MECHANISMS OF MLL REARRANGEMENT IN LEUKEMIA

白血病中 MLL 重排的机制

基本信息

批准号：
7872940
负责人：
ANDREW T. VAUGHAN
金额：
$ 39.73万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7872940
关键词：
Address Apoptosis Apoptotic BIRC4 gene Back Binding Sites Blood Blood specimen Caspase Cell Line Cell Nucleus Cells Chromatin Structure Chromosome Positioning Cleaved cell Clinical Commit Consensus DNA DNA Damage DNA Sequence Rearrangement Data Deoxyribonuclease I Derivation procedure Detection Development Disease Episome Etoposide Event Exons Exposure to Frequencies Generations Genes Genetic Genome Genomics Goals Healthcare Human In Vitro Individual Inverse Polymerase Chain Reaction Lesion Link Location MLLT3 gene Mediating Modeling Molecular Genetics Monitor Neomycin Neomycin resistance gene Neonatal Non-Hodgkin&apos s Lymphoma Nonhomologous DNA End Joining Pathway interactions Patients Process Production Proteins Recovery Research Personnel Resistance Risk Role Sampling Sequence Deletion Site Site-Directed Mutagenesis Small Interfering RNA Specificity Staging Stimulus Survivors System Techniques Testing Therapeutic Intervention Topoisomerase Topoisomerase II Topoisomerase II inhibition Topoisomerase-II Inhibitor Transfection Treatment Protocols base cell growth cell type clinically significant crosslink cytotoxic enzyme substrate fusion gene inhibitor/antagonist leukemia leukemogenesis malignant breast neoplasm novel nuclease programs repaired response vector

项目摘要

The following hypothesis is to be tested. "Transient activation ofapoptotic nucleuses Initiates MIL translocations with leukemogenicpotentiar. It is proposed that apoptotic execution may be arrested, after targeted DNA damage has occurred, in cells that survive. Preliminary data shows that apoptotictriggers initiate cleavage of MLL, which is subsequently translocated ioAF9, creating the leukemogenicMLL-AF9 fusion gene that is transcribed in cells capable of division. The relevance to health care is that this process would represent a novel pathwayfor the generation of fusion genes implicated in leukemogencsisthat would be open to therapeutic intervention. The Aims sequentially address the mechanism controllingcleavage within MLL, the growth of cells that survive and validationof these in-vitrodata with a clinical model of early stage leukemogenesis. Aim 1 Mechanism of MIX cleavage and translocation in response to apoptotic triggers. It is proposed that apoptotic cleavage of MLL is regulated by adjacent sequence motifs, including ATC tracts or topoisomerase LI binding sites. These possibilities will be tested by modifying each motif using site directed mutagenesis within an MLL containingepisome (pCEP4) that reproduces genomic MLL cleavage in cells undergoing apoptosis. Aim 2. Selection and analysis of cells that survive apoptotic activation. Cell lines will be selected using a pCEP4 episome containing a 367 bp MLL apoptotic cleavage motif adjacent to an out offrame neomycin resistance gene. Apoptotic cleavage and mis-rejoining ofthe MLL motif mayplace the neomycin back into its correct readingframe, allowing selection ofapoptotic survivors. Aim 3. Detection of. MLL translocations in patients at risk of therapy related leukemia. Blood taken from patients both before and during treatment for non-Hodgkins lymphoma or breast cancer will be examined for cells containing MLL-AF9 message or MLLrearrangements at the apoptotic cleavage site using inverse PCR. The ability of such cells to divide will be determined by the presence of duplicated genomic breakpoint junctions within each patients blood.

有待检验以下假设。 “凋亡细胞核的瞬时激活启动 MIL 具有致白血病潜能的易位。有人建议，细胞凋亡的执行可能会被阻止，之后存活的细胞中发生了针对性的 DNA 损伤。初步数据显示，细胞凋亡触发启动 MLL 的裂解，随后易位 ioAF9，产生白血病MLL-AF9 在能够分裂的细胞中转录的融合基因。与医疗保健的相关性在于，这个过程将代表一种与白血病发生有关的融合基因产生的新途径对治疗干预持开放态度。目标依次解决控制裂解的机制在 MLL 中，存活细胞的生长以及使用临床模型验证这些体外数据早期白血病发生。目标 1 MIX 裂解和易位响应的机制细胞凋亡的触发因素。据推测，MLL 的凋亡裂解受到相邻序列基序的调节，包括 ATC 束或拓扑异构酶 LI 结合位点。这些可能性将通过修改每个在含有附加体 (pCEP4) 的 MLL 中使用定点诱变的基序，可复制基因组正在经历凋亡的细胞中的 MLL 裂解。目标 2. 选择并分析凋亡后存活的细胞激活。将使用包含 367 bp MLL 凋亡裂解的 pCEP4 附加体选择细胞系与框架外新霉素抗性基因相邻的基序。细胞凋亡裂解和错误重新连接 MLL 基序可能会将新霉素放回其正确的阅读框，从而允许选择凋亡细胞幸存者。目标 3. 检测。存在治疗相关白血病风险的患者中的 MLL 易位。血取自非霍奇金淋巴瘤或乳腺癌治疗前和治疗期间的患者使用以下方法检查细胞凋亡裂解位点是否含有 MLL-AF9 信息或 MLL 重排：反向PCR。这些细胞分裂的能力将取决于重复基因组的存在每个患者血液内的断点连接。