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: 10115634
• 负责人: JAMES C MULLOY
• 金额: $ 59.45万
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115634
• 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; Genes; Genomics; HRX protein; Hematopoietic stem cells; Human; In Vitro; Instruction; Lead; Leukemic Cell; Lymphoblastic Leukemia; Lymphoid; Lymphoid Cell; MLL gene; MLL-AF4; MLL-AF9; MLL-rearranged leukemia; MLLT2 gene; MLLT3 gene; Maintenance; Mammals; Mediating; Modeling; Molecular; Mus; Myelogenous; Myeloid Cells; Nature; Nuclear; Oncogenes; Oncoproteins; Outcome; Patients; Phenotype; Play; Pre-B Acute Lymphoblastic Leukemia; Process; Proteins; Reagent; Research Personnel; Role; Sampling; Signal Pathway; Signal Transduction; Testing; Therapeutic Intervention; Transcription Elongation; Transcriptional Activation; Transplantation; Yeasts; cell transformation; cell type; cytokine; hematopoietic hierarchy; human disease; human stem cells; in vivo; leukemia; leukemogenesis; novel; novel strategies; protein complex; protein purification; self-renewal; stem cells; 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.

在急性髓系和急性白血病中，染色体带11 q23处的MLL基因经常重排。 淋巴母细胞白血病这些易位导致形成嵌合融合蛋白，所述嵌合融合蛋白含有 MLL的N-末端融合到70多种不同伴侣蛋白的C-末端。虽然有 不同MLL融合蛋白MLL-AF 4（MA 4），MLL-AF 9（MA 9）， 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配偶体蛋白，包括AF 4、AF 9和ELL，是超伸长的组成部分 复合物（SEC）在转录激活和延伸中至关重要。尽管有证据表明 MLL伴侣蛋白是这种复合物的组成部分，基因表达差异的基础仍然存在 不清楚许多研究者将MLL重排白血病称为同质实体。其不同之 融合存在于不同的谱系中。MA 4几乎仅见于pro-B ALL，MEL仅见于AML， MA 9最常见于AML，但也见于pre-B ALL。但是，不同的 融合伙伴是不确定的。到目前为止，还不可能直接比较 由于缺乏MLL-AF 4白血病的易处理模型，导致常见的MLL融合。我们已经开发出一种新颖 表达MA 4融合蛋白的方法，并使用小鼠和人产生白血病模型 造血干细胞和祖细胞（HSPC）。重要的是，我们已经产生了一个忠实的模型MA 4亲- B全部。使用我们已经生成的独特试剂，我们计划检查关键的相似性和差异 这些MLL融合蛋白之间的联系。每个MLL融合体含有三重FLAG标签，其将允许高效的融合。 蛋白质复合物的纯化和促进ChIP-seq鉴定靶基因。虽然伴侣蛋白 几年前鉴定了复合物，使用配偶体蛋白免疫沉淀复合物 而不是MLL融合。此外，这些纯化是在细胞系中进行的，而不是在细胞系中。 MLL融合诱导的原发性白血病。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HSPCs中形成，并分析它们对启动和维持 这些白血病在目标2中，我们将确定由不同的MLL融合调控的关键下游基因， 复合物在骨髓和淋巴细胞中。我们将确定每个癌蛋白的基因组占有率 通过ChIP-Seq。通过这种方式，我们希望识别出MLL共有的和每个MLL独特的目标。 MLL-融合蛋白。在目标3中，我们将确定在此ML融合模型中转换的原始细胞 系统我们将在人类干细胞和祖细胞中表达MA 4、MEL和MA 9，以确定是否每种细胞都能表达MA 4、MEL和MA 9。 癌基因能够在各种祖细胞中诱导白血病，并决定细胞起源如何影响白血病 类型.我们的研究可能会对理解这些机制产生巨大的整体影响。 MLL融合蛋白介导的转化，并将为治疗干预提供关键靶标。

项目成果

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia.

• DOI: 10.1182/blood.2021015036
• 发表时间: 2022-10-27
• 期刊: BLOOD
• 影响因子: 20.3
• 作者: Tirtakusuma, Ricky;Szoltysek, Katarzyna;Milne, Paul;Grinev, Vasily V.;Ptasinska, Anetta;Chin, Paulynn S.;Meyer, Claus;Nakjang, Sirintra;Hehir-Kwa, Jayne Y.;Williamson, Daniel;Cauchy, Pierre;Keane, Peter;Assi, Salam A.;Ashtiani, Minoo;Kellaway, Sophie G.;Imperato, Maria R.;Vogiatzi, Fotini;Schweighart, Elizabeth K.;Lin, Shan;Wunderlich, Mark;Stutterheim, Janine;Komkov, Alexander;Zerkalenkova, Elena;Evans, Paul;McNeill, Hesta;Elder, Alex;Martinez-Soria, Natalia;Fordham, Sarah E.;Shi, Yuzhe;Russell, Lisa J.;Pal, Deepali;Smith, Alex;Kingsbury, Zoya;Becq, Jennifer;Eckert, Cornelia;Haas, Oskar A.;Carey, Peter;Bailey, Simon;Skinner, Roderick;Miakova, Natalia;Collin, Matthew;Bigley, Venetia;Haniffa, Muzlifah;Marschalek, Rolf;Harrison, Christine J.;Cargo, Catherine A.;Schewe, Denis;Olshanskaya, Yulia;Thirman, Michael J.;Cockerill, Peter N.;Mulloy, James C.;Blair, Helen J.;Vormoor, Josef;Allan, James M.;Bonifer, Constanze;Heidenreich, Olaf;Bomken, Simon
• 通讯作者: Bomken, Simon