Instructive role of MLL fusion proteins in lineage determination and leukemogenesis

染色体11q23区的mll基因在急性髓系白血病和急性髓系白血病中经常发生重排。 淋巴细胞性白血病。这些易位导致形成嵌合融合蛋白，包含 MLL的N-端与70多种不同配对蛋白的C-端融合。尽管有 不同MLL融合蛋白MLL-AF4(MA4)、MLL-AF9(MA9)、 和MLL-ELL(MEL)在从患者获得的白血病细胞中显示出不同的基因表达谱。许多. 最常见的MLL伙伴蛋白，包括AF4、AF9和ELL，都是超伸长的组成部分 在转录激活和延伸过程中起关键作用的复合体(SEC)。尽管被认定为多个 MLL伙伴蛋白是这个复合体的组成部分，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，不同的是 融合在不同的血统中被发现。MA4几乎只在PRO-B ALL中存在，MEL只在AML中存在，以及 MA9最常见于AML，也可见于前B-ALL。然而，世系规范的基础不同 Fusion合作伙伴尚不确定。到目前为止，还不可能对大多数 由于缺乏易处理的MLL-AF4白血病模型，常见的MLL融合。我们开发了一部小说 MA4融合蛋白的表达及人和鼠白血病模型的建立 造血干祖细胞(HSPC)。重要的是，我们已经生成了一个忠实的MA4 PRO模型- B全部。使用我们产生的独特试剂，我们计划检查关键的相似和不同之处 在这些MLL融合蛋白之间。每个MLL融合包含一个三重标志标签，它将允许高效 纯化蛋白质复合体，便于CHIP-SEQ鉴定目的基因。虽然伴侣蛋白 复合体是几年前发现的，这些复合体是用配对蛋白进行免疫沉淀的 而不是作为MLL融合。此外，这些提纯是在细胞系中进行的，而不是在 MLL融合所致的原发白血病。在目标1中，我们将定义癌蛋白复合体的性质 在MLL融合转化的HSPC中形成并分析它们对HPC启动和维持的贡献 这些白血病。在目标2中，我们将识别由不同的MLL-融合调控的关键下游基因 髓系细胞和淋巴样细胞中的复合体。我们将确定每种癌蛋白的基因组占有率 由CHIP-SEQ.通过这种方式，我们希望确定MLL共有的和每个 MLL-融合蛋白。在目标3中，我们将确定在这个MLL融合模型中转换的起源细胞 系统。我们将在人类干细胞和祖细胞中表达MA4、MEL和MA9，以确定每一个 癌基因能够在不同的祖细胞中诱发白血病，并决定起源细胞如何影响白血病 打字。我们的研究可能会对理解心力衰竭的机制产生很大的整体影响。 由MLL融合蛋白介导的转化，将为治疗干预提供关键靶点。

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.