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

项目摘要/摘要 急性巨核细胞白血病(AMKL)是一种在四岁以下儿童中最常见的癌症。 针对AMKL的靶向治疗选择有限，存活率仍然不同。一个主要障碍是 改善AMKL的治疗方案缺乏关于AMKL发病机制的数据 白血病的发生。在AMKL已知的几种致病基因组改变中，t(1；22)易位， 编码RBM15-MKL1(RM)融合蛋白，被认为是新生儿突变，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(RBM15)是RNA募集所必需的 N6-甲基腺苷(M6A)编写器复合体和随后的表位转录修饰 成绩单。巨核细胞白血病1(MKL1)是一种转录共激活因子，参与基因 表达和巨核细胞成熟。Rm保留了这两种蛋白质的所有功能结构域，尽管我们的 对于这些蛋白质的了解，Rm本身的特性仍然知之甚少。这项提案的目标是 是在Rm的背景下研究Rbm15‘S与核糖核酸结合的分子机制 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发生了变化 通过与RNA结合促进表位转录组修饰的基因表达，从而促进 通过异常的Wnt信号导致肿瘤发生。为了验证这一假说，我们提出了两个目标。第一个目标是 用增强交联法和免疫沉淀法鉴定RM结合的RNA和修饰的m6A 测序技术。这将决定哪些转录本是RM的靶点，哪些腺苷残基是目标 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的计算集成将为rm融合靶向的rna的命运提供洞察。 蛋白质，与对照RBM15形成对比。第二个目的是研究候选蛋白在rm-1中的作用。 通过击倒生理相关小鼠中的特定候选基因来介导白血病的发生 巨核细胞系模型。对白血病发生的体外和体内评估将决定 这些候选者对于白血病细胞的体外存活和体内疾病的维持都是必需的。 这些实验将深入了解rm的转录和表位转录效应，这是至关重要的。 融合蛋白的机械功能。更好地理解RM介导的驱动机制 AMKL将极大地拓宽AMKL白血病发生的知识，并提供潜在的新疗法 改善患有这种疾病的新生儿的结局和存活率的途径。

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.