白血病发生重要的途径。 与RNA-seq的计算整合将提供对RM融合所靶向的RNA的命运的洞察 与对照RBM 15相比，第二个目的是研究候选蛋白在RM中的作用。 在生理学相关小鼠中通过敲低选择候选物介导白血病发生 巨核细胞系模型。白血病发生的体外和体内评估将确定 这些候选物是白血病细胞在体外存活和在体内维持疾病的需要。 这些实验将提供深入了解RM的转录组学和表转录组学效应， 融合蛋白的机械功能。更好地理解RM介导的机制 AMKL将极大地拓宽对AMKL白血病发生机制的认识，并提供潜在的新型治疗药物 改善患有这种疾病的新生儿的预后和存活率的途径。