Instructive role of MLL fusion proteins in lineage determination and leukemogenesis

MLL 融合蛋白在谱系确定和白血病发生中的指导作用

• 批准号: 9290731
• 负责人: JAMES C MULLOY
• 金额: $ 60.92万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9290731
• 关键词: 11q23; AF-9 protein; Acute; Acute Lymphocytic Leukemia; B-Cell Acute Lymphoblastic Leukemia; Biological Models; CD34 gene; Cell Line; Cell model; Cells; ChIP-seq; Characteristics; Chimeric Proteins; Chromatin; Chromosome Band; Complex; Consensus; Dissection; Drosophila genus; Exhibits; Fusion Oncogene Proteins; Gene Expression; Gene Targeting; Genes; Genomics; HRX protein; Hematopoietic; Hematopoietic stem cells; Human; In Vitro; Instruction; Lead; Lymphoblastic Leukemia; Lymphoid; Lymphoid Cell; MLL gene; MLL-AF4; MLL-AF9; MLLT2 gene; MLLT3 gene; Maintenance; Mammals; Mediating; Modeling; Molecular; Molecular Profiling; Mus; Myelogenous; Myeloid Cells; Nature; Nuclear; Oncogenes; Oncoproteins; Outcome; Patients; Phenotype; Play; Process; Proteins; Reagent; Research Personnel; Role; Sampling; Signal Pathway; Signal Transduction; Stem cells; Testing; Therapeutic Intervention; Transcription Elongation; Transcriptional Activation; Yeasts; cell transformation; cell type; cytokine; human disease; human stem cells; in vivo; leukemia; leukemogenesis; novel; novel strategies; protein complex; protein purification; self-renewal; targeted treatment; trend

The MLL gene at chromosome band 11q23 is frequently rearranged in both acute myeloid and acute lymphoblastic leukemia. These translocations result in the formation of chimeric fusion proteins containing the N-terminus of MLL fused to the C-terminus of more than 70 different partner proteins. Although there are similarities in gene expression among the different MLL fusion proteins, MLL-AF4 (MA4), MLL-AF9 (MA9), and MLL-ELL (MEL) exhibit distinct gene expression profiles in leukemia cells obtained from patients. Many of the most common MLL partner proteins, including AF4, AF9, and ELL, are components of a super elongation complex (SEC) that is critical in transcriptional activation and elongation. Despite the identification that multiple MLL partner proteins are components of this complex, the basis for the differences in gene expression remains unclear. Many investigators refer to MLL-rearranged leukemia as a homogenous entity. However, the different fusions are found in different lineages. MA4 is almost exclusively found in pro-B ALL, MEL only in AML, and MA9 most commonly in AML but also in pre-B ALL. However, the basis for lineage specification by the different fusion partners is uncertain. Until now, it has not been possible to perform a direct comparison of the most common MLL fusions due to the lack of a tractable model of MLL-AF4 leukemia. We have developed a novel approach to express the MA4 fusion protein and have generated leukemia models using mouse and human hematopoietic stem and progenitor cells (HSPCs). Importantly, we have generated a faithful model of MA4 pro- B ALL. Using the unique reagents we have generated, we plan to examine the critical similarities and differences between these MLL fusion proteins. Each MLL fusion contains a triple FLAG tag that will permit the efficient purification of protein complexes and facilitate ChIP-seq to identify target genes. Although the partner protein complexes were identified several years ago, the complexes were immuno-precipitated using the partner proteins by themselves and not as MLL fusions. In addition, these purifications were performed in cell lines and not in primary leukemias induced by MLL fusions. In Aim 1, we will define the nature of the oncoprotein complexes formed in MLL-fusion transformed HSPCs and analyze their contribution to the initiation and maintenance of these leukemias. In Aim 2, we will identify critical downstream genes regulated by distinct MLL-fusion complexes in both myeloid and lymphoid cells. We will determine the genomic occupancy of each oncoprotein by ChIP-Seq. In this way we expect to identify those targets that are common to MLL and unique to each of the MLL-fusion proteins. In Aim 3, we will determine the cell of origin that is transformed in this MLL-fusion model system. We will express MA4, MEL, and MA9 in human stem and progenitor cells to determine whether each oncogene is able to induce leukemia in various progenitor cells and determine how cell of origin impacts leukemia type. Our studies are likely to have a large overall impact in the understanding of the mechanisms of transformation mediated by MLL fusion proteins and will provide key targets for therapeutic intervention.

位于染色体带 11q23 的 MLL 基因在急性髓系和急性髓系细胞中频繁重排。 淋巴细胞白血病。这些易位导致嵌合融合蛋白的形成，其中包含 MLL 的 N 末端与 70 多种不同伙伴蛋白的 C 末端融合。虽然有 不同 MLL 融合蛋白 MLL-AF4 (MA4)、MLL-AF9 (MA9) 之间基因表达的相似性 和 MLL-ELL (MEL) 在从患者获得的白血病细胞中表现出不同的基因表达谱。许多 最常见的 MLL 伴侣蛋白，包括 AF4、AF9 和 ELL，是超级伸长的组成部分 复合物（SEC）对于转录激活和延伸至关重要。尽管经多方鉴定 MLL 伴侣蛋白是该复合物的组成部分，基因表达差异的基础仍然存在 不清楚。许多研究人员将 MLL 重排白血病称为同质实体。然而，不同的 融合存在于不同的谱系中。 MA4 几乎只存在于 pro-B ALL 中，MEL 仅存在于 AML 中，并且 MA9 最常出现在 AML 中，但也出现在 B 期前期 ALL 中。然而，不同的血统规范的基础 融合伙伴是不确定的。到目前为止，还无法对大多数产品进行直接比较。 由于缺乏易于处理的 MLL-AF4 白血病模型，常见 MLL 融合。我们开发了一本小说 表达 MA4 融合蛋白的方法并使用小鼠和人类生成了白血病模型 造血干细胞和祖细胞（HSPC）。重要的是，我们已经生成了 MA4 pro 的忠实模型 球。使用我们生成的独特试剂，我们计划检查关键的相似点和差异 这些 MLL 融合蛋白之间。每个 MLL 融合都包含一个三重 FLAG 标签，可以有效地 纯化蛋白质复合物并促进 ChIP-seq 识别靶基因。虽然伴侣蛋白 几年前鉴定出复合物，使用伙伴蛋白对复合物进行免疫沉淀 本身而不是 MLL 融合。此外，这些纯化是在细胞系中进行的，而不是在 MLL 融合诱发的原发性白血病。在目标 1 中，我们将定义癌蛋白复合物的性质 在MLL融合转化的HSPC中形成并分析它们对启动和维持的贡献 这些白血病。在目标 2 中，我们将识别受不同 MLL 融合调节的关键下游基因 骨髓细胞和淋巴细胞中的复合物。我们将确定每种癌蛋白的基因组占有率 通过 ChIP 测序。通过这种方式，我们希望识别出 MLL 共有的目标以及每个目标所独有的目标。 MLL-融合蛋白。在目标 3 中，我们将确定在此 MLL 融合模型中转化的细胞来源 系统。我们将在人类干细胞和祖细胞中表达 MA4、MEL 和 MA9，以确定每个细胞是否 癌基因能够在各种祖细胞中诱导白血病，并确定起源细胞如何影响白血病 类型。我们的研究可能会对理解机制产生重大的总体影响 MLL 融合蛋白介导的转化将为治疗干预提供关键靶点。